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amyotrophic lateral sclerosis ( als ) is an adult - onset motor neuron disease is characterized by selective degeneration of motor neurons in the brain and spinal cord , resulting in progressive paralysis of limb , facial , and respiratory muscles and death within few years [ 1 , 2 ] .
the pathological hallmarks of als are the atrophy of dying motor neurons and the accumulation of the lewy body - like inclusions and skein - like inclusions in the degenerated motor neurons which is surrounded by reactive astrocytes and microglia .
the exact composition of such inclusions is incompletely understood , although the protein identified so far includes ubiquitin , cu / zn superoxide dismutase 1 ( sod1 ) , dorfin ( a ring - finger - type e3 ubiquitin ligase ) , and nedl1 ( nedd4-like ubiquitin protein ligase-1 ) , which has been identified as a novel gene expressed significantly at high levels in favorable neuroblastoma relative to unfavorable ones .
nedl1 encodes hect - type e3 ubiquitin ligase and is detected specifically in human neuronal tissues , suggesting that nedl1 might be involved in the regulation of the spontaneous regression of favorable neuroblastomas caused by apoptosis and/or neuronal differentiation .
the property of nedl1 protein is also affected by binding with mutated sod1 protein .
the biological role of nedl1 involved in the pathogenesis of als is incompletely understood .
therefore , we generated the human nedl1 transgenic ( hnedl1-tg ) mice and studied the several behavioral batteries of test including hanging wire test , rotarod test , and foot print test . here , we found that the hnedl1-tg mice exhibited decreased locomotor activity compared with wild - type mice and developed als - like symptoms , including motor neuron degeneration , decreased axon , and microglia activation in the lumbar spinal cord and muscle weakness .
the plasmid that contained the human nedl1 cdna was generated by pcr , and the accession number of human nedl1 nucleotide sequence is nm_015052 in genbank / ebi data bank . the nedl1 cdna with kozak sequence that was subcloned into the pcaggs expression vector contained the regulatory elements of the cag promoter and linearized sali and bamhi sites ( figure 1(a ) ) .
transgenic mice were generated by pronuclear injection of the purified dna fragment ( 3 ng/l ) into fertilized eggs obtained from intercrosses between c57bl/6j ( clea , japan ) .
this resulted in 4 transgenic lines identified by pcr and each progeny was analyzed for the hnedl1 expression by semiquantitative rt - pcr and western blotting analyses .
a transgenic line which showed the strongest expression level of hnedl1 was used in this study .
the mice were housed in a temperature- ( 24 2c ) and humidity- ( 40%60% ) controlled room with a 12 : 12 hr light - dark cycle .
all experiments using the mice were performed according to the guidelines for animal experimentation , chiba cancer center , and chiba university .
rna was extracted from different organs of wild - type and hnedl1-tg mice by using rneasy fibrous tissue mini kits ( qiagen , valencia , ca , usa ) according to the manufacturer 's protocol ; cdna was generated from 1 g of total rna using random primers and superscript ii reverse transcriptase ( invitrogen , carlsbad , ca , usa ) .
pcr was performed using the cdna as template with the following primers : hnedl1 forward , 5-tatcagaaggtggtggctggtgtgg-3 , and reverse 5-tatgactgggagtagtcaggagagg-3 ; mouse gapdh forward , 5-atcttcttgtgcagtgccag-3 and reverse 5-gagatgatgacccgtttggc-3. pcr products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining .
protein was extracted from different organs of wild - type and nedl1-tg mice , respectively .
the extraction was performed in lysis buffer ( 25 mm tris - hcl , ph 7.5 , 137 mm nacl , 2.7 mm kcl , 1% triton x-100 , 1 mm pmsf , and protease inhibitor cocktail ) ( roche diagnostics , mannheim , germany ) .
equal amounts of protein were separated in sds - polyacrilamide gel and transferred onto pvdf membrane ( millipore corporation , bedford , ma , usa ) .
the membrane was incubated with rabbit polyclonal anti - human - nedl1 antibodies ( raised against c - terminal 17 amino acids of human nedl1 : swkpeqlgegsvpdrpg ) ( mbl , japan ) or with rabbit polyclonal anti -actin antibodies ( 2033 ) ( sigma , st .
louis , mo , usa ) , followed by incubation with the hrp - conjugated secondary antibodies ( jackson immunoresearch laboratories , west grove , pa , usa ) , respectively .
indicated proteins were visualized by using ecl system ( amersham biosciences , piscataway , nj , usa ) .
locomotor activity was measured using an animal movement analysis system ( scanet sv-10 , melquest , toyama , japan ) , as reported previously .
the side walls ( height : 60 mm ) of the enclosure were equipped with 144 pairs of photosensors located at 5 mm intervals at a height of 30 mm from the bottom edge .
a pair of photosensors was scanned every 0.1 s to detect the animal 's movements .
the intersection of paired photosensors ( 10 mm apart ) in the enclosure was counted as one unit of locomotor activity .
data collected for 120 min were used in this study . motor function and skeletal functional performance
were assessed by using rotarod ( mk-610a , muromachi kikai , tokyo , japan ) .
eleven male hnedl1-tg and twelve wild - type mice , respectively , were tested during the age of six to fifteen months old .
after getting acquainted to the apparatus , animals were placed onto the apparatus at a constant speed of 20 r.p.m .
the time which an animal could remain on the rotating rod was recorded in three consecutive attempts ; 300 seconds was chosen as the arbitrary cutoff time .
mice utilized their forelimbs to suspend their body weight on a wire stretched between two posts 60 cm and 20 cm above a foam pillow .
a score of zero was assigned if the mouse fell immediately and 60 seconds was the timeout period .
the limb paws of six - month - old mice were inked with black ( forelimb ) and blue ink ( hind limb ) , and mice were allowed to walk over a white paper strip in a straight - alley runway ( 50 cm long ) to an enclosed goal box .
tracks were collected , and the fore- and hind limbs stride length was calculated and analyzed . six - month - old wild - type and hnedl1-tg mice were sacrificed .
the segments of skeletal muscles were taken from hind legs of both wild - type and hnedl1-tg mice , fixed with 10% formalin , and embedded in paraffin blocks .
the 7 m sections were stained for hematoxylin and eosin . for neuron quantification , brain and spinal cord were isolated from mouse , fixed with 10% formalin , and embedded in paraffin blocks .
the paraffin blocks were sliced into 6 m sections , and bodian staining was performed .
the numbers of motor neuron in the total motor neuron and in the degenerated motor neuron from wild - type mice and hnedl1-tg mice were measured .
isolated spinal cord was embedded in tissue - tek o.c.t compound ( 4583 , sakura finetek japan co. , ltd . ,
the frozen spinal cord tissue was sliced into 15 m sections , and the sections were incubated 1 - 2 h with primary antibody rabbit anti - iba-1 ( cat no .
osaka , japan ) at room temperature , followed by processing the avidin - biotin - peroxidase method ( vectastain elite abc , vector laboratories , inc . ,
burlingame , ca , usa ) , as described previously [ 13 , 14 ] .
statistical analysis was performed using one - way analysis or two - way analysis of variance ( anova ) .
to understand the role of nedl1 involved in pathogenesis of familial als , we generated transgenic mice with the human nedl1 cdna driven by cag promoter that combines the cytomegalovirus enhancer and a chicken -actin promoter and intron ( figure 1(a ) ) .
the cag promoter is a very strong and ubiquitous promoter , and allows expressing foreign genes at high levels in vivo .
we obtained four independent hnedl1-tg mice lines and confirmed the hnedl1 expression in each line ( data not shown ) , and the hnedl1-tg mouse line that showed the strongest expression level of hnedl1 was used in this study .
the expression of hnedl1 in different organic tissue was examined by rt - pcr and western blotting analysis .
although the hnedl1 expression was confirmed in all tissues , a slightly higher expression of hnedl1 was confirmed in brain and skeletal muscle ( figures 1(b ) and 1(c ) ) .
since being five months old , spontaneous locomotor activity of the hnedl1-tg mice was significantly lower than that of wild - type mice ( figure 2(a ) ) .
then , we assessed the motor neuron function and the performance of skeletal muscle by rotarod test , hanging wire test , and foot print test .
as shown in the figures ( figures 2(b ) and 3(a ) ) , there are significant differences between hnedl1-tg mice and wild - type mice in the rotarod test ( f ( 1 , 21 ) = 25.392 ; p < .001 ) and hanging wire task ( f ( 1 , 21 ) = 41.173 ; p < .001 ) , during the age from six months to fifteen months .
the footprint test proved an eloquent method of quantifying the motor function ; the stride length in the hnedl1-tg mice was shortened about 2 cm compared with that in the wild - type mice ( figure 3(b ) ) .
histological study showed that there is no degenerated neuron ( figure 4 ) nor activated microglia ( data not shown ) in the frontal cortex , striatum , and hippocampus of six - month - old hnedl1-tg mice .
however , the degenerated motor neurons and decreased axon were clearly shown in the lumbar spinal cord , compared to these of the age - matched wild - type mouse ( figure 4 ) .
in addition , significant muscle atrophy was observed in the hnedl1-tg mouse ( figure 5(a ) ) .
there is no change of the number of iba-1 immunoreactive cells ( microglia cells ) in both hnedl1 transgenic mice and wild - type mice ( figure 5(b ) ) .
however , the number of activated microglia in the hnedl1 transgenic mice was significantly higher than that of wild - type mice , suggesting that microglia activation ( e.g. , inflammation ) might occur in the lumbar spinal cord of hnedl1 transgenic mice ( figure 5(b ) ) .
in this paper study , we found that the hnedl1 might be involved in pathology of als - like symptoms , such as motor neuron degeneration , decreased axon length , and skeletal muscle atrophy . in the hnedl1-tg mice
, hnedl1 is overexpressed ubiquitously in vivo since the promoter used in this study is the cag promoter .
in fact , the expression of the hnedl1 was observed not only in the central nervous system and muscle but also in the kidney and liver .
nevertheless , it should be noted that the phenotype of the hnedl1-tg mice has only been detected in muscular and nervous system .
there is increasing evidence for the ubiquitin - mediated proteasome pathway involvement in pathogenesis of neurodegenerative disease , including als , parkinson 's disease , and huntington 's disease .
these diseases are associated with degeneration of specific neuronal populations due to misfolding or aggregation of certain proteins .
these aggregates are always ubiquitin - positive and insoluble presence in the lewy - like inclusion body [ 15 , 16 ] .
generally , the inclusion bodies consist of ubiquitinated polypeptides that fail to be destroyed by the 26s proteasome , and the apparent stability may be due to decreased levels of 26s proteasomal activity , which is associated with increasing age or mutated sod1 .
ubiquitin - protein ligases e3 are to particular importance of the protein quality control system , since they determine the targeting protein specifically .
recently , immunohistochemical analysis revealed that the nedl1 and dorfin serve as e3 ligases , localized within the lewy - like inclusion body in the spinal cord of als patients and mutated sod1 transgenic mice [ 9 , 10 ] .
they specifically interacted with mutated sod1 , but not the wild type sod1 , and promoted degradation of the mutant sod1 , protected neuronal cells from the toxicity caused by mutant sod1 [ 9 , 10 ] , suggesting the potential role of nedl1 as a modifier for familial als .
surprisingly , in this study , we found that overexpression of hnedl1 in tg mice caused the neuron degeneration , neural axon decrease , and microglia activation in the lumbar spinal cord .
loss of the neuron and activation of microglia in the central neuron system , which is a common feature of neurodegenerative disease , were not observed in the brain tissue , but in the spinal cord of the hnedl1-tg mice .
mutant sod1 physically binds to the nedl1 and causes the conformational changes of nedl1 protein , increasing binding intensity to dvl 1 .
that results in degradation of dvl 1 and decrease of phosphorylated c - jun in the downstream .
the abnormality of the nedl1 e3 ligase activity is responsive for the neuron death in the spinal cord of the mutant sod1-tg mouse , and is also the considerable reason in the case of hnedl1-tg mice .
it is likely that muscular atrophy that occurred in the hnedl1 transgenic mice may be the consequence of the neuron death in the spinal cord or the extra nedl1 expression in the muscle tissues , like that atrogin-1 ( mafbx ) , a ubiquitin ligase is necessary for rapid muscular atrophy .
accumulating evidence suggests that microglial neuro inflammation plays a role in the pathogenesis of als [ 2023 ] .
a recent study suggests that the early activity of microglia is involved in the early phase of motor neuron degeneration of human mutant sod1 murine transgenic model of als . in this study
, we found that microglial activation might have occurred in the lumbar spinal cord of hnedl1 mice , suggesting that microglial activation may be involved in the behavioral abnormalities of hnedl1 mice .
it has been reported that the second - generation antibiotic drug minocycline , a potent inhibitor of microglial activation , slows disease progression in a mouse model of als [ 24 , 25 ] and that minocycline attenuates microglial activation by abused drugs [ 13 , 14 ] .
a number of trophic factors , antiinflammatory agents and inhibitors of oxidative stress have been reported to prolong survival in mouse models of als and some are now in clinical trials [ 26 , 27 ] .
therefore , it may be of interest to examine the effects of minocycline in the behavioral abnormalities of hnedl1 transgenic mice .
in conclusion
, this study suggests that overexpression of hnedl1 might be implicated in the degeneration of motor neuron in lumbar spinal cord although a further detailed study underlying the role of the nedl1 in the pathogenesis of familial als should be necessary .
furthermore , the elucidation of the mechanisms underlying motor neuron pathology in hnedl1 transgenic mice shed light on the early pathological events of familial als and might then open avenues for potential therapeutic targets in als and other motor neuron neurodegenerative disorders . | amyotrophic lateral sclerosis ( als ) is the most frequent adult - onset motor neuron disease
. approximately 20% cases of familial als show the mutation in the superoxide dismutase-1 ( sod1 ) gene .
we previously demonstrated that homologue to e6ap carboxyl terminus- ( hect- ) type ubiquitin protein e3 ligase ( nedl1 ) physically bind to mutated sod1 protein but not wild - type sod1 and promote the degradation of mutated sod1 protein through ubiquitin - mediated proteasome pathway . to further understand the role of nedl1 involved in the pathogenesis of familial als , we generated transgenic mice with human nedl1 cdna .
the transgenic mice with human nedl1 expression showed motor dysfunctions in rotarod , hanging wire , and footprint pattern examination .
histological studies indicated degeneration of neurons in the lumbar spinal cord and muscle atrophy .
the number of activated microglia in the spinal cord of transgenic mice was significantly higher than that of wild - type mice , suggesting that inflammation might be observed in the spinal cord of transgenic mice . in conclusion
, these findings suggest that the human nedl1 transgenic mice might develop als - like symptoms , showing signs of motor abnormalities , accompanied with significant reduction in muscle strength . |
two american scientists ( patients 1 and 2 ) lived and worked in the village of bandafassi in southeastern senegal in august 2008 while performing a mosquito - sampling project in surrounding villages .
patients 1 and 2 were men ( 36 and 27 years of age , respectively ) , and both had received the yellow fever 17d vaccine before their travel to senegal . during their project ,
mosquitoes in the evenings while they worked . patients 1 and 2 left bandafassi on august 21 , stayed in dakar for 2 days , and then returned to their homes in northern colorado on august 24 .
symptoms in patient 1 began on august 30 and consisted of swollen ankles , a maculopapular rash on his torso , and extreme fatigue and headache , but no fever was recorded . on august 31 , he experienced the same symptoms and light - headedness and chills , wrist and ankle arthalgia , and symptoms of prostatitis ( perineal pain and mild dysuria ) .
fatigue and rash decreased on september 1 ; only residual wrist arthralgia , headache , and prostatic symptoms persisted . on september 2
, two aphthous ulcers appeared on his lip . on september 3 , he and his wife observed signs of hematospermia ( red brown fluid in his ejaculate ) that lasted until september 7 .
patient 2 experienced his symptoms during august 29september 1 , which included a maculopapular rash on his torso , extreme fatigue , headache , and swelling and arthralgia in his wrists , knees , and ankles .
acute - phase blood samples were obtained from both patients on september 2 . in patient 3 ( a nurse and the wife of patient 1 ) similar clinical symptoms developed on september 3 , including malaise , chills , extreme headache , photophobia , and muscle pain that continued through september 6 .
she did not have detectable fever . on september 7 , a maculopapular rash developed on her torso ( figure ) that expanded to her neck and thighs on the following day , and an aphthous ulcer developed on her inside lip . on september 8 ,
patient 3 had an acute - phase blood sample drawn on september 8 . on september 11
, she visited her primary care physician , who performed a complete blood count test and studies of hepatic function ; all results were within reference ranges .
patient 2 reported wrist arthralgia for 1 month after his acute illness , and patients 1 and 3 have had recurring wrist or thumb joint arthralgia since their acute illness .
convalescent - phase blood samples were drawn on september 22 from patients 1 and 2 and on september 26 from patient 3 .
acute - phase and convalescent - phase paired serum specimens from the 3 patients were tested independently by several different laboratories .
results of virus isolation were negative for all samples when tested in vero and aedes albopictus mosquito ( c6/36 ) cell cultures and by intracerebral inoculation of acute - phase serum of patient 3 into suckling mice .
pcrs with 16 different sets of arbovirus - specific primers did not detect arboviral rna in any of the samples .
serologic analyses ( table a1 ) of samples from patients 1 and 2 showed matching results .
hemagglutination inhibition antibody titers and virus neutralizing titers were highly elevated above background levels for zikv and yellow fever virus ( yfv ) compared with other viruses tested .
these titers most often increased in the time between obtaining acute - phase and convalescent - phase serum samples .
hemagglutination inhibition , complement fixation , and virus neutralizing titers against zikv alone developed only in the convalescent - phase sample of patient 3 .
evidence suggests that patients 1 and 2 were infected with zikv , probably in southeastern senegal , by bites from infected mosquitoes .
the village of bandafassi is located in a disease - endemic area where zikv has been isolated from humans , nonhuman primates , and mosquitoes ( 4,5 ) .
serologic results suggest an anamnestic response to zikv infection , likely stemming from their vaccination with yfv .
the time between infection and the onset of clinical manifestations can be inferred to be > 9 days , given the patients travel history .
their clinical symptoms are consistent with reported symptoms of zikv - associated disease ( 3,69 ) .
exceptions are aphthous ulcers in patient 1 ( also reported by patient 3 ) , prostatitis , and hematospermia . whether these exceptions are typical but unreported symptoms or clinical anomalies is not clear .
patient 3 had never traveled to africa or asia and had not left the united states since 2007 .
circumstantial evidence suggests direct person - to - person , possibly sexual , transmission of the virus .
temperatures and mosquito fauna on the northern front range in colorado when transmission occurred do not match known mosquito transmission dynamics of zikv by tropical aedes species .
patient 3 had zikv disease 9 days after the return of her husband from senegal . however , the extrinsic incubation period of zikv in ae .
area temperatures during the week of return of patient 1 fluctuated between 10c and 31c , only ae .
vexans mosquitoes of the subgenus aedimorphus are commonly captured on the northern colorado front range , and known tropical zikv vectors are mostly from the subgenus stegomyia ( 4 ) .
mosquito sampling around the home of patients 1 and 3 at the time yielded only 7 ae .
furthermore , patients 1 and 3 reported having vaginal sexual intercourse in the days after patient 1 returned home but before the onset of his clinical illness .
it is reasonable to suspect that infected semen may have passed from patient 1 to patient 3 during coitus .
another possibility is that direct contact and exchange of other bodily fluids , such as saliva , could have resulted in zikv transmission , but illness did not develop in the 4 children of patients 1 and 3 during this time . to the best of our knowledge , human sexual transmission of an arbovirus has not been documented .
however , japanese encephalitis virus was discharged into the semen of experimentally infected boars and could infect female pigs by artificial insemination ( 11 ) . furthermore , west nile virus rna and st .
louis encephalitis virus antigen have been detected in urine of humans ( 12,13 ) , and viruria has occurred in hamsters infected with west nile virus ( 14 ) and modoc virus ( 15 ) . if sexual transmission could be verified in subsequent studies , this would have major implications toward the epidemiology of zikv and possibly other arthropod - borne flaviviruses . | clinical and serologic evidence indicate that 2 american scientists contracted zika virus infections while working in senegal in 2008 .
one of the scientists transmitted this arbovirus to his wife after his return home .
direct contact is implicated as the transmission route , most likely as a sexually transmitted infection . |
among cancers affecting women , cervical cancer has the second highest occurrence worldwide , with an incidence in 2008 of 529,800 cases ( 14.5% in developed countries and 85.5% in developing countries ) and 275,000 estimated deaths .
infection of cervical epithelial cells with high risk human papillomavirus ( hpv ) is the most important risk factor for development of cervical cancer , as first highlighted by zur hausen .
noninvasive cervical intraepithelial neoplasia ( cin ) precedes the development of invasive cancer and may progress from cin2 - 3 to ( micro)invasive cancer in 1025 years on average .
three cin grades ( cin1 , cin2 , and cin3 ) are recognized by the world health organization to distinguish the degrees of epithelial abnormality and are associated with increasing risks for invasive cancer development .
a cin lesion is , however , not a static event but a dynamic process that can persist and progress but also spontaneously regress [ 4 , 5 ] .
if left untreated , 530% of all cin2 - 3 lesions ( confirmed by a histological punch biopsy ) will develop invasive cancer . on the other hand , without cone excision , as many as 3243% of cin2 - 3 lesions
will regress spontaneously . nevertheless , in many countries including norway , all punch biopsy - confirmed cin2 - 3 lesions are usually treated with diathermic cone excision , a fairly aggressive therapy which can have serious adverse side effects .
the most serious late - complication is cervical insufficiency which can lead to late abortion and preterm delivery during the second and early third trimester of a future pregnancy [ 9 , 10 ] . until recently , regression of cin2 - 3 lesions
however , research on functional biomarkers like prb , p53 , and cytokeratin 13/14 has proven to be helpful in predicting regression , especially when combined with local immune response and hpv genotype [ 1114 ] .
furthermore , combined ki67 and prb expression can predict which cin1 lesions will progress to cin3 .
aggregated information provided by such epithelial biomarkers and local cellular immune response in the microenvironment of cin2 - 3 lesions supports prediction of regression / persistence / progression and may result in even more accurate cin treatment , as well as reducing overtreatment of patients with cin2 - 3 lesions .
unfortunately , the procedures used to obtain formalin - fixed , paraffin - embedded ( ffpe ) tissue from biopsies irreversibly degrade water - soluble proteins . a protein collection method for small punch biopsy samples that could represent not only the cellular response but also proteins from the cervical neoplasia microenvironment and intracellular compartments may further help define the biology of cin lesions ' dynamic behaviour .
we have recently described a method that can preserve and extract water - soluble proteins from punch biopsies , how a panel of 3 peaks from seldi - tof protein profiles can be used to differentiate normal tissue from cin tissue samples , and that a discrimination between cin2 and cin3 lesions could be obtained using cytokeratin 2 . in the present study we analysed protein samples from cin2 - 3 lesions with known regression / persistence status .
we have used both seldi - tof ms and bottom - up shotgun proteomics approach utilizing nanoflow liquid chromatography coupled to a ltq - orbitrap mass spectrometer .
the goal was to identify proteins that could be used in prediction of regression or persistence in cin2 - 3 .
this study is a subproject from a larger prospective study , approved by the regional medical ethics committee of helse vest , norway , the norwegian data inspection , and the health directorate of norway , numbers 33.06 , 17185 , and 07/330 .
healthy women aged 25 - 40 years , with cytological abnormal smears were followed by cervical biopsy and later cone excision . in total , 170 patients with first time onset of cin2 - 3 were included from january 2007 to december 2008 .
the interval between punch biopsy and cone excision was standardized at median 113 days ( range : 100126 ) .
this interval was chosen in view of a previous study , which showed that cin2 - 3 patients with more than 9-week punch - cone interval have a much higher chance on regression than those with < 9-week interval .
regression was defined as cin1 or less in cone histology and regression rate was 22% ( 38/170 ) .
all patients included in this study were treated according to the national norwegian population screening quality guidelines . in our cohort of patients we first analysed whether proteins and peptides detected by proteomic lc - ms ( ltq - orbitrap ) could distinguish between cin2 - 3 lesions , with and without later regression .
of the 170 patients with cervical punch biopsy samples , a random subset of 20 patients with cervical intraepithelial neoplasia lesions ( see below for reviewing details ) , 10 cin2 ( 5 with regression and 5 with persistence ) and 10 cin3 ( 5 with regression and 5 with persistence ) , were selected and defined as the learning set .
the samples were selected so that the whole sampling period was covered and the protein concentration was as close as possible to the average for the whole data set . in a second validation study ,
another 20 cin2 - 3 patients ( 10 cin2 cases , 5 with regression and 5 with persistence , and 10 cin3 , 5 with regression and 5 with persistence , defined as the validation set ) were selected to test the prognostic value of the proteins found in the learning set .
for the seldi - tof study , the sample set from a former investigation was used .
thus 2 replicates of each of the 5 regression and 40 persistent cin2 - 3 samples were included for this part of the study .
after colposcopy , punch biopsies and endocervical curettage were taken from the transformation zone and eventually premalignant mucosa .
one or two biopsies were immediately placed in polystyrene tubes ( sarsted , numbrecht , germany ) containing 5 ml rpmi-1640 ( gibco , carlsbad , usa ) tissue culture medium .
the biopsies were kept in the tissue culture medium for 24 hours at 4c before the supernatants were collected , split into aliquots of 500 l , and stored at 80c until analysis . immediately after sample collection , an additional set of biopsies
as described before after 24 hours of incubation in rpmi-1640 medium at 4c , the biopsies were routinely fixed in buffered 4% formaldehyde , embedded in paraffin , cut at 4 m , and stained with hematoxylin , eosin , and safran ( he s ) for routine histological examination .
p16 and ki67 ( mib-1 ) immunohistochemical ( ihc ) staining were used to confirm the diagnosis .
all he s and ihc sections of the 170 biopsies were reviewed by two independent pathologists , who also used the p16 and ki67 immunohistochemical information .
the participating pathologists were blinded to the original routine clinical findings , histopathological diagnosis , and follow - up . in case of discrepancies
the cases were reviewed and diagnosed on a double - head microscope by the two pathologists ( einar gudlaugsson and jan baak ) and a consensus diagnosis was obtained .
samples were subjected to seldi - tof ms profiling according to the manufacturer 's instructions ( ciphergen biosystems , fremont , ca , usa ) .
the biopsy supernatants were diluted 1 : 5 with 50 mm sodium acetate ( ph 4.3 ) and then bound to a cm10 proteinchip array .
they were incubated for two hours at room temperature on a platform shaker and then washed twice with 50 mm sodium acetate buffer , followed by two washes of 1 l energy absorbing molecule ( = eam ) solution ( consisting of 50% saturated synaptic acid dissolved in 50% acetonitrile and 0.5% trifluoroacetic acid ) .
two replicates were prepared on different cm10 proteinchips by two different technicians on two different days .
the time - of - flight spectra were generated on the protein biological system ii mass spectrometer reader ( ciphergen biosystems , fremont , ca , usa ) , using a laser intensity of 170 and a detector sensitivity of seven .
the seldi - tof ms data analyses were performed in three steps : ( 1 ) peak detection , ( 2 ) selection of peaks with the highest discriminatory power , and ( 3 ) building a multivariate model based on the selection in step ( 2 ) . the peak detection was done using the ciphergen seldi software version 3.2 after internal and external mass calibration followed by normalization ( total ion current , tic , intensity ) of all spectra as one group . the mass range from 2000 to 20000 da contained the majority of the peptides / proteins in the samples and
masses less than 2000 da were excluded as these are known to contain adducts and artifacts from the eam solution and other chemical contaminants .
the peak detection includes baseline subtraction , calibration of mass accuracy , and automatic peak detection .
each spectrum was then assigned to one of three groups , normal , regression , or persistence . to select peaks with the highest discriminatory power , the biomarker wizard ( ciphergen ) was used for peak detection and clustering of all the spectra .
this was done using a signal - to - noise ( s / n ) ratio of 5 and 15% of all spectra for the first pass detection and clustering and an s / n ratio of 2 for the second pass .
the cluster results were then imported into spss ( v17 , spss norway as , oslo , norway ) , cart ( salford , san diego , ca , usa ) , and medcalc ( medcalc software , mariakerke , belgium ) for binary logistic regression analysis . the preparation and use of the immunoaffinity column
is described in . to deplete samples of the 7 high abundance proteins , 100 l of rpmi supernatant
was diluted with 100 l tris - buffered saline ( tbs , 0.1 m tris - base containing 0.1 m nacl , ph 8.0 ) , and the solution was injected into a tbs solution with a flow of 0.2 ml / min .
the nonretained proteins were trapped on a 4 mm 2.0 mm ( inner diameter , i.d . )
, c18 security guard cartridge with 300 pore size ( phenomenex , teknolab , norway ) and were eluted by backflushing the security guard cartridge with ethanol at a flow of 0.3 ml / min .
the affinity column was washed using 0.1 m glycine at ph 2.5 with a flow of 1.2 ml / min .
both columns were reequilibrated with tbs at a flow of 0.2 ml / min for 5 minutes .
after evaporating the ethanol phase containing the nonretained protein fraction using vacuum centrifugation ( eppendorf concentrator 5301 , vwr , norway ) , 100 l 50 mm ammonium bicarbonate ph 8 was added to the samples . 1 l of 1 m dithiothreitol ( dtt ) was added to reduce the proteins .
5 l of 1 m iodoacetamide ( iaa ) was then added to alkylate the proteins followed by 5 l of dtt to stop the alkylation process .
one g trypsin ( promega ) was added , and the samples were kept at 37c for 18 hours .
after digestion with trypsin the samples were purified and concentrated using a c18 ziptip ( millipore , norway ) procedure .
the ziptips were conditioned by aspirating 30 l acetonitrile five times and equilibrated with pulling 30 l 0.1% formic acid ( fa ) in milliq water five times through the stationary phase .
approximately 10 l of the 0.1% fa solution was left above the stationary phase to avoid drying it .
each sample solution was applied on top of the stationary phase using a pipette and then pushed through the tip using air pressure from the pipette plunger .
more sample solution was added when approximately 20 l of the liquid remained so that the whole volumes of the samples were pushed slowly through the ziptip .
elution of the peptides was done in a total volume of 30 l of 80 : 20 ( v / v ) acetonitrile : milliq water by aspiring 10 l of this solution 10 times through the stationary phase .
the organic phase was then evaporated using vacuum centrifugation and , to the residual solution , 20 l 0.1% fa was added prior to the lc - ms / ms analysis . a dionex ultimate 3000 nanoflow hplc equipped with a 300 m ( i.d . )
0.5 cm length acclaim pepmap 100 c18 trap column and a 75 m ( i.d . )
15 cm acclaim pepmap 100 c18 analytical column ( dionex ) was used with a ltq - orbitrap hybrid mass spectrometer ( thermo scientific ) . 5 l of the tryptic digests was injected onto the trap column using 0.1% formic acid ( vwr ) in milliq - water at a flow of 2 l / min .
the separation was done using a gradient from 2.5% to 64% acetonitrile in 0.1% fa over 180 minutes at a flow of 300 nl / min .
the electrospray interface was a picotip emitter ( silicatip , new objective ) with a 10 m tip without coating .
full scans were performed in the orbitrap using the m / z range from 200 to 2000 .
data dependent ms / ms scans were performed in the ltq for the five most abundant masses with z 2 and intensity higher than 10,000 counts .
dynamic exclusion for 3 minutes after fragmentation of a given m / z value four times was used .
collision induced dissociation ( cid ) was used with a collision energy of 35% , activation q setting of 0.400 , and 30 ms activation time for ms .
calibration of the mass spectrometer was done weekly using the calibration solution recommended by thermo scientific .
the raw data files were analysed using the proteome discoverer 1.0 ( thermo scientific ) with the sequest algorithm to search against the homo sapiens ( tax.id : 9606 ) database at ncbi ( 531420 sequences ) with trypsin as digestion enzyme allowing for 2 missed cleavages .
all files were also searched against the human papillomavirus database ( tax.id : 10566 ) at ncbi ( 1615 sequences ) .
precursor ion tolerance was set to 10 ppm , and fragment ion mass tolerance was set to 0.8 da .
oxidation ( m ) was set as a dynamic modification and carbamidomethyl ( c ) was set as a static modification due to the use of dtt and iaa .
a high significance peptide confidence filter was set in proteome discoverer ( pd ) from thermo , which means that peptide identifications are filtered based on the following combination of charge and xcorr factor : 1.9 ( z = 2 ) , 2.3 ( z = 3 ) , and 2.6 ( z 4 ) . additional information for proteins was obtained from the uniprot database entry .
protein identifications were accepted using one peptide when certain requirements were fulfilled : the sequest xcorr factor with regard to charge had to be fulfilled according to the high significance criteria in pd . the peptide had to contain at least 7 amino acids and have at least three consecutive b- and y - ions in the ms2 spectra , and it should occur minimum three times in the same sample .
in addition , for proteins with only one identified peptide sequence , the peptide sequences were submitted for a blast search against the uniprot homo sapiens database ( htttp://www.uniprot.org/ ) to confirm that the identification matched the ncbi identification . for proteins listed as unnamed in the ncbi database , the i d mapping tool at uniprot was used to see if the protein was listed with a more descriptive annotation in this database .
only proteins identified in at least 30% of the samples in one of the groups ( regression / persistence ) were included in the remaining work .
spectral count ( spc ) results for the identified peptides were obtained and used for normalization ( see ( 1 ) ) after increasing all numbers with 1 to avoid zeros .
consider
( 1)spc normprotein x , sample x , group y = spcprotein x , sample xsum spcsample xavg .
spcgroup y. the normalized spc results were imported into sirius ( version 8.1 , pattern recognition systems , bergen , norway ) to perform a partial least - squares discriminant analysis ( pls - da ) using a target projection component , calculations of selectivity ratios ( sr ) , plotting of sr - values , and a discriminating variable ( diva ) test .
a binary response variable was added to the spc dataset to assign all samples to one of the two groups ( i.e. , regression = 0 and persistence = 1 ) , making it a supervised method .
partial least - squares discriminant analysis ( pls - da ) is a suitable method when the within group variance is comparable or dominant compared to the between group variance .
the maximum group discrimination from a pls - da model can be represented by a target projection ( tp ) component that is obtained by combining all pls components into this single tp component using a latent variable projected onto the response variable . a score value from the target projection model is calculated for each object ( sample ) with regard to the group variable . a selectivity ratio ( sr ) plot resembles a spectrum and is a plot of the ratio of explained variance to unexplained variance for each variable , where one variable in this case is an identified protein .
the discriminating variable ( diva ) test is a nonparametric test suitable for small sample sets with group heterogeneity .
a correct classification rate ( ccr ) value is calculated for each variable and will vary between 50% for a variable that provides random classification of the samples and 100% for a variable that gives a complete separation of the two groups .
the sr and ccr are closely related in that higher sr should give higher ccr .
the diva test provides a means of setting boundaries for the selectivity ratio to identify the important discriminating variables ( proteins in this case ) for a given ccr .
more in depth theoretical explanations of all these methods can be found in [ 2325 ] .
the model was cross - validated by leaving out a large percentage of the individual samples from both sets in two cross - validation steps . in an outer loop ,
this was repeated 5 times so that all samples were kept out once . in the inner loop , 25% of the samples
were kept out at a time , and this was repeated four times to keep out all samples once .
the normalized spectral count data were also imported into spss ( version 18 , spss , oslo , norway ) for a binary logistic regression analysis and cart ( salford , san diego , ca , usa ) for a classification and regression tree analysis , both used as supervised in the sense that a group variable ( regression or persistence ) was added .
the continuous variables were divided into two different subgroups , using a threshold value assessed by receiver - operating curve ( roc ) analysis ( medcalc software , mariakerke , belgium ) .
the median age of the patients at inclusion was 29.7 years ( range : 2540 ) , the interval between punch biopsy and cone excision was median , 113 days , and the mean protein concentration of the selected rpmi samples , measured by bradford , was 0.81 mg / ml ( range : 0.551.14 ) . the age , punch - cone excision interval , and protein concentration of the rpmi samples of each of the three groups of patients studied ( i.e. , lc - mc / ms learning set , validation set , and the seldi - tof set ) were consistent with the overall cohort from which our samples were selected and therefore can be regarded as representative .
a total of 40 peaks were detected in the seldi - tof spectra using the criteria described in section 2.5 .
the development of a binary logistic regression model resulted in one protein peak ( m / z 6034 ) having the best discriminatory power between the regression and persistence samples of the 40 peaks in this dataset .
figure 1 is a scatter plot showing this peak plotted against one of the peaks found as discriminatory between normal and cin2 - 3 tissue in the previous study .
the figure shows that this seldi - tof peak in fact could not discriminate between cin2 - 3 lesions with regression and persistence .
the samples were subjected to depletion of 7 high abundance proteins followed by tryptic digestion and unidimensional lc - ms / ms analysis . using the high significance peptide confidence filter in proteome discoverer and the identification criteria for proteins with only one peptide , a total of 165 protein identifications
were included ( all listed in table 1 and more detailed in supplementary tables 1 and 2 , see supplementary material available online at http://dx.doi.org/10.1155/2014/129064 ) : 57 of these were identified with two or more unique peptides and the others with only one unique peptide .
although peptides from human papillomavirus proteins were detected in all samples , none of them gave acceptable protein identification .
figure 2 shows a plot of the target projection score results from the complete dataset .
the discrimination between the regression and persistence group is 95% since all persistence samples have score values with a positive sign , and 19 out of the 20 regression samples have a negative score value .
figure 3 shows a selectivity ratio plot for all the identified proteins resulting from doing a diva test with 90% correct classification rate set as an objective goal .
this resulted in a selectivity ratio of 1.26 as the limit for a variable to be significant in discriminating the groups .
only one protein , the zinc finger protein 441 ( znf441 ) ( gi308153532 ) ( red box ) , had a selectivity ratio of 1.26 .
the cart analysis of the learning set resulted in a two - node model in which the znf441 was used as the primary group discriminator and , similar to cg12314 gene product , as the second most contributing discriminator .
this protein was identified with one peptide ( rvlitgslnwttqaiqnnr , precursor m / z : 2265.1714 da , charge : + 2 ) . a blast search against the uniprot human database with this sequence gave only one hit , phospholipase d6 ( pld6 ) ( uniprot identifier : q8n2a8 ) . a search using the id - mapping tool at the uniprot website gave no results . however , a uniprot blast search for the complete sequence from the ncbi entry resulted in a unique hit with 100% identity score , pld6 .
figure 4 shows a scatter plot using the spectral count results for these two proteins of the two sets and illustrates the discrimination obtained .
the binary logistic regression model also resulted in znf441 having highest discriminatory power ( results not shown ) .
znf441 , identified using one highly significant peptide ( qcgkalshlksfqr ) , was found in 10 and 9 of the 10 regression samples in the learning and validation set , respectively , and in none of the persistence samples .
the pld6 protein was also identified using only one high significance peptide and occurred in 7 and 5 of the 10 regression samples in the two sets .
figure 5 shows the peptide sequence , the ms2 spectrum , and the y- and b - series for the znf441 peptide . a blast search using the peptide sequence against the human uniprot database gave the two znf441 isomers as the only proteins with a 100% sequence similarity and the best scores from the search .
znf441 ( gi308153532 , uniprot : q8n8z8 ) is a nuclear protein with 693 amino acids which belongs to the krueppel c2h2-type zinc finger protein family , and it contains 19 c2h2-type zinc fingers and 1 krab - domain ( uniprot entry ) .
roc curve analysis showed that the optimal threshold for both znf441 and pld6 was 1 versus > 1 . using these thresholds , all regression and persistence cases of the learning set
set , 9 of the 10 regression and all 10 persistence cases were correctly classified .
figure 4 illustrates the power of the two proteins to distinguish between regressive and persistent cin2 - 3 lesions for all cases in both the learning and the test set .
this study describes the results from analysis of three different datasets regarding regression or persistence of cin2 - 3 lesions : one dataset from seldi - tof ms and two datasets from lc - ms / ms analysis . for the seldi - tof ms study
, supernatants from a total of 45 patient samples ( 5 cin2 - 3 with regression and 40 cin2 - 3 with persistence ) were analysed .
one discriminatory peak was found by developing a binary logistic regression model using the seldi - tof ms dataset , but no discrimination between cin2 - 3 lesions with regression or persistence could be obtained .
other binding conditions for the cm10 chip could have been used , as well as other chip types , but this was not pursued further as obtaining protein identification from a seldi - tof ms peak proved challenging .
all three multivariate statistical methods applied on the normalized spectral count results gave the same result , indicating that znf441 can discriminate between regressive and persistent cin2 - 3 lesions .
to our knowledge the exact function of znf441 has not yet been revealed , but the large family of transcriptional regulators of krab - containing zinc finger proteins are known to act as tumour suppressors . in general ,
zinc finger proteins are a highly abundant group of proteins that varies in both structure and function .
they are involved in several cellular activities , including development , differentiation , and tumour suppression .
a zinc finger is a peptide domain whose secondary structure is stabilized by a bound zinc ion and a zinc finger protein can contain between 1 and 40 such domains .
zinc fingers were originally considered only as dna - binding domains , but their role in protein - protein interactions has eventually been recognized .
proteins with multiple zinc fingers can have two to three different types of binding activity through different fingers .
the krueppel - associated box ( krab - domain ) is located near the n - terminal end of the protein , spans across 5075 amino acids , and is divided into two boxes ( a and b ) .
krab - containing proteins are transcriptional repressors and use the zinc fingers to bind dna .
krab - containing proteins are critical to cell differentiation , proliferation , apoptosis , and neoplastic transformation . increased expression of the znf23 has been found to induce apoptosis in ovarian cancer cell lines .
znf431 functions as a transcriptional repressor for patched1 ( ptch1 ) through binding to the target promoter sequence .
ptch1 is a member of the hedgehog ( hh ) family and acts as a negative regulator of the hh pathway .
this pathway is important during embryonic development but has also been shown to be active during cancer development in adults . .
repression of ptch1 in a gastric cell line was shown to correlate with high level of methylation of cpg islands at regulatory sequences and this could be associated with the development of gastric cancer .
another zinc finger protein , znf411 , was found to suppress the map kinase signalling pathway , which is important for cell cycle checkpoints .
overexpression of this pathway has been reported in different squamous cell carcinomas [ 36 , 37 ] .
the relationship between cin grade and the map kinase pathway has also been investigated and was found to be an early marker for cervical carcinogenesis but not related to virus clearance .
furthermore , the oncogenic e6 and e7 , expressed in high risk hpv and known to play an important role in cin tumour progression , also contain zinc finger domains , as recently reviewed by ruttkay - nedecky et al . .
in fact , new cell - permeable artificial zinc finger proteins ( azps ) have been launched as potential antiviral drug candidates that are able to reduce hpv replication [ 40 , 41 ] .
phospholipase d6 ( pld6 ) was only reported by the cart analysis as contributing to the discrimination . in general ,
phospholipase d ( pld ) proteins have been implicated in membrane trafficking [ 42 , 43 ] , cytoskeletal reorganization , endocytosis , exocytosis , cell migration , and cell proliferation .
the mouse homologue zucchini ( mzuc ) , also known as pld6 , has been shown to possess single strand - specific nuclease activity .
this endoribonuclease has been shown to be essential for primary pirna biogenesis [ 46 , 47 ] .
pirnas are a distinct class of small rnas , called piwi - interacting rnas , and have been discovered in both mammalian and drosophila germline .
they cluster at transposon loci in male germline stem cells and it has been suggested that pirnas and their associated piwi proteins are involved in epigenetic mechanisms like methylation and chromatin modifications . a pirna population has also been identified in the he - la cervical cancer cell line . in germline stem cells
furthermore , pirnas have been detected in human cancer and somatic cells , and epigenetic disruption of the piwi / pirna pathway is indeed a hallmark for cancer development in testis .
diminished pirna expression has been found in testicular tumours as compared to normal testis . in the current study ,
pld6 was found to be expressed in most regression cases ( 12/20 ) but not in the persistent cases .
the exact mechanisms for the epigenetic silencing exerted by the pirna - piwi pathway components remain unsolved and identification of additional protein components is crucial for a better understanding of the role of pirnas in cancer .
this study and a previous study show that cin biopsies shed a complex mixture of proteins into a cell culture medium when placed at 4c for 24 hours . for the lc - ms / ms study , supernatants from two series of 20 patient samples
each ( 10 cin2 - 3 with regression and 10 with persistence in each series ) were analysed using a bottom - up shotgun proteomics approach in which the proteins were digested into smaller peptides using trypsin .
samples were pretreated by an immunoaffinity adsorbent which was previously validated by sds - page and lc - ms / ms ( supplementary figure 1 ) . despite the depletion of seven high abundance proteins including immunoglobulins and albumin , peptides from these proteins
in addition , not unexpectedly , hemoglobins constitute a relatively large part of the identified proteins ( cervical tissues with cin2 - 3 are usually richly vascularised ) and should be included in future depletion work .
further fractionation of the depleted samples prior to the lc - ms / ms analysis would be an advantage to increase the supernatant proteome coverage and also possibly the sequence coverage of the identified proteins .
this fractionation could be obtained using 2d - gel separation of the protein mixture or , for example , a cation exchange fractionation of the peptides after digestion .
another option is enrichment of subproteomes like phosphorylated proteins or glycosylated proteins . the results in this study
should of course be validated by analysing a larger number of samples and also by analyses using other methodology like immunohistochemistry .
using three different statistical methods to analyse normalized spectral count data , this study has identified zinc finger protein 441 as a highly discriminating factor between cin2 - 3 regressive and persistent lesions .
zinc finger proteins constitute the largest family of transcriptional regulators in mammals with important dna binding domains and are also involved in protein - protein interactions .
their ability to induce apoptosis has been shown , as well as their function as nuclear transcriptional repressors of genes involved in signal transduction important for development of carcinogenesis .
pld6 is involved in biogenesis of pirnas , small noncoding rnas involved in hypermethylation events and important for transcriptional , epigenetic , and signalling pathways alterations . in line with these findings , both pld6 and znf441
were almost absent in the persistent cin2 - 3 cases contrary to the regression cases ( figure 4 ) .
the depletion of seven high abundance proteins followed by a unidimensional separation of tryptic digests of nondepleted protein mixtures shows the potential of the described method for collection of proteins from cin biopsies . from a prognostic aspect ,
the findings are promising tools for further investigation and understanding of the biology behind regression of precancerous cervical lesions . | regression of cervical intraepithelial neoplasia ( cin ) 2 - 3 to cin 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde - fixed paraffin - embedded ( ffpe ) biopsies .
proteomic analysis of water - soluble proteins in supernatants of biopsy samples with lc - ms ( ltq - orbitrap ) was used to identify proteins predictive of cin2 - 3 lesions regression .
cin2 - 3 in the biopsies and persistence ( cin2 - 3 ) or regression ( cin1 ) in follow - up cone biopsies was validated histologically by two experienced pathologists . in a learning set of 20 cin2 - 3 ( 10 regressions and 10 persistence cases ) ,
supernatants were depleted of seven high abundance proteins prior to unidimensional lc - ms / ms protein analysis .
mean protein concentration was 0.81 mg / ml ( range : 0.551.14 ) .
multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent cin2 - 3 .
the findings were validated in an independent test set of 20 cin2 - 3 ( 10 regressions and 10 persistence cases ) .
multistep identification criteria identified 165 proteins . in the learning set , zinc finger protein 441 and phospholipase d6 independently discriminated between regressive and persistent cin2 - 3 lesions and correctly classified all 20 patients .
nine regression and all persistence cases were correctly classified in the validation set .
zinc finger protein 441 and phospholipase d6 in supernatant samples detected by ltq - orbitrap can predict regression of cin2 - 3 . |
with the use of emd , protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants .
zirconia has adequate mechanical properties similar to those of stainless steel , such as high flexural and tensile strength , high resistance to abrasion and corrosion , low thermal conductivity.1,2 survival rate and fracture load after chewing simulation of zirconia abutments showed to be similar to that of titanium abutments.3 the biocompatibility of zirconia is as comparable to that of titanium .
accumulation of bacteria on y - tzp proved to be satisfactorily lower than titanium , which is an important characteristic of zirconia in maintaining prosthodontic restorations free of periodontal problems.4,5 stable soft tissue attachment which has influence on the periodontal health is affected by the material composition of the implant abutments.6,7,8 to obtain the most esthetic results as in maxillary anterior implant restorations , soft tissue regeneration may be needed in cases with even zirconia abutments . in this study
, we discuss positive effects of enamel matrix derivatives on soft tissue regeneration around zirconia abutments with which periodontal tissue formation can be promoted .
enamel matrix derivative ( emd , emdogain , straumann , malm , sweden ) , is a commercially available extract of enamel matrix proteins , introduced for periodontal regeneration .
emd is predominantly composed of amelogenins and partly of peptides such as enamelin , tuftelin , ameloblastin , and proteases .
enamel matrix proteins secreted by cells of hertwig 's epithelial root sheath are deposited on the developing root surface and start the formation of acellular cementum , a process known as cementogenesis .
development of periodontal ligaments and alveolar bone follows subsequently.9,10,11,12 emd can be employed on denuded root surfaces to stimulate regenerative responses and restore periodontal attachment successfully.13 therefore , emd facilitates the adhesion , proliferation , mineralization , and differentiation of osteoblasts and contribute to enhanced osteogenesis and periodontal regeneration.14,15,16,17,18,19 practically , the use of emd may offer positive benefits in treatment of periodontal defects and guided bone regeneration.20,21,22,23,24 thus , beneficial outcomes of emd on periodontal regeneration have been proven through clinical and histological data .
gingival fibroblasts are major cells of the gingival connective tissue and play a major role in the soft tissue sealing in the trans - mucosal part of implants . based on the previous reports of emd on the treatment of gingival recession and the ability to improve clinical parameters,25,26,27,28,29 we can speculate that emd may have direct benefits on the gingival fibroblasts . studies on emd
have reported that emd has concentration dependent stimulatory effects on human gingival fibroblasts and the amount of matrix produced.30,31 studies on proliferation and attachment characteristics of human gingival fibroblasts on titanium surfaces have been reported.32,33,34 however , promoted attachment of human gingival fibroblasts may be achieved on zirconia surfaces also.35,36 with the additional use of emd , protective barrier between attached gingiva and transmucosal abutment with zirconia surface may be enhanced leading to final esthetic results with implants .
this study was designed to characterize the proliferation , attachment , and gene expression of immortalized human gingival fibroblasts on zirconia by means of mtt assay , sem , and real - time polymerase chain reaction ( rt - pcr ) .
the sixty zirconium discs ( 12 mm 4 mm height ) were sectioned from a cylinder block of zirconia ( lava , 3 m espe , st .
zirconia discs were washed twice in absolute alcohol and once in demineralised water ultrasonically , and sterilized in an autoclave .
the immortalized human gingival fibroblasts ( hgf ; applied biological materials inc . , richmond , bc , canada ) were grown at 37 in an atmosphere of 5% co2 using dulbecco 's modified eagle 's medium ( dmem , gibco brl , grand island , ny , usa ) supplemented with 10% fetal bovine serum ( fbs ) and 1% antibiotics .
the cell proliferation of cells cultured under different concentrations of emd on cell proliferation was examined with an mtt test assay ( sigma , st .
, cells were cultured with dmem , 10% fbs , and 1% antibiotics only ; in the emd 25 group , emdogain ( 25 g / ml ) was added to the control group media ; in the emd 100 group , emdogain ( 100 g / ml ) was added to the control group media .
the substance used for the mtt test was a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl - tetrazolium salt , which turns into a blue formazan product due to the viable mitochondria in active cells .
hgf cells were seeded at a density of 210 cells / ml on the discs .
cells were cultured at 37 in 5 % co2 for 4 , 24 , and 48 hours .
the discs were moved to well plates after incubation , and new media were added .
diluted mtt ( 5 mg / ml ) solution was added , and the incubation was continued at 37 in 5% co2 for 4 hours . after the incubation medium removed , 400 l of isopropanol with 0.04 n hcl was added to each well .
the resulting formazan crystal products , which reflect the level of cell metabolism , were dissolved .
the absorbance of the formazan product at 490 nm was measured in triplicate with a microplate reader ( bio - kinetics reader , el312e , winooski , vt , usa ) .
hgf cells were seeded at a density of 2 * 10 cells / ml and grown for 4 , 24 and 48 hours in an incubator of 37 in 5% co2 .
the experiment wells were rinsed twice with a 0.1 m phosphate buffered saline ( pbs ) buffer ( ph 7.4 ) after loosely bound cells were aspirated and removed .
the excess glutaraldehyde solution was removed from the cells by rinsing with pbs one more time .
dehydration in ethanol baths of progressively higher concentrations ( 50 , 60 , 70 , 80 , 90 , 95 , and 100% ) for 10 min in each bath was performed .
the cells were dried to a critical point and the discs were gold sputter coated with a thickness of 100 nm using an ion coater ( eiko ib-3 , eiko engineering ltd . ,
the discs with the cultured cells were evaluated and the images were recorded by sem ( tescan vega ii lmu , brno , czech republic ) at 100 and 500 magnification . to quantify the expression levels of collagen type i , osteopontin , fibronectin , and tgf-1 , real - time polymerase chain reaction ( rt - pcr ) was done .
a thermal cycler and a laser with fiber optics were used with the 384-well plates ( ab gene , hamburg , germany ) for intensification of the fluorescent signals on this system . the fluorescence emission from each sample
is collected by a charge - coupled device - camera and the quantitative data were analyzed using the abi prism 7900 sequence detection system ( applied biosystems , san francisco , ca , usa ) and the sequence detection system software ( sds version 2.2 , pe applied biosystems ) .
reaction regimes were composed of mixtures 10 pmol/l of the primers , 2x sybr green pcr master mix ( applied biosystems ) with hotstartaqt dna - polymerase included , the dntp mix with dutp added , the sybrs green i fluorescent dye , and rox dye as a passive reference . to produce the relative standard curves of each gene , serial dilutions ( 1 , 1/2 , 1/4 , 1/8 , and 1/16 ) of cdna were included in the real - time quantitative pcr plates .
glyceraldehyde-3-phoshpate dehydrogenase ( gapdh ) was used as the housekeeping gene . the amplification program consisted of 50 for 2 min and 95 for 10 min followed by 40 cycles of initial denaturation at 95 for 30 seconds , followed by annealing at 60 for 30 seconds and extension at 72 for 30 seconds .
real - time pcr cycle numbers were modified to molecular size ( ng ) according to the equation .
the name and primer sequences of the genes used are listed ( table 1 ) .
the mean ( mv ) and standard deviation ( sd ) values of the mtt test were presented .
kruskal wallis test was conducted for statistical evaluation of the differences between the groups . the subsequent multiple comparisons between the groups
the cell proliferation of control , emd 25 , and emd 100 group were improved as the culture time increased .
after 4 hours of cell culture , hgf proliferation of emd 100 group decreased in comparison with the control group .
after 24 hours of culture , cell proliferation of emd 100 group increased to be comparable with the control group .
after 48 hours of cell culture , cell proliferation of emd 100 group considerably increased compared to that of the control group .
cellular viability of emd 25 group showed to be the greatest of all groups at 4 , 24 , and 48 hours of cell culture .
cell proliferation of the control group and emd 25 group was found to be statistically significant ( fig .
1 ) . after 4 hours of culture , emd 25 group showed more cellular attachments with lamellipodia and spreading of the cellular matrix ( fig .
2 ) . the control group and the emd 100 g / ml group showed stretching of the cells .
both experimental groups showed more flattening of the cells in a concentration - dependent manner compared to the control group ( fig .
however , emd 100 group showed more flattened cells compared to the control and emd 25 group with coherent vesicles of emd with extracellular matrix covering the cells and the surface of the matrix ( fig . 4 and fig . 5 ) .
at 48 hours of culture , cells of all control and experimental groups showed higher adhesion properties . in the emd 25 group , cellular processes showed to be longer and thinner ( fig .
higher magnification , hgf of experimental groups showed to be more flat and more cell to cell contact with each other was shown in the emd 100 group ( fig .
the amount of type i collagen expression was remarkably increased in a concentration - dependent manner and was found to be statistically significant ( p<.05 ) .
fibronectin and integrin both decreased with emd 25 group and then increased with emd 100 group , but did not show any significant differences .
tgf-1 showed to be decreased with emd 25 group and then increased with emd 100 group with statistical significance ( fig . 8) .
for long - term stability of dental implants , biological seal of soft tissue and implant interface is very important .
transmucosal part of dental implant requires sufficient connective tissue attachment and inhibition of bacterial invasion and proliferation .
titanium is the predominant metal used because of its biocompatibility and stability of the attachment of surrounding hard and soft tissues.37 however , titanium abutments may sometimes exhibit esthetic problems , especially in the anterior region . to improve the esthetics of dental implants , zirconia abutments with the biocompatibility and mechanical strength of titanium
can be used.6,7,8,38,39,40 previous investigations support this and thus , zirconia abutments are now used successfully in clinical use .
raffaeli suggested that zirconia is a more biocompatible material compared to feldspathic porcelain and can strongly increase the adhesion competence and the proliferation rate of fibroblasts.41 tet proposed that zirconia showed collagen fiber orientation that was similar to that of machined titanium surface and demonstrated less plaque formation resulting in periodontal health and improved esthetics.42 recent studies on zirconia surfaces with hgf show good attachment and cell proliferation and support the clinical significance of zirconia abutments .
however , soft - tissue regeneration procedures for further esthetics and treatment of peri - implantitis may lead to need for increased attachment and growth of hgf on zirconia surfaces .
thus , enamel matrix proteins , which have been found to have positive effects on regeneration of periodontal defects , may be applied to enhance fibroblast attachment and proliferation .
previous research has reported that enamel matrix derivative may enhance periodontal wound regeneration which has direct effect on the gingival fibroblasts and human periodontal ligament cells.25,26,27,28,29 rincon reported that emd of 20 g / ml enhanced wound healing by promoting proliferation and migration of gingival and periodontal ligament fibroblast.43 in this study , the concentration of emd was based on the study of gestrelius et al.44 in which emd 25 group showed the highest cellular proliferation.14,30 our results of mtt assay of human gingival fibroblasts at 4 hours show that the concentration of emd 25 group showed the greatest cellular viability whereas emd 100 group showed decreased cellular viability . these in vitro results imply that high concentration may not have positive effect at short cell culture period .
human gingival fibroblasts cultured on the samples with emd were more spread out and evenly attached to the surface of the zirconia discs .
the spreading of cytoplasm between lamellipodia showed to be remarkable in the emd 25 group . from the sem results
, it can be speculated that lower concentration of emd has more positive influence on initial cellular attachment .
type i collagen exist in extracellular matrix of gingival fibroblasts and promote the attachment of gingival fibroblasts.45 in this study , expression of type i collagen in the experimental groups with emd significantly improved in a concentration dependent manner .
the results of this study support previous studies where emd increased the number of gingival fibroblast and the amount of cellular matrix produced.30 osteopontin is a cell adhesion protein expressed in bones which is biosynthesized by preosteoblasts , osteoblasts , and osteoclasts .
however , elevated mrna expression of osteopontin may be used to identify periodontal fibroblasts with the potential to facilitate periodontal regeneration.46 in this study , expression of osteopontin was suppressed in the emd 25 group but increased significantly in the emd 100 group .
previous studies have indicated possibilities of differentiation and mineralization of nonosteogenic cells which might have inherent roles in formation of cementum or bone.47 the results of our study comply with these findings and emd of high concentration may stimulate osteogenic induction of human gingival fibroblasts .
fibronectin is a protein related to cellular attachment . normally , with increased expression of this protein
the gene expression results of our study showed that although cellular morphology for attachment was favorable , expression levels of fibronectin were decreased for emd 25 group .
increased expression level of fibronectin is known to be related to rapid cell attachment.48 therefore , at lower concentration of emd , cell attachment may occur slowly and at higher concentration of emd , cell attachment may occur rapidly .
tgf-1 is a growth factor controlling cellular proliferation , differentiation , apoptosis , and integrin expression.49 in this study , mrna expression of tgf-1 was stimulated in the emd 100 group .
these data comply with previous studies where emd had stimulatory effect on expression of tgf-1 of hgf.14 emd had stimulatory effect on type i collagen in a concentration dependent manner .
however , as gene expression of proteins investigated in this study was promoted with higher concentration of emd , the adequate concentration of emd effective on attachment and proliferation of gingival fibroblasts on zirconia surface needs further investigation .
also , further study must be directed on whether high concentration of emd would possibly transfer human gingival fibroblasts into cells with osteogenic potential and possibility of bone and tissue regeneration with gingival fibroblasts .
through this short term culture of hgf on zirconium discs , we conclude that enamel matrix derivative contribute to proliferation and attachment of hgf cells . also , enamel matrix derivative stimulates production of extracellular matrix collagen , osteopontin , and tgf-1 in high dose .
but more investigation is required to determine appropriate concentration of enamel matrix derivative for utmost cell proliferation and attachment and further investigation on differentiation potential of hgf to osteoblast - like cell would be necessary . | purposethe objective of this study was to investigate the biologic effects of enamel matrix derivative ( emd ) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts ( hgf ) to zirconia surfaces.materials and methodsimmortalized human gingival fibroblasts ( hgf ) were cultured ( 1 ) without emd , ( 2 ) with emd 25 g / ml , and ( 3 ) with emd 100 g / ml on zirconia discs .
mtt assay was performed to evaluate the cell proliferation activity and sem was carried out to examine the cellular morphology and attachment .
the mrna expression of collagen type i , osteopontin , fibronectin , and tgf-1 was evaluated with the real - time polymerase chain reaction ( rt - pcr).resultsfrom mtt assay , hgf showed more proliferation in emd 25 g / ml group than control and emd 100 g / ml group ( p<.05 ) .
hgfs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on emd 25 g / ml group and emd 100 g / ml group after 24h culture .
after 48h of culture , cellular attachment was similar in all groups .
the mrna expression of type i collagen increased in a concentration dependent manner .
the genetic expression of osteopontin , fibronectin , and tgf-1 was increased at emd 100 g / ml . however , the mrna expression of proteins associated with cellular attachment was decreased at emd 25 g / ml.conclusionthrough this short term culture of hgf on zirconium discs , we conclude that emd affects the proliferation , attachment , and cell morphology of hgf cells .
also , emd stimulates production of extracellular matrix collagen , osteopontin , and tgf-1 in high concentration levels.clinical relevancewith the use of emd , protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants . |
in november 2013 , the us food and drug administration ( fda ) created shock - waves around the world by issuing a cease and desist order against 23andme , the dominant provider of direct - to - consumer ( dtc ) genomic services.1 23andme , in common with several other companies worldwide , offered genetic testing services directly to consumers via online platforms , without physician involvement and at increasingly reduced prices .
the fda 's move was a dramatic culmination of heated controversies that such services had provoked among clinicians , bioethicists , regulators and ordinary citizens .
first , the company 's failure to comply with the agency 's requirements for authorisation , regarding which it had applied and entered into negotiations with the fda .
of course , the fda 's order needs to be understood and evaluated in the specific context of the us health regulatory framework and the course of conduct on which 23andme had embarked .
but , as the excited flurry of commentary in the media and blogosphere illustrated , the second reason offered by the fda resonated more broadly among those concerned with dtc genomics
. indeed , concerns about the potential harmfulness of dtc genomic services had been repeatedly voiced by regulatory and other expert bodies , both in the usa and europe , for some time prior to the fda 's momentous decision.2
3 these harms include risks of inappropriate dosage adjustment , decisions to undergo unwarranted diagnostic procedures , the creation of needless anxiety or false reassurance , and the invasion of consumer 's privacy .
in addition to these harms to consumers , other concerns included the danger of ill - founded requests for diagnostic services from such consumers overburdening the healthcare system and unnecessarily depleting scarce healthcare resources . in 2010 ,
the fda had already foreshadowed its concerns about the risk of harm entailed by dtc genomics in letters to several dtc genomics providers.4 in a similar vein , a number of professional medical societies had issued policy recommendations spelling out serious concerns about dtc genomics , in particular , reservations about their analytic validity and clinical utility , and advising consumers to refrain from undergoing such tests . in light of such concerns , many critics have called for the strict regulation , and even the outright prohibition , of dtc genetic services.3 given the supposed lack of adequate supervision by a physician or genetic counsellor in the dtc genetics model , the main strands of the harm argument are twofold : ( 1 ) consumers can be misled into believing the information that they receive is sufficiently reliable to be medically actionable ; ( 2 ) even if any information they receive is medically actionable , in principle , consumers may not be equipped to use that information in a way that benefits them .
these risks can have various undesirable knock - on effects , such as bad decision making about diagnostic procedure or lifestyle , needless distress , inappropriate use of resources , and so on .
however , this scepticism about dtc genomic services is only one side of a highly polarised debate.5 proponents of such services have defended them primarily as enhancing the autonomy of their users , providing them with valuable information that empowers them to make valuable health and lifestyle decisions .
such claims can be interpreted as directly contradicting the harm - based arguments noted above , because one salient way of interpreting harm is as a threat to autonomy .
this is not to suggest that all harms that are potentially associated with dtc genomics are automatically and comprehensively translatable into autonomy harms .
however , most of the supposed harms of dtc begin with the consumer deciding ( for good or bad reasons ) to obtain genomic information dtc and then how best to act upon it . whether the consumer will unnecessarily burden the healthcare system ( which is not per se an autonomy harm ) has its roots in the concern that the consumer has been given misleading information which , in turn , signals that his autonomy has been undermined .
therefore , autonomy is central in the assessment of dtc and it is important to clarify the notion of autonomy in play , in order to determine whether dtc is a threat or , instead , a means of realising that value . for the purposes of this paper , i shall draw on the influential conception of personal autonomy elaborated by joseph raz in the morality of freedom as part of a broader liberal perfectionist theory of politics .
on the razian interpretation , autonomy is the value that consists in becoming the part - author of one 's life through making a series of life - shaping choices in the course of navigating within a given social context.6 according to raz , autonomy is conditioned upon the presence of appropriate mental abilities , the existence of an adequate range of options and independence from others ( p.372):if a person is to be maker or author of his own life then he must have the mental abilities to form intentions of a sufficiently complex kind , and plan their execution .
these include minimum rationality , the ability to comprehend the means required to realize his goals , the mental faculties necessary to plan actions etc . for a person to enjoy an autonomous life he must actually use these faculties to choose what life to have .
finally his choices must be free from coercion and manipulation by other , he must be independent .
all three conditions , mental abilities , adequacy of options and independence admit of degree.(p.373 ) if a person is to be maker or author of his own life then he must have the mental abilities to form intentions of a sufficiently complex kind , and plan their execution .
these include minimum rationality , the ability to comprehend the means required to realize his goals , the mental faculties necessary to plan actions etc . for a person to enjoy an autonomous life he must actually use these faculties to choose what life to have .
finally his choices must be free from coercion and manipulation by other , he must be independent .
all three conditions , mental abilities , adequacy of options and independence admit of degree.(p.373 ) the razian view focuses on an important dimension to autonomy by requiring the presence of an adequate range of options which are morally acceptable. raz argues that the value attributed to autonomy stems at least partly from the fact that it enables individuals to choose freely from valuable options that must exhibit sufficient variety .
so , for example , someone with only a multiplicity of trivial , short - term options to choose from whether to eat now or later , whether to scratch their left or right ear cannot realise personal autonomy in their lives .
equally , someone whose choices are driven entirely by the overriding need to avoid some imminent threat to their life may have valuable options , but will not enjoy a life of personal autonomy because the options do not exhibit the requisite variety , since they are all dominated by the life - preserving aim .
it is an important implication of this conception of autonomy that we can enhance a person 's autonomy by providing them with additional morally worthwhile options , but that we can not do so by presenting them with additional worthless or morally bad options .
it is important to note that raz 's view differs from an understanding of autonomy that is common in modern bioethics .
the latter focuses on an individual realising their capacity to make choices , and it requires that these choices be informed and free of coercion and undue influence . the existence of options from which to choose is assumed but not usually addressed or subjected to quality control . with all the focus being on independent choice , often it is the validity ( or lack of ) informed consent that becomes the sole criterion for whether autonomy is served .
critics of dtc genomics were quick to point out that dtc consumers autonomy suffers due to the inadequate informed consent processes employed by current companies .
characteristically , the remedies to address any autonomy deficits have primarily focused on strengthening consent procedures.7 however , the limitations of approaching autonomy exclusively via the informed consent route are well documented . as o'neill has rightly observed : informed consent procedures protect choices that are timid , conventional , and lacking in individual autonomy ( variously conceived ) just as much as they protect choices that are self - assertive , self - knowing , critically reflective and bursting with individual autonomy ( variously conceived).8 even ideal informed consent processes can , at best , ensure one dimension of autonomy , namely the existence of independent choice .
there is a broad range of conceptions of autonomy elucidating several of its dimensions , and although there is a similarly wide range of views about the value of such conceptions , it is unlikely that one dimension alone fully captures this value .
raz 's conception avoids this pitfall by extending the requirements of autonomy beyond the minimum condition of self - fulfillment through independent choice .
it sets stricter criteria for achieving autonomy by demanding certain qualities in the available options , and rendering such qualities integral to the value of choice .
he makes a plausible case that not all kinds of options serve one 's interests , and those options that fail to promote one 's interests do not enhance one 's autonomy . what underpins raz 's view is the notion that autonomy is key to a good life , at least in modern societies , and therefore , any setbacks to autonomy are harms to an individual .
moreover , the achievement of a good life in this account is not just an individualistic undertaking independent of social practices . by linking autonomy to the quality of options that are on offer in a certain social context , options that are socially created , defined and sustained
it is further worth noting that using the razian conception enables us to formulate the autonomy - based critique of dtc in its strongest form , since much concern about dtc has precisely centred on the quality of the options it generates .
it is , therefore , all the more significant if that critique can be countered on its most hospitable razian terrain .
in common with other understandings of autonomy , raz 's independence condition precludes others subverting one 's decision making through various forms of manipulation , such as misleading advertising .
one autonomy - based critique of dtc genomics involves the claim that dtc genomics companies are engaged in precisely such manipulation . on this view
, companies fail to provide adequate information to consumers about the services they offer . for example
, they exaggerate the potential clinical utility of such services , while also providing limited information about the research uses to which they put the data that is collected , the assignment of intellectual property rights , and the data access given to third parties.914 this objection , characteristically formulated as the failure to meet a condition of informed consent , obviously raises serious concerns
. however , it can be replied that it does not identify a problem that is either inherent or unique to the provision of dtc genomic services.15
16 instead , dtc genomics should comply with consumer protection legislation that is more generally applicable . admittedly , the distinctive nature of dtc genetics , that is , the fact that it is a combination of a product ( kit ) , laboratory test ( eg , single - nucleotide polymorphism ( snp ) analyses ) and a service ( interpretation of results relating to health but not also to other considerations , such as ancestry ) , poses challenges in subsuming it under existing regulatory standards.17 however , existing regulations regarding medical devices , in vitro diagnostic tests , clinical laboratory standards , and consumer law , offer valuable starting points in fashioning a regulatory regime adapted to the distinctive nature of dtc genomic services . is there any reason to suppose that there is an insuperable barrier to dtc genomics companies satisfying informed consent requirements , including requirements to provide accurate information about their services ? one argument centres on the fact that most dtc genomics services are provided online .
this creates difficulties in verifying the identity , and competence to consent , of the individual consumer.18 however , this objection equally applies to other services provided online , it is not specific to dtc genomics services . a more radical argument , however , is that misleading advertising is inherent to dtc genomics , because such advertising necessarily makes the false claim that it generates options that are medically actionable in valuable ways.11 if advertisements for dtc genomics were truthful , the objection goes , they could not claim to be offering a valuable or commercially viable product .
this radical objection feeds into the second condition on razian autonomy . establishing the autonomy - enhancing character of dtc genomics within a razian framework , requires showing that it generates worthwhile options .
precisely this feature of autonomy , however , has been invoked by cathleen kaveny in important recent work to show that not only does dtc genomics not enhance autonomy , it may even pose a serious threat to it.19 there are two main strands of argument in her detailed and wide - ranging critique which echo the key concerns that have already been put forward by others .
the first is that the information yielded by dtc genomics does not generate valuable options because it is not medically actionable , that is , it does not generate clinical options for improving the consumer 's health .
the second is the claim that , even supposing that information that is in principle medically actionable is generated , its potential medical value can not be actualised by ordinary dtc consumers .
one main reason for this is the probabilistic nature of the information combined with the serious difficulties ordinary people experience when engaging in probabilistic reasoning .
these difficulties may lead them to make harmful decisions , a risk exacerbated by their lack of genetic literacy .
what kaveny has done is to elaborate the commonly rehearsed arguments against dtc within the razian framework of autonomy , thereby giving those arguments a deeper and more compelling basis .
bearing in mind that this is a controversial area in which expert opinion divides , kaveny and other critics are perfectly justified in raising questions about the medical actionability of the information generated by dtc genomics.20 however , it is arguable that their skepticism is exaggerated . to begin with
, the medical actionability of dtc genomics is not monolithic but rather depends on a number of key variables .
most dtc genetics companies use snp genotypes , a lower - cost technology with certain limitations .
a smaller number offer whole genome or exome sequencing which generates more comprehensive data and can , in principle , lead to greater medical actionability . as
sequencing becomes increasingly less expensive , it is likely to be more commonly adopted within the dtc genomics industry . another important
variable is whether what is being tested are high - penetrance genes , which have high predictive value , such as testing for brca1 and brca2 mutation ( linked to breast cancer ) or mutations linked to the lynch syndrome , a serious risk factor for colon cancer.2123 therefore , a blanket skepticism about the medical actionability of dtc genomics is unwarranted .
this conclusion is strengthened by the fact that medical actionability is likely to increase over time , especially as more people undergo genetic testing and more data become available for analysis , which will lead to a better understanding of genetic susceptibility to disease . even assuming , however , that dtc generates information that is , in principle , medically actionable in a sufficient range of cases , it may be objected that consumers are typically not equipped to use this information in a way that meaningfully enhances their autonomy .
a common concern is that such information is probabilistic in character , and that ordinary people typically have serious difficulties in handling probabilities without expert assistance .
translates the results , and places them in the context of other health data , although a well - established feature of clinical genetic testing , is mostly absent in dtc genetics .
it is notable , however , that as the dtc genetics model has evolved , it has increasingly incorporated some form of counselling.24 nevertheless , it has not been shown that its absence seriously impairs autonomous decision making.2527 while evidence is not conclusive , most studies that have been conducted so far show that consumers do not suffer psychological harms , nor do they take action to alter their lifestyle in response to their genomic profiles.28
29 studies have also shown that there is no increase in the use of health services attributable to the consumers receiving dtc genomic data.30 dealing with probabilistic data is a recurrent feature of decision making well beyond the medical context .
it is hardly a problem uniquely posed by genetic information , and to present it as such smacks of genetic exceptionalism .
for example , easily accessible data such as one 's body mass index , or blood cholesterol readings , or more complex information resulting from family history , has predictive value about one 's health .
such information often has greater predictive weight than genetic information , but no argument regarding autonomy impairment has been made in this context , even if the understanding of such predictions on the part of the individual may be limited.31 ultimately people integrate such information into decisions they make about their lives ( by acting upon it or ignoring it ) . if our limited abilities to process probabilistic information imperil our autonomy ,
surely the correct first - line response is to adopt measures to improve these abilities rather than to prohibit access to the information.32 such measures are not limited to , and need not necessarily involve , genetic counselling .
empirical studies examining patients understanding of consent documents , advertising materials and so on , systematically establish ways to mitigate the risk of misunderstanding through better designed information documents , aided by pictorials , web - based tools , and so on.33 it is therefore hasty to conclude that dtc genomics fails to generate valuable medical options that can be meaningfully engaged with by its consumers .
however , a further important point needs to be made under raz 's valuable options condition .
critics have assumed that the value of the options generated by dtc genomics should be assessed exclusively from the point of view of medical actionability .
although an option of this kind is certainly desirable , enabling health improvement is only one way through which value may be acquired .
there are other ways in which an option may be valuable , for example , satisfying one 's curiosity , increasing one 's genetic literacy , enabling participation in research and contributing thereby to science knowledge , or as a means of exploring one 's ancestry
. indeed , there is increasing evidence that dtc genetics users are motivated by a variety of reasons in undergoing testing , including those described above.34
35 engaging with genetic testing also has an educational component insofar as individuals get a hands - on experience with genetics and its role and limitations in health and disease.36
37 it is noteworthy that in the aftermath of the fda 's order , although 23andme ceased offering health - related information to its users , it continued selling ancestry services and raw genetic data ( without interpretation ) . in short , worthwhile options generated by dtc genomics include
personal utilities that go beyond health improvement.38 of course , some of these utilities have also attracted skepticism .
is there something deeply problematic in conceiving of the self as a genetic self , perhaps one detached from other important aspects of identity?39 the answers to these questions remain open , but it is unclear why they pose a special autonomy - based problem for dtc genetics . and , even if they do pose such a problem , it may be that this cost is outweighed by other considerations .
if , for example , the personal utility arising from searching for genetic relatives on a data base and comparing genomes comes at a cost , such as fostering a conception of identity in terms of genetic make - up alone , this needs to be set against potential benefits , for example , discovering the existence of half - siblings or genetic relations to parents .
it can also be outweighed by the value of research participation which contributes to the common good .
these personal utilities are important and have independent significance , hence they are among several considerations that need to be balanced against other benefits and costs .
it is reasonable to suppose that even if in general the clinical utility of dtc genomics is low , the personal benefits generated through such services work to expand the range of valuable options , and to hence , satisfy raz 's third requirement of autonomy .
an intriguing issue concerns results that have a high predictive value for currently non - preventable or non - treatable conditions .
again , it has been argued that this information lacks value due to the absence of treatment options.19 this argument unduly minimises the fact that people seeking this information often have salient family history , and may be living with agonising uncertainty about their own future .
the option of acquiring certainty , for those who wish it , about something so important , and around which they may design life strategies , can not lack value.4042 it has been further objected that if one knows enough about one 's future , the life options are not those of the authentic self ( the one that does not know).43 for this argument to stand , it needs to be shown why the one with insights about the future is less authentic than the one without them . first , those who seek dtc information about such diseases are doing so because they want to receive this information ( they are not forced to obtain it ) and presumably this is an act of their authentic self
. some will choose to change their life plans , for example , decide not to have children , while others may choose to do so sooner than later .
the variation in responses to such information may be more indicative of people maintaining their authenticity rather than losing it .
bearing in mind that this is a controversial area in which expert opinion divides , kaveny and other critics are perfectly justified in raising questions about the medical actionability of the information generated by dtc genomics.20 however , it is arguable that their skepticism is exaggerated . to begin with , the medical actionability of dtc genomics is not monolithic but rather depends on a number of key variables .
most dtc genetics companies use snp genotypes , a lower - cost technology with certain limitations .
a smaller number offer whole genome or exome sequencing which generates more comprehensive data and can , in principle , lead to greater medical actionability . as
sequencing becomes increasingly less expensive , it is likely to be more commonly adopted within the dtc genomics industry .
another important variable is whether what is being tested are high - penetrance genes , which have high predictive value , such as testing for brca1 and brca2 mutation ( linked to breast cancer ) or mutations linked to the lynch syndrome , a serious risk factor for colon cancer.2123 therefore , a blanket skepticism about the medical actionability of dtc genomics is unwarranted .
this conclusion is strengthened by the fact that medical actionability is likely to increase over time , especially as more people undergo genetic testing and more data become available for analysis , which will lead to a better understanding of genetic susceptibility to disease . even assuming , however , that dtc generates information that is , in principle , medically actionable in a sufficient range of cases , it may be objected that consumers are typically not equipped to use this information in a way that meaningfully enhances their autonomy .
a common concern is that such information is probabilistic in character , and that ordinary people typically have serious difficulties in handling probabilities without expert assistance .
translates the results , and places them in the context of other health data , although a well - established feature of clinical genetic testing , is mostly absent in dtc genetics .
it is notable , however , that as the dtc genetics model has evolved , it has increasingly incorporated some form of counselling.24 nevertheless , it has not been shown that its absence seriously impairs autonomous decision making.2527 while evidence is not conclusive , most studies that have been conducted so far show that consumers do not suffer psychological harms , nor do they take action to alter their lifestyle in response to their genomic profiles.28
29 studies have also shown that there is no increase in the use of health services attributable to the consumers receiving dtc genomic data.30 dealing with probabilistic data is a recurrent feature of decision making well beyond the medical context .
it is hardly a problem uniquely posed by genetic information , and to present it as such smacks of genetic exceptionalism .
for example , easily accessible data such as one 's body mass index , or blood cholesterol readings , or more complex information resulting from family history , has predictive value about one 's health .
such information often has greater predictive weight than genetic information , but no argument regarding autonomy impairment has been made in this context , even if the understanding of such predictions on the part of the individual may be limited.31 ultimately people integrate such information into decisions they make about their lives ( by acting upon it or ignoring it ) . if our limited abilities to process probabilistic information imperil our autonomy , surely the correct first - line response is to adopt measures to improve these abilities rather than to prohibit access to the information.32 such measures are not limited to , and need not necessarily involve , genetic counselling . empirical studies examining patients understanding of consent documents , advertising materials and so on , systematically establish ways to mitigate the risk of misunderstanding through better designed information documents , aided by pictorials , web - based tools , and so on.33
it is therefore hasty to conclude that dtc genomics fails to generate valuable medical options that can be meaningfully engaged with by its consumers .
however , a further important point needs to be made under raz 's valuable options condition .
critics have assumed that the value of the options generated by dtc genomics should be assessed exclusively from the point of view of medical actionability .
although an option of this kind is certainly desirable , enabling health improvement is only one way through which value may be acquired .
there are other ways in which an option may be valuable , for example , satisfying one 's curiosity , increasing one 's genetic literacy , enabling participation in research and contributing thereby to science knowledge , or as a means of exploring one 's ancestry .
indeed , there is increasing evidence that dtc genetics users are motivated by a variety of reasons in undergoing testing , including those described above.34
35 engaging with genetic testing also has an educational component insofar as individuals get a hands - on experience with genetics and its role and limitations in health and disease.36
37 it is noteworthy that in the aftermath of the fda 's order , although 23andme ceased offering health - related information to its users , it continued selling ancestry services and raw genetic data ( without interpretation ) . in short , worthwhile options generated by dtc genomics
personal utilities that go beyond health improvement.38 of course , some of these utilities have also attracted skepticism .
is there something deeply problematic in conceiving of the self as a genetic self , perhaps one detached from other important aspects of identity?39 the answers to these questions remain open , but it is unclear why they pose a special autonomy - based problem for dtc genetics . and , even if they do pose such a problem , it may be that this cost is outweighed by other considerations .
if , for example , the personal utility arising from searching for genetic relatives on a data base and comparing genomes comes at a cost , such as fostering a conception of identity in terms of genetic make - up alone , this needs to be set against potential benefits , for example , discovering the existence of half - siblings or genetic relations to parents .
it can also be outweighed by the value of research participation which contributes to the common good .
these personal utilities are important and have independent significance , hence they are among several considerations that need to be balanced against other benefits and costs .
it is reasonable to suppose that even if in general the clinical utility of dtc genomics is low , the personal benefits generated through such services work to expand the range of valuable options , and to hence , satisfy raz 's third requirement of autonomy .
an intriguing issue concerns results that have a high predictive value for currently non - preventable or non - treatable conditions .
again , it has been argued that this information lacks value due to the absence of treatment options.19 this argument unduly minimises the fact that people seeking this information often have salient family history , and may be living with agonising uncertainty about their own future .
the option of acquiring certainty , for those who wish it , about something so important , and around which they may design life strategies , can not lack value.4042 it has been further objected that if one knows enough about one 's future , the life options are not those of the authentic self ( the one that does not know).43 for this argument to stand , it needs to be shown why the one with insights about the future is less authentic than the one without them .
first , those who seek dtc information about such diseases are doing so because they want to receive this information ( they are not forced to obtain it ) and presumably this is an act of their authentic self .
some will choose to change their life plans , for example , decide not to have children , while others may choose to do so sooner than later .
the variation in responses to such information may be more indicative of people maintaining their authenticity rather than losing it .
as we saw at the outset , a recurrent concern about dtc genomics is that it poses an unacceptable risk of physical and mental harm to consumers , as well as potentially placing serious burdens on the entire healthcare system .
this harm rationale has been invoked by regulatory bodies in europe and the usa in recommending a restrictive approach to the governance of dtc genomics .
this paper has dealt exclusively with the harm rationale articulated as a threat to the autonomy of consumers , that is , to the exercise of their capacity to make choices from a range of valuable options . precisely on this autonomy - based understanding of harm , for example ,
critics of dtc genomics have urged a restrictive approach to the regulation of such services . because autonomy features so prominently in the dtc debate , it is important to clarify whether it is imperilled . in this article , i have challenged the thesis that dtc genomics is , in principle , harmful in virtue of the supposed threat it poses to autonomy . on the contrary , using a razian framework , it is plausible to regard it as potentially enhancing autonomy .
this is especially so in light of the fact that it generates valuable options , although it is important to bear in mind that the value derives from plural sources , and not exclusively from medical actionability .
however , the capacity of dtc genomics to enhance autonomy depends upon its satisfaction of a number of requirements , including those governing consumer protection , for example , accurate advertisement , informed consent requirements , privacy protections , providing aids to probabilistic reasoning , the option of some form of genetic counselling and so on . accordingly
, this defence of dtc genomics in principle is not to be confused with the defence of any existing dtc genomic services , which might be deficient in various ways .
i have not argued that there is a positive societal obligation to provide dtc genomic services . nor am i committed to the claim that the autonomy - based case is all - things - considered conclusive for the question of legal permissibility , though it seems to me to create a strong pro tanto case for permissibility .
autonomy is just one , albeit very important , evaluative consideration in shaping an adequate regulatory scheme .
for example , raz interprets the harm principle , which licences state coercion only to prevent harm to others , in terms of threats to autonomy .
if the line of argument pursued in this paper is correct , and if we subscribe to a regulatory approach based on the harm principle , then dtc genomics does not of its very nature constitute such a threat .
it therefore should not be legally prohibited , contrary to what is currently the case in several countries.44 however , if the harm principle is not the exclusive justificatory basis of regulatory constrains , my argument does not provide a conclusive case for the permissibility of dtc genomics .
first , because autonomy - based harm is likely to be a central consideration in any assessment of the legal permissibility of dtc genomics and in the regulation of biotechnology more broadly .
it is therefore worth weighing dtc genomics on the scales of autonomy , even if other scales will also be needed before we answer the question of whether to allow it and under what conditions .
second , although a non - harm - based case for prohibiting dtc can not be ruled out , i am doubtful as to its prospects of success in light of the autonomy - based case advanced here .
appeals to the potential distress caused to dtc consumers , or to the morally inappropriate conception of personal identity supposedly fostered by dtc genomics , seem to me highly questionable bases for prohibition given the existence of an autonomy - based case for permissibility .
the aim of this article is to make progress in the controversy surrounding dtc genomics by addressing some of the underlying ethical and political questions that it raises .
the verdict of autonomy , i believe , is that there is at the very least a pro tanto case in favour of the legal permissibility of dtc genomic services .
the next phase in the debate requires opponents of dtc genomics either to challenge that case , or else to show that autonomy - based concerns are trumped by countervailing considerations of other kinds .
if , as i am inclined to believe , the case for outright prohibition or heavy restrictions does not succeed , we should then move on to the more fertile question of the precise regulatory conditions under which dtc genomic services should be permitted .
the answer to this question may legitimately vary to some extent from one jurisdiction to the other , in light of such matters as established legal doctrine and varying cultural and social conditions .
but in any modern liberal society , the autonomy - based argument developed in this paper should form a central part of the framework for elaborating those conditions . | direct - to - consumer ( dtc ) genetic services have generated enormous controversy from their first emergence .
a dramatic recent manifestation of this is the food and drug administration 's ( fda ) cease and desist order against 23andme , the leading provider in the market .
critics have argued for the restrictive regulation of such services , and even their prohibition , on the grounds of the harm they pose to consumers .
their advocates , by contrast , defend them as a means of enhancing the autonomy of those same consumers .
autonomy emerges as a key battle - field in this debate , because many of the
harm arguments can be interpreted as identifying threats to autonomy .
this paper assesses whether dtc genomic services are a threat to , or instead , an enhancement of , personal autonomy .
it deploys joseph raz 's account of personal autonomy , with its emphasis on choice from a range of valuable options .
it then seeks to counter claims that dtc genomics threatens autonomy because it involves manipulation in contravention of consumers independence or because it does not generate valuable options which can be meaningfully engaged with by consumers .
it is stressed that the value of the options generated by dtc genomics should not be judged exclusively from the perspective of medical actionability , but should take into consideration plural utilities .
finally , the paper ends by broaching policy recommendations , suggesting that there is a strong autonomy - based argument for permitting dtc genomic services , and that the key question is the nature of the regulatory conditions under which they should be permitted .
the discussion of autonomy in this paper helps illuminate some of these conditions . |
tooth brushing plays an important everyday role in personal oral hygiene and effective plaque removal . toothbrushes may become heavily contaminated with microorganisms and these microorganisms may originate not only from the oral cavity but also from the environment where the toothbrushes are stored .
this contamination implicates in the possibility of reinfection of a patient by toothbrushes harboring pathogenic microorganisms . as early as 1920 , cobb et al .
found that toothbrushes can be heavily infected by mutans streptococci after 24 h. according to glass et al . , microorganisms not only adhere to and reproduce on used toothbrushes but also have the ability to transmit organisms responsible for both local and systemic diseases .
procedures for the decontamination of toothbrushes would prevent the risks of reinfection or infection by other pathogenic microorganisms from the environment .
several bactericidal agents have been promoted to reduce the possibility of toothbrush bacterial contamination between uses .
these include the use of chlorhexidine , brushtox , and several dentifrices . while all these have shown varying degrees of efficacy , none are widely used as a home - based application .
a possible reason for the noncompliance with these methods is that they are time consuming and may result in unwanted product residues .
recently , few studies indicated that the use of microwave and ultraviolet ( uv ) light is the most effective household method to sanitize the toothbrushes after contamination . furthermore , due to the ease of use
however , the extent of bacterial decontamination using the microwave and uv light has not been determined in a clinical setting .
therefore , the present study was designed as an investigator - blinded , controlled , microbiological study to compare the efficacy of microwave and uv light in decreasing toothbrush bacterial contamination .
thirty male dental graduates residing in a common hostel ( with a common source of water for daily use ) , with at least 28 teeth and age ranging from 22 to 28 years ( mean age 25 2.6 years ) were enrolled into this study .
inclusion criteria included subjects in good general health , who were able to give informed consent and comply with the study protocol , having at least 14 natural teeth per arch , and brushing their teeth twice daily .
exclusion criteria included the clinical evidence of gross caries or periodontal disease , the presence of systemic diseases or conditions that would affect the oral cavity such as uncontrolled diabetes mellitus , use of any medications associated with xerostomia or any antibiotic therapy within 90 days prior to the start of the study protocol .
subjects were randomly assigned to either control ( n = 10 ; group 3 ) or experimental ( n = 20 ) groups . experimental group comprised the microwave group ( n = 10 ; group 1 ) and the uv group ( n = 10 ; group 2 ) . before the start of the study ,
each subject was given a new , identical multitufted toothbrush with soft nylon tufts ( ajay quest toothbrush , raghav lifestyle products , new delhi , india ) with a tube of toothpaste ( colgate total toothpaste , colgate - palmolive india pvt . ltd . ,
two unused toothbrushes ( control ) were cultured to check for any microbial growth in packed toothbrushes before starting the study . furthermore , the toothbrush rinsing water from the common tank intended to be used was also subjected to a microbial check before the start of the study .
the study was conducted in three phases that included contamination procedures including bacterial culture , sanitization procedures , and postsanitization evaluation .
subjects were trained and/or instructed to use the standardized modified bass technique for brushing their teeth for 3 min , twice daily for 1 week .
each subject was instructed to rinse the used toothbrush under running tap water without mechanical manipulation for at least 30 s. subjects were also instructed to keep their toothbrushes in their living room within the provided aerated box at room temperature .
after 1 week of use , each toothbrush was collected in a sterile paper bag and sent to the central food technological research institute ( cftri ) laboratory , mysore , within 4 h after collection in the morning .
various selective and nonselective media used in the study included trypticase soy agar for total counts , mitis salivarius agar for total streptococci , mitis salivarius agar with 2 iu / ml of bacitracin for mutans streptococci , macconkey agar with 1% lactosec for escherichia coli and other coliforms , and rogosa sl agar for lactobacilli . for bacterial extraction ,
the toothbrushes were individually placed in prelabeled , sterile , 50-ml centrifuge tubes containing 10 ml of the trypticase soy broth ( tsb ) to immerse the bristles , then vortexed vigorously for 1 min , squeezed against the side of the tube to drain , rinsed with 5 ml tsb , and drained again .
a series of undiluted and 10-fold dilutions of each sample were prepared and plated onto the surface of selective and nonselective media .
a duplicate series of plates was then incubated aerobically or anaerobically at 37c for 24 days , until colony formation was visible .
the number of colonies , measured as colony - forming units ( cfus ) , was counted using a colony counter .
a new set of standardized toothbrush was once again given to each subject and subjects were instructed to use it for one more week following the same instructions as given earlier .
after 1-week usage , the toothbrushes were once again collected and subjected to sanitization procedures .
the experimental group toothbrushes were randomly assigned to either the uv light or microwave group .
toothbrushes from the uv light group were sanitized by placing the brush in the uv light toothbrush sanitizer ( violight tooth brush sanitizer , violight inc . ,
sanitization was carried out by placing the brush in the receptacle and exposing the head for 12 min to uv radiation ( manufacturer 's recommendation , 6 min ) .
toothbrushes from the microwave group were sanitized by placing the brush in a microwave oven ( 2450 mhz ; kenstar microwave oven , kenstar kitchen appliances india limited , mumbai ) .
the wet brush was placed on the revolving table along with a glass of distilled water to protect the magnetron .
once again each toothbrush belonging to different groups was collected in a sterile paper bag and sent to the laboratory for further microbial culture and colony count procedure .
data obtained for all the microbial counts were log transformed to normalize their distributions prior to analysis .
logs of the total bacterial count ( log cfu ) after toothbrush contamination and decontamination were compared and analyzed using one - way anova , tukey 's post hoc procedure for multiple comparisons , and paired t - test for the effect of the pre and postsanitization procedure on the microbial count in specific groups .
subjects were trained and/or instructed to use the standardized modified bass technique for brushing their teeth for 3 min , twice daily for 1 week .
each subject was instructed to rinse the used toothbrush under running tap water without mechanical manipulation for at least 30 s. subjects were also instructed to keep their toothbrushes in their living room within the provided aerated box at room temperature .
after 1 week of use , each toothbrush was collected in a sterile paper bag and sent to the central food technological research institute ( cftri ) laboratory , mysore , within 4 h after collection in the morning .
a standard bacterial culture method was followed in the study . various selective and nonselective media
used in the study included trypticase soy agar for total counts , mitis salivarius agar for total streptococci , mitis salivarius agar with 2 iu / ml of bacitracin for mutans streptococci , macconkey agar with 1% lactosec for escherichia coli and other coliforms , and rogosa sl agar for lactobacilli . for bacterial extraction ,
the toothbrushes were individually placed in prelabeled , sterile , 50-ml centrifuge tubes containing 10 ml of the trypticase soy broth ( tsb ) to immerse the bristles , then vortexed vigorously for 1 min , squeezed against the side of the tube to drain , rinsed with 5 ml tsb , and drained again .
a series of undiluted and 10-fold dilutions of each sample were prepared and plated onto the surface of selective and nonselective media .
a duplicate series of plates was then incubated aerobically or anaerobically at 37c for 24 days , until colony formation was visible .
the number of colonies , measured as colony - forming units ( cfus ) , was counted using a colony counter .
a new set of standardized toothbrush was once again given to each subject and subjects were instructed to use it for one more week following the same instructions as given earlier .
after 1-week usage , the toothbrushes were once again collected and subjected to sanitization procedures .
the experimental group toothbrushes were randomly assigned to either the uv light or microwave group .
toothbrushes from the uv light group were sanitized by placing the brush in the uv light toothbrush sanitizer ( violight tooth brush sanitizer , violight inc . ,
sanitization was carried out by placing the brush in the receptacle and exposing the head for 12 min to uv radiation ( manufacturer 's recommendation , 6 min ) .
toothbrushes from the microwave group were sanitized by placing the brush in a microwave oven ( 2450 mhz ; kenstar microwave oven , kenstar kitchen appliances india limited , mumbai ) .
the wet brush was placed on the revolving table along with a glass of distilled water to protect the magnetron .
once again each toothbrush belonging to different groups was collected in a sterile paper bag and sent to the laboratory for further microbial culture and colony count procedure .
data obtained for all the microbial counts were log transformed to normalize their distributions prior to analysis .
logs of the total bacterial count ( log cfu ) after toothbrush contamination and decontamination were compared and analyzed using one - way anova , tukey 's post hoc procedure for multiple comparisons , and paired t - test for the effect of the pre and postsanitization procedure on the microbial count in specific groups .
thirty subjects with an age ranging from 22 to 28 years ( mean age 25 2.6 years ) were enrolled in this study .
all the subjects were male and shared the common living environment ( common hostel ) for more than 4 years .
all the subjects were able to return their toothbrushes on days 7 and 14 in sealed labeled bags as instructed .
moreover , no bacterial growth was observed on culturing the rinsing water obtained from the common tank .
the mean microbial growth on toothbrushes in terms of log cfu is shown in table 1 .
descriptive statistics of the measured variables of the various groups one - way anova was used to test for differences in mean microbial cfu counts among three groups of toothbrushes sanitized by their respective methods . the mean microbial cfu count done before the sanitization procedure demonstrated no significant difference , f ( 2 , 27 ) = 0.344 , p = 0.712 , among the groups whereas the mean cfu count done after sanitization procedures differed significantly across the all the three groups , f ( 2 , 27 ) = 267.219 , p < 0.001 .
tukey 's post hoc comparisons of the three groups after the sanitization procedure indicated that the microwave group ( m = 1.49 , 95% ci [ 1.2729 , 1.7071 ] ) gave a significantly lower cfu count than the uv radiation group ( m = 3.22 , 95% ci [ 2.93 , 3.50 ] , p < 0.001 ) and the control group ( m = 5.88 , 95% ci [ 5.49 , 6.26 ] , p < 0.001 ) .
comparisons between the uv radiation group and the control groups also showed statistically significant reduction in the microbial cfu count at p < 0.001 [ table 2 , figure 1 ] .
the mean difference is significant at the 0.05 level the paired t - test was also conducted to analyze the microbial cfu count difference between pre- and postsanitization procedures .
the results of the paired t - test of the microwave group revealed that there were significant differences in log cfu counts , between the pre- ( m = 5.82 0.89 ) and postsanitization procedure ( m = 1.49 0.303 ) , with t ( 9 ) = 16.18 and p < 0.001 .
similarly , the uv radiation group also showed significant differences with t ( 9 ) = 6.77 and p < 0.001 , between the pre- ( m = 5.53 0.974 ) and postsanitization procedures ( m = 3.22 0.40 ) whereas for the control group , the difference between pre- ( m = 5.79 0.68 ) and postsanitization procedures ( m = 5.8 0.54 ) was not statistically significant , with t ( 9 ) = -0.599 , p = 0.564 .
the present study was undertaken to quantitatively analyze the microbial growth after toothbrush contamination and to compare the efficacy of two different sanitization techniques ( uv light and microwave irradiation ) after the contamination procedure .
the result outcome revealed that the contaminated toothbrushes harbored an increased number of aerobic and facultative anaerobic bacteria species .
this finding is in accordance with the results of previous studies that indicated that an actual risk of recolonization exists after each brushing . in the recent years
, there has been an increasing interest in the interrelationship between contaminated toothbrushes and systemic reinfections .
several studies have also stressed on the role of contaminated toothbrushes and its causation in systemic infections . in this regard , brook and gober suggested that contaminated toothbrushes contributed to the persistence of group a beta - hemolytic streptococci in the oropharynx and to the failure of penicillin therapy in some cases of pharyngotonsillitis . in another study ,
significant bacteremia has also been reported after tooth brushing , especially in patients with severe periodontitis .
therefore , a concern has been raised that the microbial load on toothbrushes might have a significant impact in periodontal patients under therapy .
discussions on the modern toothbrushes have suggested the problem of toothbrush construction as a factor of toothbrush contamination .
the nylon , multitufted toothbrush has been cited for its design of tufts set too closely to accommodate easy cleaning .
the filaments are collected into bundles , bent in half with a metal anchor in the center , and driven into premolded holes in the toothbrush head at a high speed . in toothbrushes , the bristles can harbor inherent microorganisms , further increasing the bacterial contamination .
several studies have suggested the need for toothbrush disinfection to reduce the number of microorganisms on the bristles using different methods , including uv radiation , microwave irradiation , boiling water , and chemical agents , such as listerine , plax , cepacol , and chlorhexidine .
in addition , some authors have also attempted to incorporate antimicrobial agents , such as silver , chlorhexidine , and others to the toothbrush bristles as a coating layer during the manufacturing process . despite evidence demonstrating that chemical rinses and dentifrices can reduce the total bacterial load on a toothbrush , these methods are not widely used .
therefore , recently , devine et al . raised a need of disinfection methods that are rapidly effective , cost - effective , nontoxic , and can be easily implemented .
the present study was mainly sought to compare two sanitization techniques used for the decontamination procedure .
our result showed that there was a significant reduction in microbial contamination in both microwave and uv groups compared to the control group .
furthermore , our results also showed a significant reduction in the microbial count in the microwave group when compared with the uv group .
our results confirm the findings of chibebe et al . where previously contaminated toothbrushes when exposed to microwave irradiation at 2450 mhz were reported to get inactivated by the action of microwave .
a possible explanation for the effect of microwave irradiation upon formed microbial assemblage on brushes can be validated by the fact that many bacterial species are responsible for biofilm formation on different surfaces like toothbrushes , and microwave irradiation can disrupt the biofilm structure .
data suggest that the destruction of microorganisms by microwave is mainly due to a thermal effect of microwave exposure on the microorganism cellular content resulting in cell lysis .
another general indication for heat damage is the cell membrane rupture resulting in a leakage of nucleic acid and protein from cells . in this context
, some studies have reported that microwave - injured cells often release ninhydrin - positive material , purines , and pyrimidines into a suspension .
the presence of these materials in a suspension , in previous studies , has demonstrated the possibility of damage to cells by microwave at the membrane level . in the present study ,
the uv radiation group showed a significant differential reduction in the microbial count compared to the control group .
however , the microbial count did not significantly reduce as compared to the microwave irradiation group . although we exposed toothbrushes for 12 min to uv radiation ( 6 min , manufacturer 's specification ) , the result was not significant as compared to microwave irradiation .
previous studies have revealed that the longer exposure to uv light is necessary to ensure a complete inactivation of all microorganisms .
the long exposure of uv light inactivates microorganisms by damaging the dna and disrupting the chemical bonds that hold the atoms of dna together in the microorganism .
if the damage is severe enough , the bacteria can not repair the damage and are inactivated .
however , despite long exposure , a previous literature review has questioned the potential of low - intensity uv radiation in microbial deactivation , and the authors concluded that low - intensity uv rays are not effective against certain microbes and molds .
furthermore , tightly packed bristles and other areas are not in direct exposure and have no chance of disinfection . in the present study , these factors might be the reason for the uv radiation to be less efficient in toothbrush sanitization compared to microwave irradiation .
in contrast to our results , boylan et al . have reported that a uv light toothbrush holder can effectively reduce an average of 86% total cultivatable bacteria on a toothbrush compared to controls .
our result is not in agreement with this result as our result showed only 42% reduction in the microbial count after the uv sanitization procedure .
therefore , further microbial studies are required to verify the efficacy of the uv light toothbrush holder in the reduction of the microbial content from contaminated toothbrushes . in the present study
, we instructed all the subjects to store their toothbrushes at room temperature in the provided aerated box when not in use .
reported that the number of microorganisms in the toothbrushes kept under aerated conditions was lower than that in toothbrushes stored in plastic bags .
several authors have reported that bacterial contamination can be reduced by washing toothbrushes after use , and drying under aerated conditions .
therefore , as time increases between one tooth brushing and another , more microorganism development can occur in the toothbrushes stored in a wet / moisture environment .
our study demonstrated that significant microbial colonization was present after 1 week of repeated use of toothbrushes .
however , data suggest that the time necessary for colonization is contradictory varying from 1 to 30 days . according to cesco et al . , the colonization on toothbrushes by mutans streptococci occurs in a short time period , since after a single tooth brushing , they found the development of the microorganism in 24% of the cases .
svanberg reported the presence of mutans streptococci on toothbrushes after 3 days and colonization by mutans streptococci was observed on bristles after five consecutive days of toothbrush use .
our study also showed similar findings where cultivatable microorganisms were present on the bristles after a short - term ( 1 week ) usage with aerated storage conditions .
our results suggested that microwave irradiation is the better option for the sanitization of tooth brushes as compared to uv radiation .
however , further studies are required for determining the optimum temperature and duration for the complete eradication of the organisms and spores , thereby achieving sterilization instead of sanitization . moreover
, the duration of uv sanitization also needs to be reassessed to achieve optimum results .
our results clearly suggest that there is a definite contamination of the toothbrushes after use ; hence need arises for either improving sanitization measures or frequent changes of toothbrushes especially after any infections .
the evidence presented in this study suggests that microwave irradiation is an effective disinfectant agent for the microbiota present on the toothbrushes
. it may be an important and efficacious oral health measure not only for the debilitated but also for healthy individuals .
further in vivo trials are anticipated on more specific bacteria and biofilm of the oral cavity .
however , there seem to be good reasons for the daily use of a toothbrush sanitization even before the results of these further trials are available . | background : toothbrushes are rapidly contaminated with different microorganisms representing a possible cause of infection or reinfection especially in the periodontal patients under therapy . the purpose of this study was to evaluate the sanitization of toothbrushes previously contaminated by various oral microorganisms using a domestic microwave oven and commercial ultraviolet ( uv ) light toothbrush sanitizer.materials and methods : thirty male dental graduates were randomly assigned to control or experimental groups and received standardized toothbrushes for home use .
each subject was instructed to use it with the standardized modified bass technique for 1 week and submit it to the investigator after use .
collected toothbrushes were cultured and analyzed for the number of colony - forming units ( cfus ) . in the next phase ,
once again a new set of toothbrush was given to each subject and instructed to use it for one more week and follow the same instructions as given earlier .
subsequently , the used toothbrushes were again collected and were sanitized by microwave irradiation , uv radiation , or were not sanitized ( control group ) .
after the sanitization procedure , toothbrushes were again cultured for the number of cfus .
the collected data of the presanitized and postsanitized cfu count were log transformed to normalize their distributions prior to analysis . furthermore , log cfu data were compared and analyzed by one - way anova , tukey 's post hoc procedure , and paired t - test for the difference in the mean at p<0.05.results : result showed that after the sanitization procedure , there was a significant ( p<0.001 ) reduction in microbial contamination in both microwave and uv group toothbrushes compared to control group toothbrushes whereas the microbial count in the microwave group was significantly less ( p<0.001 ) compared to the uv group.conclusions:the evidence presented in this study suggests that microwave irradiation is an effective disinfectant agent for bacteria and fungi on toothbrushes . |
since ancient times , electrical stimulation has been used to modulate the nervous system and to treat some neurological disorders ( rossi , 2003 ) .
scribonius largo , physician of the roman emperor claudius , in his text compositiones medicamentorum
( 46 ad ) suggested the application of electric ray ( torpedo torpedo and torpedo nobiliana ) on the cranial surface as a remedy for the headache .
these fishes are known for being capable of producing an electric discharge and their scientific name comes from the latin
torpere , to be stiffened or paralyzed ( but also to be numb , insensitive ) , referring to the effect on someone who handles them ( debru , 2006 ) .
electric fishes were later used for the treatment of seizures , depression , and pain until the eighteenth century ( kellaway , 1946 ; schwalb and hamani , 2008 ) . in the early ninetieth century giovanni aldini ( 17621834 ) ,
nephew of the discoverer of animal electricity luigi galvani ( 17271798 ) and professor of physics at the university of bologna , performed electrical stimulations on the exposed human cerebral cortex of recently decapitated prisoners . in 1804 , aldini reported that cortical stimulation evoked horrible facial grimaces .
this finding led him to conclude that the cortical surface could be electrically stimulated ; supporting that electricity could have therapeutic effects in the treatment of many neuropsychiatric disorders ( aldini , 1804 ; boling et al .
aldini s experimentations and hypotheses led to direct research into two strands that would later developed during the ninetieth and twentieth century : on the one hand the use of brain stimulation for neurophysiologic investigation ( initially on animals and then on humans ) to understand the functioning of the brain , on the other hand the use of the techniques of brain stimulation for therapeutic purposes .
concerning neurophysiologic research , in 1809 luigi rolando ( 17731831 ) first used galvanic current to stimulate the cortical cortex of animals ( rolando , 1809 ) , highlighting the functions of brain areas , while in 1870 gustav fritsch ( 18381927 ) and eduard hitzing ( 18381907 ) showed that electrical stimulation of specific cortical areas evoked muscle contractions in dogs ( fritsch and hitzing , 1870 ) . in 1872 david ferrier ( 18431924 ) identified monkey s cerebral cortex points whose stimulation was related to specific movements of the animal ( ferrier , 1873 ; gross , 2007 ) . in 1874 the american physician robert bartholow ( 18311904 ) was the first to report findings from studies of electrical stimulation of the cerebral cortex in an awake human ( bartholow , 1874 ) . in 1882 the italian neuropsychiatrist ezio sciamanna ( 18501905 ) performed a series of systematic experiments of electrical stimulation on a trepanned patient who had a traumatic brain injury ( sciamanna , 1882 ; zago et al . , 2008 ) .
in 1883 the italo - argentine surgeon alberto alberti ( 18561913 ) conducted an experiment lasting more than 8 months of cerebral stimulation in a woman in whom an eroding tumor of the skull allowed easy access to the dura mater surface , like in bartholow s case ( alberti , 1886 ) .
unfortunately , the contribution of these researches in determining the motor topography of the human brain nonetheless remained poorly exploited except to confirm the electrical excitability of the cortex and demonstrate the contralateral cortical hemispheric representation of motor functions ( zago et al . ,
more precise and systematic observations on the topography of the brain had been made in 1887 by the british surgeon victor horsley ( 18571916 ) ( vilensky and gilman , 2002 ) , but we should wait for until 1950 when fundamental studies of the neurosurgeon wilder penfield ( 18911976 ) were published before the brain stimulation of the human cortex could give a real accurate representation of the human brain functions , including motor and somatosensory areas ( cortical homunculus ; penfield and boldrey , 1937 ; penfield and rasmussen , 1950 ) .
electroshock , introduced by ugo cerletti ( 18771963 ) in 1938 was the first modern example of therapeutic application of brain stimulation for the treatment of severe psychosis ( cerletti , 1940 ; kalinowsky , 1986 ) .
the application of an electric current on the skull evoked an epileptic seizure that roughly remodeled the neural connections , providing a clinical improvement to the patients .
despite opposing opinions about this technique , this method had a more solid foundation rather than the dubious experiments of electrical brain stimulation for the treatment of schizophrenia and other mental illness conducted at tulane university in the 1960s ( baumeister , 2000 ) .
brain stimulation for pain control , used as early as 1950 with good effects through temporary electrodes implanted into brain regions , after a first experimental phase , found its explanation in the gate control theory developed by melzach and wall in 1962 ( rezai and lozano , 2002 ) .
these previous studies were the basis that led to the development of new techniques of neurostimulation : transcranial magnetic stimulation , cortical brain stimulation , and deep brain stimulation ( dbs ) .
transcranial magnetic stimulation produces a magnetic field to modulate the excitability of the brain cortex . unlike electroshock
, it can stimulate only a specific area ( selectivity ) through an eight - shaped magnet and it does not involve loss of memory and/or seizures .
it is mainly used for the treatment of psychiatric diseases , as obsessive compulsive disorder and depression , and recently in one case of minimally conscious state ( wassermann et al . , 2008 ; piccione et al . ,
cortical brain stimulation involves the application of chronic low - frequency electrical pulses on the motor cortex through intra- or extra - dural implantation of one or more electrodes connected to a generator with a battery located in the chest .
it is particularly used for the treatment of some forms of parkinson s disease , epilepsy , and dyskinesia , but also for the pain control and in patients afflicted with stroke ( pagni et al .
deep brain stimulation is a surgical procedure that allows implanting microelectrodes precisely in some brain areas through a combination of stereotactic and neuroimaging techniques .
deep brain stimulation is an evolution of functional stereotactic neurosurgery techniques , initially used to produce selective lesions of specific deep brain structures ( thalamic and cerebellar nuclei ) .
thus , a new balance of damaged neural circuits could be found , removing the tremor in patients suffered from medically refractory parkinson s disease and motor disorders of dyskinesias . in 1947
ernst spiegel and henry wycis , modifying the original apparatus of clarke and horsley ( 1906 ) , produced the first human stereotactic frame that using pneumoencephalogram allowed to determine cartesian coordinates of structures around ventricles ( basal ganglia ) for identifying the precise localization of the targets that had to be destroyed by radiofrequency ( spiegel et al .
intra - operative electrical stimulation of these structures was systematically used for the exploration and the localization of the deep cerebral nuclei and for confirming target ( guiot et al .
these observations led to suggest that these stimulations of deep cerebral nuclei could be used not only as a method for diagnostic purposes but also as a therapeutic method itself .
thus , the evolution from lesional to stimulating functional neurosurgery was determined ( porta and sironi , 2009 ) .
the origins of this technique are linked to the discovery of the effects of electrical stimulation of the deep brain areas , conducted during the stereotactic lesional functional neurosurgery to identify the correct position of coagulant electrodes for the treatment of dyskinetic disorders and tremor in parkinson s disease ( schwalb and hamani , 2008 ) .
thanks to the spread of stereotactic method , various studies demonstrated that , while low - frequency stimulation ( 510 hz ) could enhance tremor and other correlated symptoms , high - frequency stimulation ( 50100 hz ) resulted in a reduction of symptoms ( albe fessard et al . , 1963 ; blomstedt and hariz , 2010 ) .
( 1952 ) , bekthereva et al . ( 1963 ) , sem - jacobsen ( 1965 ) , and cooper ( 1978 ) .
deep electrical stimulation of brain structures was originally introduced as a therapeutic option to treat behavioral disorders or chronic pain . in 1952 ,
the spanish neuroscientist jos m. delgado , basing on his experience of deep neurophysiologic electrical stimulation in animals , first described the technique of implantation of intracranial electrodes in humans , indicating the importance of this method for diagnosis and its possible therapeutic role in patients with mental disorders ( delgado et al . , 1952 ) .
over the next two decades , he implanted radio - equipped electrode arrays that he called stimoceivers , in cats , monkeys , chimpanzees , gibbons , bulls , and even humans , and he showed that he could control subjects mind and bodies with the push of a button .
his experiments on animals were often very theatrical . for example , in 1963 he demonstrated the possibility to stop a bull from charging in response to a radio - signal of one electrode implanted in the brain of animal .
however , the critics contended that the stimulation did not quell the bull s aggressive instinct , as delgado suggested , but rather forced it to turn to the left ( horgan , 2005 ) . at that time , he implanted electrodes in 25 human subjects , most of them schizophrenics and epileptics . in 1969
he described his brain stimulation researches and discussed critical aspects and ethic implications in the book physical control of the mind : toward a psychocivilized society , where he showed the tremendous opportunities but also the great risks derived from neurotechnology ( delgado , 1969 ) .
the first to use chronic depth stimulation as a therapy in motor disorders was natalia petrovna bekthereva , neuroscientist at the institute of experimental medicine and the academy of medical sciences in leningrad . in 1963
she published a work on the use of multiple electrodes implanted in sub - cortical structures for the treatment of hyperkinetic disorders ( bekthereva et al . , 1963 ) .
however , since her papers were written in russian , her works were not well known around the world . in her therapeutic electro - stimulation , as she named this method , she used electric stimulation with high - rate pulses of suprathreshold current , achieving excellent results ( bekthereva et al . , 1975 ) .
the norwegian neurophysiologist and psychiatrist carl wilhelm sem - jacobsen initially used depth electrodes implanted for recording and stimulation in patients with epilepsy and psychiatric disorders .
he successfully implanted multiple electrodes in the thalamus to stimulate the targets in order to identify the best lesional site in parkinson s disease .
these electrodes were often left into the patient s brain for several months , without any side effects . as he wrote :
these electrodes could then be used , following stimulation responses , to make incremental staged lesions in the target area
( sem - jacobsen , 1965 , 1966 ; blomstedt and hariz , 2010 ) . by the early 1970s
, there were some reports of chronic dbs system implanted in the thalamus for the treatment of chronic pain ( hosobuchi et al .
, 1973 ; mazars et al . , 1974 ) , and isolated experiences in the patients with persistent vegetative state ( hasserl et al . , 1969
the experience of the american neurosurgeon irving s. cooper in placing electrodes over the cerebellum and into the deep thalamic nuclei for central palsy , spasticity and epilepsy was more extensive and continuous . in 1977
he reported its excellent results from chronic cerebellar stimulation in over 200 patients ( cooper , 1978 ) .
the lack of correlation of opinion of efficacy between the patients and clinicians led cooper and other scientists to perform double - blind studies on cerebellar stimulation for spasticity .
the results of these studies did not try to show a real efficacy of this procedure ( schwalb and hamani , 2008 ) .
after the introduction of l - dopa in the late 1960s , there was a sharp decline of the surgical treatment of parkinson s diseases and the ablative procedures continued , only targeting ventral intermediate nucleus ( vim ) and globus pallidus .
dbs progressed as a technique through its use in psychiatric and pain control surgery . despite the sharp decline of surgery for parkinson s disease given the use of l - dopa , many groups continued to perform thalamotomy for tremor of various etiologies ( schwalb and lozano , 2004 ) .
the positive effect of thalamic stimulation on tremor was well known ( due to the diagnostic neurostimulation maneuver done prior to coagulation to be sure of being in the right target ) , but the idea to use chronic stimulation as a therapeutic method did not emerge until benabid s preliminary report in 1987 on stimulation of the vim nucleus ( benabid et al . , 1987 ) .
in 1991 , both benabid and blond and sigfried groups reported their results on thalamic dbss for tremor ( benabid et al .
subsequent studies found that the dbs of thalamus was safer than thalamotomy and especially bilateral thalamotomy .
likewise , the stimulation of globus pallidus was demonstrated safer than pallidotomy , originally proposed by laitinen for medically refractory parkinson s disease in the early 1990s ( laitinen et al . , 1992 ) .
the major safety of the dbs of these areas led a gradual abandonment of lesional techniques . in 1994 pollak s group began to stimulate a new target , involved in parkinson s disease : the sub - thalamic nucleus of luys ( stn ; pollak et al . ,
in particular , the dbs of this area has been found to be effective for bradykinesia , tremor , and rigidity .
moreover , the stimulation of snt and globus pallidus was explored for the treatment of both generalized and segmental dystonia ( yu and neimat , 2008 ) .
in addition to movement disorders , dbs was also mostly used and explored for treatment of chronic pain , subsequently approved by us food and drug administration in 1989 . concerning the future of dbs for movement disorders , although multiple studies demonstrated its efficacy , many questions still require answers . in deed , as above reported , dbs has been showed to be effective in patients with medically refractory parkinson s disease in both motor function and quality of life , but it is unclear what the effect of these techniques are on non - motor aspects of this pathology .
furthermore , we should exactly know when a patient could be considered as drug unresponsive and whether a early dbs could slow the progression of the disease .
concerning dystonia , a rigorous trial was conducted on the efficacy of dbs of globus pallidus for primary dystonia , but the report of dbs for secondary dystonia consists of small case series . on contrary , the efficacy of stimulation of snt is still clearly defined for this disorder ( holloway et al . , 2006 ; benabid , 2007 ; schwalb and hamani , 2008 ) .
bds in the treatment of refractory epilepsy has gotten the attention from epileptologists due to its well - documented success in treating movement disorders .
early results of the sante trial should lay the foundation for widespread implementation of dbs for epilepsy targeting the anterior thalamic nucleus .
other hopeful target seems to be the caudate nucleus , the sub - thalamic nucleus , the cerebellum , the centro - median nucleus of the thalamus , and the hippocampus , even if the results are non - conclusive ( halpner et al .
recently , the indication of the use of dbs has been extended to new diseases , so new interesting perspectives for future therapies seem to be opened . bilateral thalamic stimulation has been used for the treatment of refractory tourette syndrome , a complex pathology characterized by multiple motor tics and one or more phonic / vocal tics lasting longer than 1 year .
the first results are positives with improved clinical features ( porta et al . , 2010 ) .
dbs has been also indicated for the treatment of serious psychiatric disorders , such as refractory depression and obsessive compulsive disorder .
in addition to psychiatric diseases , dbs has also been suggested as a potential therapy for obesity , eating disorders , and drug resistant hypertension ( mayberg et al . , 2005 ;
although dbs for pain has been largely abandoned , the group of milan has explored dbs of the posterior hypothalamus for cluster headache ( franzini et al . , 2003 ;
moreover , a recent study has re - explored dbs for the minimally consciousness state after severe traumatic brain injury ( schiff et al . , 2007 ) .
since the introduction of dbs , almost 20 years ago , there has been an immense resurgence of interest in the neurosurgical technique for the treatment of more neurological and psychiatric disorders .
the reversible nature of stimulation technique is an attractive feature and clinical conditions that were not believed to be surgically tractable are now being considered suitable for dbs therapy .
the success of dbs in the treatment of refractory parkinson disease is evident , while for the other motor disorders ( primary tremor , dyskinesias , medically refractory tourette s syndrome ) good results are waited .
the major psychiatric diseases ( refractory depression , obsessive compulsive disorder ) , cluster headache , epilepsy , eating disorders ( obesity ) , and drug resistant hypertension are the new field where the dbs seems to have interesting therapeutic possibilities .
what other neurological and psychic disorders can dbs be applied to ? what should be the criteria for selecting candidates ?
only when they are or are considered as ? for some diseases should dbs be used regardless of pharmacological therapy ? could an early use of dbs change the natural history of some kinds of disease ?
in addition , an objective and statistically valid assessment of long - term results and of possible technique - related complications is still central .
, it is fundamental to the involvement of family members , in addition to their direct involvement with the informed consent .
the procedure must be supported by interdisciplinary teams of neurosurgeons , neuroscientists , psychiatrist , psychologist , and other health professionals who can help assess patients suitability for dbs and continuously monitoring them over time .
the dbs is not a modern form of psychosurgery and for this reason technical and ethical mistakes , that historically characterized this terrible chapter of history of neuroscience , should be avoided ( kringelbach and aziz , 2010 ) . while psychosurgery was a lesional unselective and irreversible manipulation of a brain area ( e.g. , the lobotomy proposed by egas moniz since 1935 and its subsequent variants ) , the primary goal of dbs is to rebalance the damaged neuronal circuits through a electrical selective and reversible manipulation ( stimulation ) of targeted brain structures , whose alteration may determine , along with neurological deficits , also behavioral problems ( e.g. , tourette s syndrome ) . concerning the psychiatric indications ( refractory depression and obsessive compulsive disorder ) , the evidences of organic alterations underlying these events provide an adequate explanation for the fact that the rebalancing of specific neurophysiologic substrates through the dbs can improve these behavioral disorders , harmonizing the physical and psychological expressions of these subjects .
the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | this paper briefly describes how the electrical stimulation , used since antiquity to modulate the nervous system , has been a fundamental tool of neurophysiologic investigation in the second half of the eighteenth century and was subsequently used by the early twentieth century , even for therapeutic purposes . in mid - twentieth century the advent of stereotactic procedures has allowed the drift from lesional to stimulating technique of deep nuclei of the brain for therapeutic purposes . in this way , deep brain stimulation ( dbs ) was born , that , over the last two decades , has led to positive results for the treatment of medically refractory parkinson s disease , essential tremor , and dystonia . in recent years , the indications for therapeutic use of dbs
have been extended to epilepsy , tourette s syndrome , psychiatric diseases ( depression , obsessive compulsive disorder ) , some kinds of headache , eating disorders , and the minimally conscious state .
the potentials of the dbs for therapeutic use are fascinating , but there are still many unresolved technical and ethical problems , concerning the identification of the targets for each disease , the selection of the patients and the evaluation of the results . |
responses to applied medicines can be different and personalized treatment requires collection of the maximum number of data .
one of them is the metabolic profile of the patient reflecting the present state of the organism . for this purpose
spectroscopic techniques are applied , such as raman spectroscopy , mass spectrometry maldi imaging and high - resolution magic angle spinning nuclear magnetic resonance ( hr mas nmr ) .
these techniques allow determination of the structure of chemical compounds and biomolecules , and are applied in cancer tissue examination .
the purpose of these studies is identification of metabolites characteristic for cancer diseases and correlation of changes in metabolic profiles with extent of disease and probable prognosis .
raman spectroscopy is based on irradiation of a sample by means of laser and recording of scattered radiation .
a significant difference was observed in the raman spectra of benign and malignant tissues . for cancer tissues bands corresponding to c - c
( 1150 cm ) and c = c ( 1520 cm ) stretching vibrations of carotenoids and symmetric and asymmetric vibrations of the c - h at 28502940 cm for lipids were not observed ; however , these bands were present in the spectra of non - cancerous tissues .
diagnostic algorithms were developed on the basis of raman spectra tissues and tests of imaging were undertaken .
mass spectrometry is based on ionization of the sample and separation of ions according to their mass to charge ratio ( m / z ) . in analysis of tumor tissues the two - dimensional imaging of cells and tissues by means of maldi imaging technique
- dimensional maps of peptides and proteins containing 350 - 400 peaks within the range of m / z from 2,000 to 20,000 were recorded .
two peptide ions , m / z 4,027 and 4,355 , showed significant overexpression in prostate cancer in comparison with benign changes .
the peptide ion at m / z 4,274 showed expression in benign tissues adjacent to epithelial and stromal cells while for prostate cancer it showed only a little or no expression .
the peptide ion at 4,355 was identified as a fragment of mitogen - activated protein kinase mekk2 . for breast cancer tissues
there were detected six peaks in the range of m / z from 8345 to 8640 , more intense in the presence of receptor her-2 .
the peak at m / z 8404 was identified as cysteine - rich intestinal protein 1 ( crip1 ) , strongly associated with her-2 overexpression .
application of maldi imaging allowed prediction of her-2 status with a sensitivity of 83% , a specificity of 92% , and an overall accuracy of 89% .
the protein profiles corresponding to early stages of gastrointestinal cancer disease were significantly different from advanced stage tumors .
signals identified as -defensin-1 , -defensin-2 , and calgranulin a and b showed overexpression . sensitivity and specificity of this method were 93.8% and 95.5% , respectively .
maldi imaging was applied for 48 tissues from 25 grade iii and iv ovarian cancer patients and 23 benign tumors .
one of the identified peptides , pa 28 ( corresponding to 84 amino acid residues from 11s proteasome activator complex ) , can be used as a cancer marker .
pa 28 showed a high level of expression in carcinoma , especially in epithelial cells .
nuclear magnetic resonance ( nmr ) is a universal technique that allows one to study liquid , solid and gaseous samples placed in the magnetic field .
nmr spectroscopy is a non - destructive technique in comparison with mass spectrometry and allows histopathological analysis after measurement .
today magnetic resonance imaging ( mri ) has become a more widely accessible diagnostic technique .
the bridge between nmr measurements of liquids and magnetic resonance spectroscopic imaging ( mrsi ) in vivo by means of mri scanners is high - resolution magic angle spinning nmr , which was introduced in 1996 .
the first publication on studies of tissues of lipoma and liposarcoma utilizing this method appeared in 1997 . in 2009
two review articles about tissue spectra registration , identification of metabolites , determination of their concentration and relaxation times ( t1 and t2 ) were published [ 10 , 11 ] .
the nmr spectrum is a graph correlating signal intensity and the frequency of radiation absorbed by the atomic nucleus in the case of tissues , proton atoms .
chemical shifts depend on the structure of the molecule and are expressed in ppm ( parts per million ) .
high resolution of the spectrum in hr mas technique is obtained by spinning the sample around an axis inclined 54.7 to the direction of the static magnetic field [ 12 , 13 ] .
figure 1 illustrates the various methods of examination of the same tissue by nmr technique : 1h mrsi image and spectrum recorded using a scanner for the selected prostate fragment , which was then excised and re - analyzed .
the spectrum recorded in vitro provides more information about the kind and concentration of metabolites than the mrsi spectrum .
a t2-weighted mri image of prostate ; b mrsi spectrum taken from the voxel shown in a ; c 1h hr mas nmr spectrum of excised tissue ; d histological picture of examined specimen , in 60% contain stromal tissue
magn res med 2003 ; 50 : 944 - 954 ] in hr mas nmr technique the samples are spun with a spin rate of 45 khz . too fast spinning may cause mechanical changes and destruction of the sample , while too slow spinning decreases the resolution of spectra .
it was verified that during prolonged storage at this temperature concentration of metabolites did not change .
hr mas nmr measurements usually are performed at 14c , but also at room temperature .
tissues taken from rat fresh excised liver and earlier frozen renal cortex were examined at 17c and 30c .
no changes were observed in nmr spectra re - recorded over 5 h after excision .
however , significant changes in spectra of the renal cortex recorded after 10 min and 240 min were observed .
intensities of signals coming from triglycerides and trimethylamine n - oxide were decreased probably due to enzymatic degradation or autolysis .
no significant changes in concentrations of n - acetylaspartate , lactate and acetate in rat brain tissues ( 012 min after excision to freezing ) were observed . it was concluded that degradation of glucose and glycogen to lactate occurred during the excision of tissue .
reduction of temperature during measurement to 2c reduced changes in concentration of metabolites below 5% . in the hr mas nmr method analyzed tissues were placed in special vessels called rotors .
the diameter and volume of the rotor were 4 mm and 50 l , respectively . for analysis 840 mg of sample is necessary ; thus tissues collected by biopsy may also be analyzed .
for nmr measurement rotors were filled up with d2o or pbs solution in d2o [ 23 , 24 ] .
application of hr mas nmr will be discussed in the example of breast cancer tissues because the most attention was focused on this issue .
it was found that an increased ratio of lactate and choline concentrations is associated with an increase in tumor grade .
it was shown that an increase of t2 relaxation time of phosphocholine can help to distinguish grade ii and grade iii of ductal carcinomas .
identified numerous metabolites : amino acids ( mainly glycine , alanine , valine , leucine , lysine , isoleucine , tyrosine and histidine ) , metabolites associated with consumption of energy ( lactate , glucose ) and choline components ( glycerophosphocholine and phosphocholine ) .
principal component analysis showed increased levels of lactate , creatine , glycine and phosphocholine from specimens with high numbers of tumor cells .
two groups of samples were studied : tumors smaller than two centimeters ( t1 ) and bigger tumors ( t2t4 ) .
higher intensity ratios of glycerophosphocholine to phosphocholine and glycerophosphocholine to choline were found for non - involved samples than for tumors , whereas the intensity ratio of phosphocholine to choline was lower for non - involved specimens . on the basis of microarray studies , basal molecular subtype of breast cancer , an erbb2-overexpressing group and two or three luminal subtypes a , b and c were distinguished .
these subtypes indicated differences in gene expression profile and in prognosis and course of disease [ 28 , 29 ] .
hr mas and microarray analysis were performed for 46 samples of breast cancer . the quality of rna obtained after hr mas analysis for 11 tissues was insufficient ; therefore for microarray analysis adjacent tissue from the same tumors was used .
the examined tissues were diagnosed as invasive ductal carcinoma with er receptor expression which belong to luminal a subgroup .
two strategies were applied to combine the gene expression and metabolic data : multivariate analysis to identify different groups , and study of the correlation between levels of metabolites and biological processes . on the basis of gene expression
36 tumors were classified as luminal a subtype , one as basal , one as erbb2 , seven normal - like and one unclassified .
on the basis of hr mas spectra 31 tissues were classified as luminal a subtype but within this subtype three subgroups were distinguished : a1 , a2 and a3 . in subgroup a2 glucose and alanine concentrations
the intensity of -hydrogen amino acid signals was significantly lower for a1 and higher for a3 .
signals of lipid residues were significantly higher and signals of myo - inositol were significantly lower in a1 than in a2 and a3 .
moreover , subgroup a2 was enriched for a gene related to the cell cycle and dna repair .
the a2 group 's metabolic and transcriptional features indicate a higher warburg effect and proliferation compared with the other luminal a samples . in the second strategy
the correlations between concentrations of myo - inositol , taurine , creatine , glycerophosphocholine , phosphocholine , glycine and choline and all transcripts in microarray were investigated .
concentrations of the amino acids alanine , glycine , leucine , isoleucine , lysine , valine , histidine , tyrosine , glutamate , phenylalanine , taurine and creatine were determined in 80 samples of breast cancer from patients from the d region by the hr mas nmr method . moreover ,
the presence of metabolites such as myo - inositol , scyllo - inositol , choline , phosphocholine , glycerophosphocholine , lactate , acetate and -glucose was proven . a significant increase in the concentrations of creatine , choline , phosphocholine and glycerophosphocholine
significantly higher concentration of pc was found for patients with higher degree of malignancy . a similar insignificant tendency exists for glycerophosphocholine , phenylalanine and glucose . after the division of samples into three categories ( g1 , g2 and g3 ) , differences in concentration of phosphocholine disappear and remain only as a strong trend . there was no significant correlation between concentration of phosphocholine and ki 67 proliferation antigen ; however , there is a very evident trend .
lower cr and cho concentrations were observed for tumors with metastases in axillary lymph nodes .
reduced amounts of gpc , myo - inositol , glutamate and acetate were found for tumors with estrogen receptor .
increased level of myo - inositol and glutamate accompanies the presence of keratin ck5/6 ( tendency for scyllo - inositol and isoleucine ) .
higher concentrations of cr , gpc , acetate and glutamate were found for tumors with protein cyclin e present . a similar tendency was observed for alanine , choline , taurine , glycine , myo - inositol , leucine , lysine and reverse for histidine .
there are no significant differences in metabolite concentrations depending on the triple - negative phenotype ( tendency towards higher concentrations for phosphocholine and glutamate ) .
size of tumor , presence of her2 receptor as well as overall survival do not correlate with any metabolite concentration .
it can be expected that the development of nuclear magnetic resonance scanners and higher accessibility of hr mas nmr measurements would allow us to improve the diagnosis of cancers based on the metabolic profile .
new applications of nuclear magnetic resonance spectroscopy in chemistry , biology , pharmacy and medicine ( 68/e-61/bwsn-0126/2008 ) . and grants ds / i-18/17/2011 ( institute of organic chemistry , lut ) , 503/1 - 034 - 03/503 - 1 ( department tumour pathology , mul ) and 503/1 - 034 - 02/503 - 1 ( department of chemotherapy , mul ) . | the personalized approach in cancer treatment stimulates the search for new analytical techniques , including spectroscopic methods such as raman spectroscopy , mass spectrometry maldi ( matrix - assisted laser desorption / ionization ) imaging and high - resolution magic angle spinning nuclear magnetic resonance ( hr mas nmr ) .
the purpose of these studies is determination of metabolic profiles of cancer tissues , and their application in diagnostics and therapy of cancers .
the review is mainly focused on application of hr mas nmr technique .
qualitative and quantitative analysis of metabolites by means of this method is described for breast cancer tissues . in the near future
hr mas nmr in vitro studies of metabolic profiles combined with in vivo studies using mri scanners may be applied as a new diagnostic tool . |
the ewing 's sarcoma / primitive neuroectodermal tumour ( es / pnet ) family of tumours is part of a rare group of malignant neoplasms with small round - cell morphology .
es / pnet is an uncommon primary malignancy of the bone primarily affecting children and young adults with more than 80% of patients being younger than 20 years of age at the time of presentation .
less frequently , es / pnet arises from soft tissues and then the neoplasm is classified as extraskeletal es / pnet .
involvement of the mediastinum as the primary site of origin is extremely uncommon in the literature , only four cases were reported in a span of 20 years in a large case series from a tertiary care centre in india .
a rare case of primary pnet tumour of the anterior mediastinum mimicking massive pleural effusion in a young male is reported here .
a 30-year - old male , carpenter by profession , presented with dry cough and progressive shortness of breath for two months , right - sided severe chest pain for one and half month and low - grade fever for one month .
the patient was a non - smoker , non - alcoholic and non - diabetic .
clubbing was absent and there was no peripheral lymphadenopathy . on examination of the respiratory system features of right hemithoracic volume enlargement were present .
percussion note was dull all over the right hemithorax with presence of tenderness on superficial percussion .
on auscultation , a tubular breath sound was heard near the right infraclavicular area parasternally , in all other areas on the right side vesicular breath sound was markedly diminished .
examination of other systems including the lymphoreticular system and testicular examination were all within normal limits .
chest x - ray postero - anterior view revealed right - sided homogenous opacity involving all three zones with loss of costophrenic and cardiophrenic angles along with contralaterally shifted mediastinum [ figure 1 ] , suggesting right - sided massive pleural effusion ; but on thoracentesis , only 50 ml hemorrhagic fluid could be aspirated from the right infrascapular area after repeated attempts .
pleural fluid study yielded reddish - coloured , predominantly lymphocytic , exudative fluid with high adenosine de - aminase ( ada ) [ total cell count 170/cmm , lymphocyte- 80% , neutrophil-15% , ada-122 iu , protein-3.7g / dl , sugar- 86 mg / dl ] and pleural fluid for pap stain was negative .
subsequently contrast - enhanced ct ( cect ) scan of the thorax was done and showed a huge non - homogenous mass of heterogeneous density with multiple foci of necrosis occupying the entire right hemithorax , possibly arising from the anterior mediastinum , causing shifting of the mediastinum to the left and compression of the superior venacava ( svc ) , there was minimal pleural effusion [ figure 2 ] .
chest x - ray pa view showing homogenous opacity on the right side involving all zones with obliteration of the costophrenic angle and contralateral mediastinal shift ct scan of the thorax ( cect ) showing a large mass of heterogeneous density with foci of necrosis occupying the entire right hemithorax ct - guided fine needle aspiration cytology ( fnac ) was performed and the pathologist opined it as small round - cell tumour , possibilities : non - hodgkin 's lymphoma or small - cell carcinoma of lung [ figure 3 ] .
ct - guided tru - cut biopsy of the lesion was performed next and it revealed a tumour composed of round , oval or fusiform cells with hyperchromatic nuclei and scanty cytoplasm , mitotic figures were present [ figure 4 ] and it was reported again as small round - cell tumour , possibly small - cell carcinoma of the lung .
subsequently , fibreoptic bronchoscopy was done and it showed narrowing of the right bronchial tree possibly due to external compression but no intraluminal growth was noted .
on immunohistochemistry of the histology block of tru - cut biopsy , tumour cells expressed mic-2 and were immunonegative for cytokeratin , synaptophysin , chromoganin a , cd 20 , cd 3 , tdt , and the findings were consistent with primitive neuroectodermal tumours ( pnets ) .
cytology of ct - guided fnac slide under high - power field showing highly cellular smear with small round cells with hyperchromatic nuclei and scanty cytoplasm ( hematoxylin - eosin , 400 ) histopathology of ct - guided tru - cut biopsy slide under high - power field showing sheets of small round or oval cells with hyperchromatic nuclei and variable amount of cytoplasm , tumour cells showing rosette formation at places ( hematoxylin - eosin , 400 ) finally , a diagnosis of pnet of the mediastinum was made .
so it was an extremely rare case of peripheral pnet arising from the anterior mediastinum .
in 1973 , hart and earle first coined the term primitive neuroectodermal tumour ( pnet ) , which drew attention to the primitive nature of the tumour rather than histogenesis .
this entity is primarily a disease of childhood and young adults with a median age at diagnosis being nine years , and 80% of the cases are under 20 years of age .
pnets are highly malignant , undifferentiated neoplasms of the es / pnet family , arising from the germinal matrix cells of the primitive neural tube .
pnets are a variety of small round blue cell tumors ( srbct ) and may involve more than one site at presentation .
when located in extraosseous sites , supratentorial brain , spinal canal , and soft tissues of the thigh are the usual sites ; primary involvement of extracranial sites like the mediastinum , chest wall , kidney is very rare . in a study of extraskeletal es by ahmad et al .
primary mediastinal pnets are mostly located in the posterior mediastinum like other neurogenic tumours , primary involvement of the anterior mediastinum is extremely uncommon .
these neoplasms are usually seen in young males who predominantly present with chest pain as in our case .
radiologically , it is very difficult to differentiate these neoplasms from other common anterior mediastinal tumours , ct scan of the thorax reveals an ill - defined , heterogeneous mass with necrotic foci within , calcification is usually absent .
smears are generally highly cellular and are composed of both single cells and groups of loosely cohesive cells with a high nuclear / cytoplasmic ratio , hyperchromatic nuclei without prominent nucleoli , distinctively smooth nuclear membrane contour , finely granular chromatin , one or two small nucleoli and scanty but almost always present perinuclear clear cytoplasm suggesting epithelial differentiation .
in atypical es and pnet the cellular and nuclear atypia is more marked than in conventional es , rosette- like structures are more common , the distinction between large light and small dark cells less obvious and cells with thin cytoplasmic processes are present . in atypical cases diagnosis on fnac is quite challenging and the extraskeletal variant of es / pnet is more often difficult to diagnose on cytopathology , and a histology with immunohistochemistry can only clinch the diagnosis in these cases.the classic histological pattern of es / pnet consists of solid sheets of small uniform primitive cells with round nuclei and scanty cytoplasm that lack significant differentiation . in more differentiated es / pnet
, homer - wright rosettes may be identified . due to the lack of characteristic morphologic features , es
/ pnet is difficult to distinguish from histologically similar small round - cell tumours including rhabdomyosarcoma , desmoplastic small round - cell tumour , poorly differentiated synovial sarcoma , mesenchymal chondrosarcoma , neuroblastoma and lymphoma .
immunohistochemical expression of the mic2 gene product ( cd99 ) in es / pnet is helpful in separating this entity from other small round - cell tumours .
however , cd99 expression is not specific for es / pnet , as it can be expressed by other sarcomas , including poorly differentiated synovial sarcoma and mesenchymal chondrosarcoma
. other ancillary techniques , including cytogenetic analysis , reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization may provide further aid in confirming the diagnosis of es / pnet .
demonstration of the t(11;22)(q24;q12 ) chromosomal translocation ( ews - fli1 gene rearrangement ) is highly specific for es / pnet as it is encountered in more than 90% of the neoplasms .
management is usually multimodal : surgery followed by ifosfamide or doxorubicin - based chemotherapy and radiotherapy .
overall , pnets are aggressive neoplasms that have less than 20% five - year survival , but the overall prognosis of extraskeletal es / pnet seems to be better .
a retrospective study of 24 patients with extraskeletal es showed an overall five - year survival rate of 61% .
wide tumor - free resection margins in conjunction with multi - agent chemotherapy are necessary for good clinical outcomes . although , a rare entity , extraskeletal es / pnet should be considered in the differential diagnosis of primary mediastinal neoplasms , especially in children or young adults and if a primitive small round - cell tumour is encountered in cytology or histology . histopathology with immunohistochemistry , cytogenetic analysis and other molecular tests to identify chromosomal translocations , are invaluable to establish specific diagnosis . | a 30-year - old male , carpenter by profession , presented with a history of dry cough and progressive shortness of breath for two months along with right - sided chest pain for one and a half months .
the clinico - radiological picture was suggestive of right - sided massive pleural effusion .
computed tomography ( ct ) scan of the thorax showed a huge mediastinal mass occupying the entire right hemithorax with very small amount of pleural effusion .
ct - guided fine needle aspiration cytology and tru - cut biopsy from the mass both revealed small round - cell tumour , possibly small cell carcinoma of the lung .
however , on immunohistochemistry tumour cells expressed mic-2 and it was consistent with a diagnosis of primitive neuroectodermal tumour . |
squamous cell carcinoma ( scc ) is the most common malignant mucosal neoplasm to affect the head and neck region .
occasionally , variants of scc may be encountered , which includes verrucous , exophytic or papillary , spindle cell ( sarcomatoid ) , basaloid and adenosquamous types that make up an aggregate of 10%15% of all sccs .
spindle cell carcinoma ( spcc ) is a relatively rare malignancy affecting the upper aerodigestive tract comprising up to 3% of scc .
the most common site of origin in the head and neck region is larynx and hypopharynx .
spcc , also called lane tumor , pseudosarcoma , carcinosarcoma , sarcomatoid carcinoma , collision tumor and pleomorphic carcinoma , is an uncommon poorly differentiated type of scc .
it is a biphasic tumor with a carcinoma that has surface epithelial changes and an underlying spindle - shaped neoplastic proliferation .
there has been confusion over the basic nature of the sarcomatoid element , that is , whether it is benign or malignant and mesenchymal or epithelial in origin . the epithelial nature of the sarcomatoid component of spcc is clearly revealed by a combination of immunohistochemical staining for keratins and electron microscopic demonstration of tonofilament - like filaments and/or desmosome - like structures .
however , who classification of tumor has placed this entity under malignant epithelial tumors of scc .
moreover , to the best of our knowledge , there is only one case report in literature documenting the malignant transformation of oral verrucous leukoplakia into spcc .
we report here a case of spcc arising in the buccal mucosa in a patient who was previously diagnosed with oral leukoplakia and was under medical management for same .
a 35-year - old female patient reported to the outpatient department of oral medicine and radiology with the chief complaint of pain in the left cheek for past 8 days . according to history of presenting illness , the patient was provisionally diagnosed as homogenous leukoplakia with respect to left buccal mucosa 6 months back [ figure 1 ] . at that time , an incisional biopsy was done from left buccal mucosa which was suggestive of hyperkeratosis .
the patient was then prescribed antioxidant once daily and was advised to report for regular follow - up .
after 6 months , she reported with the chief complaint of pain in the left buccal mucosa past 8 days .
personal history showed that she had history of areca nut chewing , 23 times in a day for 67 months and had quit the habit 4 years back .
extraoral examination showed the presence of left submandibular lymphadenopathy which was tender and fixed to the underlying soft tissues .
intraoral hard tissue examination showed grossly decayed 18 , root stumps of 14 , 46 , 47 and 48 , carious 13 , 14 , 23 and 35 and missing 27 , 36 and 37 .
intraoral soft - tissue examination revealed diffuse , thick , nonscrapable white patch on the left buccal mucosa extending from commissure region up to the retromolar region anteroposteriorly and from the level of the occlusal plane to buccal vestibule superoinferiorly [ figure 2 ] .
a punched out ulcer was seen on the left buccal mucosa approximately 1 cm 1 cm in size located 1 cm lateral to the commissure of mouth [ figure 2 ] .
on palpation , it was well defined with indurated margins and covered with a yellowish base .
based on history and examination findings , provisional diagnosis of homogenous leukoplakia with malignancy in relation to left buccal mucosa was made .
the patient was advised to get routine blood investigation and contrast - enhanced computed tomography ( cect ) scan of the head and neck . clinical photograph showing homogenous leukoplakia on the left buccal mucosa before appearance of ulcer clinical photograph of the patient showing homogenous leukoplakia with an ulcer after 6 months of initial lesion cect revealed heterogeneously enhancing soft - tissue thickening in the left buccal space with cervical lymphadenopathy involving level ib and bilateral upper jugular nodes [ figure 3 ] .
histopathological sections showed hyperparakeratinized stratified squamous dysplastic epithelium with a focal area of invasion in the underlying connective tissue [ figure 4 ] .
the lesional tissue was composed of streaming fascicles of anaplastic spindle cells [ figure 5 ] , which showed marked pleomorphism and strong resemblance to atypical mesenchymal cells .
the cells present in the fasciculated pattern were elongated with elliptical nuclei and showed hyperchromatism and altered nuclear - cytoplasmic ratio [ figure 6 ] .
numerous mitotic figures and abnormal mitoses were seen throughout the lesional tissue . in the deeper layer ,
spindle cells had invaded the muscle tissue and had caused degenerative changes in the muscle fibers .
sections showed that 90% of the tumor cells were positive for pancytokeratin [ figure 7 ] and 5% of the tumor cells were positive for vimentin [ figure 8 ] .
the patient was referred to the department of oral and maxillofacial surgery where it was planned to carry out surgical excision with wide margins along with neck dissection .
contrast - enhanced computed tomography section showing enhanced and enlarged bilateral cervical lymph nodes ( arrow ) photomicrograph ( 10 ) showing dysplastic epithelium with focal area of invasion in underlying connective tissue photomicrograph ( 10 ) showing streaming fascicles of anaplastic spindle cells ( arrow ) photomicrograph ( 40 ) showing marked pleomorphism and strong resemblance to atypical mesenchymal cells with hyperchromatism and altered nuclear - cytoplasmic ratio photomicrograph ( 10 ) showing positive immunohistochemical expression for pancytokeratin in majority of tumor cells and inset ( 40 ) showing cytoplasmic positivity for pancytokeratin ( arrow ) photomicrograph ( 10 ) showing positive immunohistochemical expression for vimentin in very few tumor cells and inset ( 40 ) showing cytoplasmic positivity for vimentin
they are relatively uncommon in the oral cavity accounting for < 1% of all tumors in this region .
spcc is a rare variant of scc with both malignant squamous cells and spindle cells of epithelial origin .
moreover , the diagnosis can be challenging , especially when scc component is not obvious .
thus , to establish a correct diagnosis , any clue of epithelial component should be carefully sought in suspected lesions .
four factors are considered as predisposing factors , namely , tobacco , alcohol , poor oral hygiene and previous irradiation to area of tumor .
it commonly affects men than women , usually in the middle or later decades of life with a mean age of 57 years . in the present case , the patient is a female in the third decade of life .
although the patient did not have any previous history of irradiation , there was known history of areca nut chewing .
clinical presentation of oral spcc can vary from an exophytic polypoid mass with an ulcerated surface to a frankly infiltrative ulcer , frequently affecting lower lip , tongue and alveolar ridge . in the present case ,
the patient presented with leukoplakia which had transformed into ulcer over a period of 6 months on buccal mucosa , rare site of presentation .
histologically , it is characterized by the presence of two distinct epithelial - derived components : a carcinomatous or scc component and a sarcomatoid or dysplastic spindle cell component .
the carcinomatous component comprises a minor portion of the tumor mass and is represented by dysplasia , carcinoma in situ or frankly invasive carcinoma .
areas of squamous differentiation are most consistently identified at the base of polypoid lesion at advancing margin or within invaginations at the surface where epithelium is not ulcerated or denuded .
the spindle cell component comprises major portion of this tumor and imitates a number of different mesenchymal processes .
it can be arranged in diverse array of appearance : storiform , cartwheel or whorled : resembling a malignant fibrous histiocytoma ; interlacing bundles or fascicles similar to leiomyosarcoma or malignant peripheral nerve sheath tumor ; chevron or herringbone similar to fibrosarcoma . in the present case ,
areas of elongation and spindling seem to arise from basal epithelial cells , making demarcation between surface epithelial cells and underlying tumor indistinct .
the epithelial and spindle components share a common pathway of tumorigenesis despite their divergence at the phenotypic level .
however , through positive keratin immunostaining and demonstration of desmosomes and tonofilaments in the cells , it has been proven that spindle cell elements are epithelial in origin .
immunohistochemically , the most sensitive and reliable epithelial markers to be used are keratin and epithelial membrane antigen .
these findings suggest that these cells have acquired mesenchymal properties both morphologically and functionally through metaplastic changes .
double labeling with keratin and vimentin reflects the versatility of intermediate filament phenotype . in the present case
spcc in the oral cavity and oropharynx is potentially aggressive and seems to recur easily and tends to metastasize .
although it is difficult to predict biologic behavior , those with deeply invasive tumors tend to have a poor prognosis , whereas those with early stage tumors usually have an excellent prognosis .
it is common for potentially malignant disorders such as leukoplakia to undergo malignant transformation into scc but is rare to transform into spcc , a variant of scc .
spcc is a biphasic malignant tumor which is aggressive in nature , tends to recur easily and metastasize .
interesting part of this tumor is that it mimics other connective tissue sarcoma and malignancy at microscopic level , so its histopathologic differentiation is a must for timely diagnosis and management .
| spindle cell carcinoma ( spcc ) , a variant of squamous cell carcinoma , is a biphasic malignant neoplasm that occurs mainly in the upper aerodigestive tract .
it is uncommon in oral cavity , accounting for < 1% of all tumors in oral cavity .
furthermore , it is uncommon for oral potentially malignant disorders such as leukoplakia to undergo transformation into spcc . in this paper
, we are reporting a case of spcc of buccal mucosa in a 35-year - old female who was previously diagnosed with oral leukoplakia past 6 months . |
after the pediatrician and the newborn health screener technician , pediatric otolaryngologists and audiologists are parents ' first professional contact . typically , after the diagnosis of hearing loss , they make the referrals to the early education specialists .
training for medical / audiological professionals typically is focused on identification and collection of quantitative data about the child 's physiological hearing loss .
most have not studied the impact of deafness on language and emergent reading development or have critically examined the benefits of another communication option for deaf children with cochlear implants ( deaf / ci ) , that of early sign bimodal / bilingualism [ 15 ] .
these areas are typically relegated to the speech - language pathologist , deaf educator , and reading specialist .
the purpose of this paper is to cross disciplines to inform medical and audiological professionals about an alternative communication option sign bimodal / bilingualism for deaf children with cochlear implants . related to the theme of this special issue
, we suggest that pediatric medical / audiological professionals can accentuate the pearls or benefits of the implant ( i.e. , improved speech production ) and decrease the pitfalls , of the implant .
we see one pitfall as the limiting of the deaf / ci childs language and literacy learning potential to one language ( monolingualism ) instead of broadening their language - learning base with sign bimodal / bilingualism ( two languages ) .
indeed , every audiologist already knows that even with early hearing detection intervention ( ehdi ) in place , still many deaf newborns are missed . as a result
moreover , many early identified children with even the best surgical outcomes and professional and parent training may never develop spoken language or emergent literacy skills in the same manner as hearing children do [ 68 ] .
we speculate that sign bimodal / bilingualism may be a viable solution for interested parents for late implanted and even early implanted deaf children because it opens both pathways visual and auditory to language learning as early as possible [ 4 , 5 ] .
it means that the individual uses both languages for everyday purposes . indeed , by introducing american sign language ( asl ) ( bilingualism ) and sign - supported speech ( bimodalism ) as another language avenue in addition to spoken english
the advantages of early sign bimodal / bilingualism are that it enriches the deaf child 's early capacity to think and communicate with parents , extended family and friends and learn to read and access the school curriculum [ 1013 ] .
another advantage is that signs can enhance the learning of spoken english as deaf children can piggyback their speech vocabulary skills onto their sign language vocabulary skills [ 14 , 15 ] .
a disadvantage of bilingualism is that if both languages remain underdeveloped , then the child becomes semilingual or weakly proficient in both languages .
semilingualism has tremendous negative consequences especially if the semilingual deaf youth or deaf adult gets caught in the criminal justice system .
they can not participate in their criminal defense because of their weak sign skills and weak english skills thereby jeopardizing obtaining their constitutional rights of due process .
sign bimodal / bilingual users utilize both code - switching and code - mixing strategies .
code - switching is moving from one language to another , inside a sentence or across sentences , [ 17 , page 699 ] .
it refers to the subtle and purposeful way in which bilinguals switch between the two languages [ 17 , page 37 ] .
in contrast , code - mixing has been defined as the mixing of two languages within a sentence or across sentences [ 17 , page 699 ] .
code - switching and code - mixing have also been linked to language contact studies which examine concepts such as language transfer , borrowing , and interference [ 17 , pages 51 , 7 ] .
we differentiate our communication definition of cs from other cs definitions with adult language users who have full access and proficiency in both languages such as interpreters , teachers , and deaf adults who had deaf parents . while alice , the child in this case , study had neither full access nor native proficiency in both languages , we still view her as an emerging bimodal / bilingual who wears a cochlear implant and who used both codeswitching and code - mixing strategies in two languages to enrich her communication , literacy , and language learning . using pinker 's (
fingerspelling is shown in hyphenated capital letters and simcom or codemixing is shown with sign capitalized and voice made at the same time in smaller case letters .
for example , cat : asl sign , cat : spoken word , c - a - t : fingerspelled word , cat / cat : ( sc ) simcom sign and spoken word at the same time . )
hidden mental worlds , we take our readers down the rabbit hole to see how alice develops both of her languages by looking at two data sets : ( 1 ) her language assessment profile and ( 2 ) her responses to the storytelling experiments .
in this study , we utilize qualitative descriptive tools used in reading research [ 19 , 20 ] .
these narrative accounts are used by observers to make verbatim accounts of the child 's use of language while reading a text or answering questions from the reading teacher [ 21 , 22 ] .
observers then find trends or patterns in the data to identify thinking and reading processes [ 19 , 22 ] .
the dependent variables are reading and language behaviors that can be described and quantified ( i.e. , retelling of events in storybook or number of times the student codeswitches and codemixes while using the two languages and the independent variable is the intervention , the use of storybook reading to faciltiate early reading development ) .
transcripts of the spoken and signed conversations of alice were transcribed into english print with an 80% agreement noted by an independent transcriber .
( 1 ) how does alice use her sign bimodal / bilingualism , code - switching , and code - mixing strategies during general camp activities ( i.e. , play , speech lessons , auditory listening sessions , art , and at lunch time ) ? ( 2 ) what sign bilingual code - switching and code - mixing strategies did she use during reading activities while attending to a story told in sign only and speech only and in retelling wordless picture books when read to her in speech only and in sign only ? wordless picture books and books with repetitive and predictable phrase and sentence patterns were used : frog where are you ? by mayer [ 23 , 24 ] .
we also used the book , there was an old lady who swallowed a fly , that had repetitive and predictable sentence patterns .
the setting of the study was a summer reading camp located at a preprofessional university training institution .
bachelors level , master 's and doctoral level students in communication sciences , speech - language pathology , audiology and deaf studies / deaf education preparing for careers in working with deaf children with cochlear implants were observers and participants .
our case study , alice , attended camp for four weeks , four days a week from 9:00 am to noon .
deaf and hearing staff and visitors ate lunch with the six children in order to promote additional conversation skills .
mason has conducted a series of intervention studies and has reviewed other studies which show causal links between storybook reading to children and emergent and later reading achievement .
thus , we utilized a deaf native signer to provide the asl telling and reading of the story and a speech - language pathologist to provide the stories in spoken english only ( see for similar procedures ) .
following up activities included discussion of concepts and vocabulary , relating story to students ' personal experiences , art and play - acting experiences using both languages . upon this rich language base ,
children in groups or individually met with the speech - language pathologist or audiologist to listen to stories told in oral language and to build spoken and auditory language skills . just as yoshinago - itano piggy - backed
speech onto sign at the word level for deaf / ci infants , we aimed to investigate the effects of piggy - backing spoken english stories onto stories told / read using american sign language for purposes of examining code - switching and code - mixing behaviors .
alice , a caucasian girl , was born deaf from unknown causes . at age three , she began an early intervention program in a deaf regional day school and she started to learn sign language provided by a hearing parent - infant educator .
her primary caretaker is her grandmother who knows little signs and has a high school diploma .
on wesby 's play and theory of mind scale ( adapted from ) , the team evaluators placed her at level ix ( age 3.5 to 4 years ) which observes play activities in the camp .
related to corresponding language behaviors at this level , she used conjunctions ( and ) and began to respond to why and how questions that require reasoning about perception .
these language behaviors were observed in american sign language ( asl ) , spoken english , and in a mixture of signs and speech .
the speech - language pathologists and audiologists could not test alice for speech reception without visual cues .
alice 's score of 37 db hearing loss refers to the sound field pure tone average with her implant . at the 500 hz
, alice scored 40 db ; at the 1000 hz , she scored 35 db ; at the 2000 hz , she scored 30 ; at the 4000 hz , she scored 30 .
it is important to note that the degree of hearing loss does not equate in a linear way with the child 's ability to hear conversational speech and comprehend language in real - world environments such as noisy classrooms , on a playground , or in the school cafeteria [ 2 , 3 ] .
the degree of hearing loss expressed in decibels may show impressive decreases from 100 db without the implant to 37 db with the implant .
however , such an average inaccurately predicts the child 's functional hearing in noisy real - world environments or the ability to comprehend and produce language through audition alone .
prior to age 3.5 when she was implanted , alice was nonverbal and only used signs for communication . with the implant and training
, she did make marginal gains in speech production . however , her speech was for the large part unintelligible .
alice was administered the sd / pbk , a speech discrimination testing using the phonetically balanced kindergarten word lists .
alice was also administered the wipi , the word intelligibility by picture identification test ( wipi ) .
she was also given the six - sound ling test , and without visual cues , she was able to recognize the sounds : ah , ee , sh , s , and m with 100 percent accuracy .
the ppvt is a single - word vocabulary test where the child views the four picture plates and identifies the word the test administrator says .
she scored in the 4-year - old range for receptive spoken vocabulary , 3.5 years behind her chronological age of 7 years , 5 months .
she was fitted with an implant made by cochlear corporation with a nucleus freedom bte speech processor .
she was mapped 10 months prior to the reading camp at a hospital in a larger metropolitan city , 80 miles away . when reading words and paragraphs on the reading assessments , alice translated the printed english words into signing as she codeswitched from english to signing .
when she did not know a work in a text , she would fingerspell it .
she was given an informal reading inventory and scored at the first - grade instructional level , kindergarten independent level- and 2nd - grade frustration level .
when reading a story about a pet dog on the iri , alice would sign word for word in the paragraph . while she could match printed words in the sentence with manual signs with accuracy and facility , when she was asked questions about the story as a whole , she could not answer them .
thus , she was able to code - switch from print to sign at the word level , but she could not integrate her understanding of the specific word into the context of the sentences or the complete story .
she was administered the test of early reading-3 for deaf and hard of hearing ( quotient 109 , 75 percentile rank ) on a test that measures alphabet knowledge , conventions , and meanings of print . on the terra-3
when asked to decode or figure out the meaning of a printed word , she did not use any phonemic awareness or phonics , but relied on visual strategies .
alice made miscues or mistakes in reading based on letter visual similarities such as identifying the sign brown for the word , down , long for log , and friend for then .
we presented alice with a list of words and we asked her to sound out the words , but she could not do this task . based on her audiogram denoting a 37 db loss with her implant , we thought she could use sounding - out strategies for word decoding , but she could not phonologically decode familiar words , unfamiliar words , or pronounce nonwords .
related to her expressive writing behaviors , alice could label her drawings with single words .
she was not able to write complete sentences unless she copied them from the whiteboard model during our reading camp activities .
we determined that alice knew the fingerspelled alphabet and could spell short three- to four - letter words using fingerspelling .
we also observed alice using sign - supported speech with hearing adults and asl with the deaf adults at our summer reading camp .
the cpvt contains many signed english signs , so it does not measure asl exclusively .
alice scored above her age range on this test . the native deaf evaluators on our staff administered to alice the kendall conversational proficiency level assessment .
this is a rating scale that assessed alice 's sign language communication competency across three levels from 0 to level 7 .
alice was able to converse in sign language about topics which interested her , refer to a number of objects and actions , and communicated about the location of objects .
she could discuss characteristics of people and objects , communicate about what she owns , link her conversations to others , and use sign to affirm the presence of objects , identify pictures using signs , and use at least two syntactically related components in sign language .
our videotaping data , field notes , and notes from her files revealed that alice used code - switching or code - mixing from one language to another ( asl to speech , asl to print , and sign - supported speech to print ) with different communication partners , in various settings , and for various reasons .
for the most part , she used speech and sign - supported speech with their hearing parents , siblings , and hearing staff members of the camp that could not sign .
when walking across campus on a field trip , she would often stop at environmental signage and alice would stop and attempt to read the print .
she did the same when she was looking at print in books ; when they came across a word they did not know , they would fingerspell the word . during therapy time with the audiologist and the speech pathologist , she would code - switch to speech or sign supported - speech . on the playground ,
the alice liked to use sidewalk chalk to print their names and draw pictures and label them with letters and words .
she would use asl to explain these pictures and print words to her teachers and classmates . on the playground and at lunch , alice would code - switch from asl to speech when using social words such as please , thank you or sorry , or excuse me , when they were in emotional or physical pain ( i.e. , falling down on the playground ) or expression of surprise ( uh - oh , oh , no ) and to use emotion words such as sorry and i love you . she would code - switch to speech to emphasize , repeat , or reinforce a point or express a strongly felt attitude in a story , and yell out loud , yes or no and to imitate hearing people using cell phones or singing into a microphone .
she would code - switch from asl conversation to fingerspelling when coming across a word she did not know in a storybook or on environmental signage around campus , when labeling pictures in a book or retelling a story .
she would codemix asl and sign - supported speech when telling something that happened at home or on a family outing
. she would codes - witch from asl or sign - supported speech to fingerspelling when using a name sign to get the attention of a staff or peer member of the camp , when working with the slp or the audiologist who did not knew little or no sign , or when communicating with the native deaf storytellers at camp and the deaf graduate students who joined them for lunch .
when attending to a story from the native deaf storyteller , she would answer questions in asl and when asked to retell segments of stories .
she would code - switch to sign - supported speech when reading word for word in a storybook .
the first session was presented in asl by a native deaf signer , then the next story was presented in spoken english by the speech - language pathologist .
todd , the native deaf signer , signed the whole story in asl pointing to the pictures as he goes along . here
are some excerpts from todd 's asl storytelling with students ' responses ( box 1 ) . in box 2 ,
two speech - language pathologists , ann and faye , tell this same story in spoken english only . in the asl storytelling rendition , two of the camp children bailey and alice used speech and sign ( codemix ) to answer a question and to label a picture ( spider / spider crawl ) .
bailey uses code - mixing to emphasize a point ( catch / catch ) .
alice and bailey code - switch to speech ( no ) to express disgust at a woman eating the spider .
alice switches to speech to make the animal sounds of a cat ( meow , meow ) .
bailey codeswitches to speech to excitedly answer todd 's predictive question of what lies ahead ( dog ! ) .
bailey codeswitches to speech to make the sound of a dog ( woof , woof ) .
alice switches to speech to todd 's asl classifier ( puffed cheeks ) and says fat .
alice answers todd 's question and says horsey . in the spoken - language story rendition ,
the two girls principally used speech to label pictures of animals , to label actions , to label sounds that animals and birds made , and to show enjoyment of the story ( vocalizations during laughter ) or to express negation ( no ) .
after the spoken repetition of the line in the story , i do not know why she swallowed the fly !
said by one of the hearing story readers ( ann ) , one of the girls ( alice ) said in a complete sentence : i do not know why .
the girls also used signs to support their speech , either using signs and speech simultaneously or separately .
( alice : bugs / bugs ) , ( bailey : bird / bird ) .
todd utilizes a number of strategies similar to those found with deaf parents reading to their deaf children .
he used signing to build background knowledge , to share personal experience tied to book theme , and to persons and objects in pictures .
he also asked questions using signing to check the children 's comprehension . not using fingerspelling extensively ,
he frequently paused and waited for reply , he maintains visual attention and recovered attention when the children became visually distracted .
he did use some code - switching from sign to fingerspelling and while pointing at print and giving the sign language equivalent .
having laid the concepts for the story using asl by todd , faye and ann presented the same story in spoken language .
similar to todd , faye and ann used a number of strategies using speech to make the storytelling enjoyable and entertaining for the children as well as to teach them speech skills .
faye used signs ( simcom ) to support the children 's learning of speech . both clinicians used speech to help the children label pictures in books , to support children 's learning of words , to ask questions , to praise , to model the speaking of complete sentences , to redirect questions , and to encourage turn - taking .
ann used speech to set up contrasts of concepts for spoken words ( old versus young ) .
the children used code - switching and code - mixing with their storytellers to emphasize a point .
they used speech to express emotion such as excitement or disgust and to enhance their storytelling by making the sounds of animals .
the girls used code - mixing to support their use of speech and their use of asl .
alice would view an asl classifier ( puffed cheeks ) , then use the spoken word , fat . during the spoken story ,
in the next experiment , alice was read four wordless picture books and predictable books in asl and in spoken language only .
table 1 shows the data on the number of incidences where she used asl only and speech only in her retellings of the four stories .
the table also shows the number of times she codeswitched and codemixed during her retellings of each story .
regarding alice 's expressive speech language , her speech utterances numbered 11 after the asl rendition of the first story and increased to 23 after the spoken english rendition of the new story .
alice made 24 and 22 codemixes after the stories were told in asl and decreased to only 3 codemixes with the last story being told in speech only .
the asl utterances remained low for both after the asl storytelling and the spoken english storytelling .
from alice 's data and from the other five deaf / ci children in the camp , we developed a four - point scale filled out by three hearing and three deaf evaluators who worked with the children in the camp .
they collectively placed alice at level three ( box 3 ) . from our observations ,
if we climb down the rabbit hole with alice and observe her language and reading processes , we see her sign bimodal / bilingualism has rich , expressive power .
viewed thusly , we can get a more complete picture of alice 's language and literacy - learning potential in both languages .
we , capitalizing on the pearls or benefits of cochlear , implant technology ( improved speech production ) and avoid one pitfall of being limited to one language - learning pathway .
we speculate that sign bimodal / bilingualism through parent and teacher storybook reading may even benefit those deaf infants who are identified early and receive early intervention because it opens both pathways to language learning and enhances the cognitive , language , emergent literacy , and psycho - socioemotional capabilities of deaf children [ 4 , 5 ] .
more interdisciplinary research conducted by teams of early childhood educators , speech - language pathologists , educational audiologists , deaf educators , and reading specialists are needed to substantiate this speculation .
such research would enhance the potential of pediatric otolaryngologists and audiologists in providing information about communication and language options for families of deaf children with cochlear implants . | alice , a deaf girl who was implanted after age three years of age was exposed to four weeks of storybook sessions conducted in american sign language ( asl ) and speech ( english ) .
two research questions were address : ( 1 ) how did she use her sign bimodal / bilingualism , codeswitching , and code mixing during reading activities and ( 2 ) what sign bilingual code - switching and code - mixing strategies did she use while attending to stories delivered under two treatments : asl only and speech only .
retelling scores were collected to determine the type and frequency of her codeswitching / codemixing strategies between both languages after alice was read to a story in asl and in spoken english .
qualitative descriptive methods were utilized .
teacher , clinician and student transcripts of the reading and retelling sessions were recorded .
results showed alice frequently used codeswitching and codeswitching strategies while retelling the stories retold under both treatments .
alice increased in her speech production retellings of the stories under both the asl storyreading and spoken english - only reading of the story .
the asl storyreading did not decrease alice 's retelling scores in spoken english .
professionals are encouraged to consider the benefits of early sign bimodal / bilingualism to enhance the overall speech , language and reading proficiency of deaf children with cochlear implants . |
hyperglycemia and insulin resistance are common in critically ill patients and more pronounced among patients having sepsis , burns , trauma , and cardiothoracic surgery.1 hyperglycemia was previously considered an adaptive response essential for survival and was not routinely controlled in intensive care units ( icus).2,3 however , more recent evidence suggests that hyperglycemia in critically ill patients is associated with worse outcomes .
multiple randomized control trials have evaluated the role of intensive insulin therapy in intensive care patients.417 from these studies controversy exists with regards to the target blood glucose level during insulin therapy .
several studies have evaluated glycemic control in surgical , medical and mixed icu patient populations .
the largest surgical trial was performed by van den berghe and colleagues and was a randomized study of 1548 adult surgical patients receiving mechanical ventilation on icu ( 62% had undergone cardiothoracic surgery ) who were randomized to conventional blood glucose management or intensive insulin therapy.9 insulin therapy was initiated when the blood glucose concentration reached 12 mmol / l in the conventional group versus 6.1 mmol / l in the intensive treatment group and the target range was 1011.1
mmol / l ( 180 to 200 mg / dl ) in the conventional group compared to 4.46.1 mmol / l ( 80 to 110 mg / dl ) in the intensive insulin therapy group .
all patients were given 200 to 300 g of intravenous glucose during the first 24 hours of icu admission , with aggressive nutritional support to reach the caloric goal by icu day 2 ( the majority with mixed enteral and parenteral nutrition ) .
a 12 month risk reduction in mortality of 43% ( p < 0.036 ) was demonstrated in the intensive therapy cohort .
risk reductions in this group were also seen for other outcomes including a 46% risk reduction in bloodstream infections .
first , the overall icu hospital mortality rate was large in the control group , which may have confounded the benefit of intensive insulin therapy .
in addition , the large infusion of glucose during the first 24 hours of icu admission followed by aggressive nutrition may have additionally benefited the intensive insulin group as they were rescued by early aggressive therapy .
the leuven medical was the only large randomized trial that evaluated the effect of glycemic control in medical icu patients and assigned 2000 patients to intensive insulin therapy or conventional glucose management.10 as in the surgical trial , intensive therapy targeted a blood glucose level of 80 to 110 mg / dl , whereas conventional therapy targeted a blood glucose level of 180 to 200 mg / dl .
however , the intensive insulin therapy arm had a higher mortality when icu stay was shorter than 3 days but was associated with reduced mortality if longer than 3 days .
intensive therapy was also associated with reduced acute kidney injury rate , duration of mechanical ventilation , icu length of stay , and total hospital length of stay .
the most robust trial to date is the multicenter normoglycemia in intensive care evaluation survival using glucose algorithm regulation ( nice - sugar ) trial which raised the question of potential treatment - related harm and also documented significant hypoglycemia .
the nice - sugar trial randomly assigned 6022 medical and surgical icu patients to either intensive insulin therapy ( target blood glucose level of 81 to 108 mg / dl [ 4.56 mmol / l ] ) or conventional glucose control ( target blood glucose of < 180 mg / dl [ < 10 mmol / l]).9 although the conventional glucose group was defined only by a maximal blood glucose target , the insulin infusion was reduced and then discontinued if the blood glucose level dropped below 144 mg / dl ( 8.0 mmol / l ) .
the intensive insulin therapy group had a significantly higher 90 day mortality and significantly higher incidence of severe hypoglycemia , defined as blood glucose < 40 mg / dl ( 2.2 mmol / l ) .
therefore , a blood glucose target of 8.010 mmol / l ( 144180 mg / dl ) rather than a more stringent target ( eg , 81108 mg / dl [ 4.56.0 mmol / l ] ) or a more liberal target ( eg , 180200 mg / dl [ 1011.1 mmol / l ] ) , avoids marked hyperglycemia , while minimizing the risk of both iatrogenic hypoglycemia and other harms associated with a lower blood glucose target .
the purpose of the study was to record blood glucose concentrations , in patients admitted to a general icu , for the first 48 hours after icu admission and compare the results to an audit standard of 8.010.0 mmol / l , based on the nice - sugar study and international recommendations for glycemic control in critically ill patients.10 we aimed to identify reasons and periods of time where deviations in blood glucose control are most likely and to make recommendations on how to improve this .
we decided to undertake this clinical audit because the findings of the van den berghe studies were not replicated by other investigators and we were concerned that the widely accepted van den berghe approach for intensive blood glucose control increases mortality , as reported by the nice - sugar trial and recent meta - analyses.11,18 our aim : to change current practice and patient care in accordance with the latest recommendations and evidence based medicine .
a retrospective review of 20 patients receiving mechanical ventilation in an eight - bedded general ( medical - surgical ) icu of a district general hospital was conducted .
an audit tool was developed to evaluate the pattern of glycemic control over time and data was collected from the icu observation charts for the first 48 hours after icu admission .
blood samples for glucose measurement were obtained by means of arterial catheters , and intravenous infusion of insulin in saline was used for control of blood glucose .
all these patients received intravenous fluids in a traditional manner and after vital signs became stabilized , enteral nutritional support was provided as soon as possible .
sixty - five percent ( 13 ) of these were male and the majority of the patients ( 55% ) were aged between 70 and 80 years old .
fifteen percent ( 3 ) of these patients were diabetics and 45% ( 9 ) were surgical patients .
twenty percent of the patients received intravenous infusion of insulin in order to achieve glycemic control .
only 22.2% of the patients with blood glucose level greater than 10 mmol / l , received insulin .
only 15% of the patients with blood sugar level less than 8 mmol / l received enteral nutrition .
one case of hypoglycemia was seen ( blood glucose level , 40 mg / dl [ 2.2 mmol / l ] ) in a non - diabetic patient being treated with an insulin infusion .
figure 1 shows the pattern of glycemic control in our icu for the first 48 hours after icu admission .
a target blood sugar range is difficult to meet and may be leading to more morbidity due to hypoglycemic episodes . for hyperglycemic critically ill patients we recommend a blood glucose target of 8.010
we suggest that use of intravenous fluids that contain glucose should be minimized and administration of insulin be commenced if blood glucose exceeds 10.0 mmol / l ( 180 mg / dl ) , and adjusted when needed to maintain blood glucose of 8.010.0 mmol / l ( 144180 mg / dl ) . our purpose is to design a successful protocol for glycemic control in our institution .
the majority of suggested protocols demand a minimum of hourly glucose monitoring for successful glycemic target achievement while reducing the risk of hypoglycemia.19 for the implementation of insulin protocols for strict glycemic control in our icu , there is a need for an automated continuous vascular blood glucose monitor which is still undergoing evaluation in the united kingdom.20 the strategy of glycemic control should be carefully coordinated with the level of nutritional support and metabolic status of the critically ill patient .
we suggest an uninterrupted feeding regime in order to avoid hypoglycemia , which is an independent risk factor for death after adjustment for age , severity of illness , diabetes , renal failure , and mechanical ventilation.21 thus , glucose control protocols should include specific algorithms for managing hypoglycemic events in critically ill patients .
early glucose control has the potential to reduce icu or hospital length of stay for all patients admitted to the icu . by ensuring compliance , developing protocols and incorporating a multidisciplinary approach
, clinicians will achieve blood glucose control in the critically ill population and improve outcomes . | critically ill patients are predisposed to stress - induced hyperglycemia .
recent evidence suggests that uncontrolled hyperglycemia is associated with poor outcomes within the population of surgical and medical intensive care units .
we retrospectively audited our practice in the management of hyperglycemia in the critically ill , in order to identify reasons and periods of time that deviations in blood glucose control are most likely , and to make recommendations on how to improve this .
our study showed poor compliance with the current recommendations for glycemic control in the critically ill and highlighted the need for a successful protocol for glycemic control in our institution .
that should be carefully coordinated with the level of nutritional support and metabolic status of the acutely ill patient . |
oral cancer is commonly preceded by premalignant lesions and conditions grouped under common terminology as potentially malignant disorders .
the precursor lesions that precede oral cancer are defined as an altered epithelium that shows a variety of cytological and architectural changes that have been traditionally brought under the common denominator dysplasia with an increased likelihood for progression to malignancy , squamous cell carcinoma .
the presence of oral epithelial dysplasia ( oed ) is generally accepted as one of the most important predictors of malignant development in premalignant disorders .
silverman et al . in their study have reported malignant transformation in 36% of cases with oed .
lichen planus a unique chronic inflammatory mucocutaneous disorder was considered as a precancerous condition by who in 1978 .
the etiopathogenesis in oral lichen planus ( olp ) appears to be complex , with interactions between and among genetic , immune , environmental , and lifestyle factors .
olp affects 1 - 2% of the general adult population affecting women more than men ( 1.4:1 ) and occurs predominantly in adults over 40 years of age , although occurrence of this disease in younger adults and children is not unusual .
according to location , 72.9% of lesions occurred on posterior buccal mucosa with a 1.03% concurrent occurrence of buccal mucosa and tongue .
the common denominator of the various neoplastic processes is the anarchic and autonomous increase in the number of their constituent cells .
consequently , mutations and other molecular alterations that impede physiologic apoptosis can play a decisive part in the pathogenesis and progression of neoplasms .
this is exemplified by the high frequency of mutations affecting the tumor proliferation gene ki-67 , tumor suppressor gene p53 and its regulators or the oncogene bcl-2 and bax ; a bcl-2 related protein that counteracts bcl-2 and is required for p53 mediated apoptosis in diverse types of cancer .
the malignant potential of these lesions are still debatable and is a matter of serious controversy .
recent techniques , such as immunohistochemistry ( ihc ) and molecular biology have been contributing to evaluate the expression of proteins related to the regulation of the cellular cycle . to detect early cancer development and prevention of disease progression to malignancy ,
loss of bcl-2 in basal cells of potentially malignant and malignant oral epithelial lesions and loss of bax in poorly differentiated oral squamous cell carcinoma ( oscc ) is not associated with mutations in the coding regions of these genes .
the aberrant expression of bcl-2 and bax suggests a role , in cooperation with other molecular changes in the progression of oral dysplasia and oscc .
as both apoptosis and abnormal proliferation play an important role in tumor formation , this study was conducted to suggest the malignant transformation rate in olp and oed cases using immunohistochemical expression of the tumor markers ki-67 , p53 , bcl-2 , and bax .
a cross - sectional descriptive analytical study was done to observe the expression of ki-67 , p53 , bcl-2 , and bax antigens in basal keratinocytes and to evaluate the malignant transformation by using ihc procedure in normal oral mucosa , olp and oed .
the study was conducted among 70 samples ( group 1- controls , group 2- olp and group 3- oed ) in the department of oral pathology and microbiology , panineeya mahavidyalaya institute of dental sciences , hyderabad .
study group comprised a total of 60 formalin fixed archival blocks after histopathological confirmation , which were divided into two groups ( 30 each of olp and oed ) and a control group of 10 normal healthy controls .
though , it was challenging to obtain the normal individuals , we could motivate them and procure the tissues only after they gave us the consent voluntarily .
cases of clinically and histopathologically diagnosed olp and oed , without any previous history of treatment for any type of oral diseases and subjects who were not on any sort of medication were included in the study .
pregnant women with olp and oed , subjects with olp along with oed , immune compromised patients and patients with multiple oral and / head and neck lesions were excluded .
five sections ( 4 m thick ) were obtained from each of the formalin fixed , paraffin embedded tissue blocks .
the first section was stained with hematoxylin and eosin for histological confirmation , while the remaining four sections were taken on 3- aminopropyltriethoxysilane ( apes ) coated glass slides and were stained with monoclonal antibodies p53 , ki-67 , bcl-2 , and bax and analyzed .
the ihc method used was a new direct technique ( enhanced polymer one - step staining method ) by dako ( india ) .
tissue sections were made from paraffin blocks stored in neutral buffered formalin using a microtome .
these sections of 4 m mounted on apes coated slides were then deparaffinized in xylene followed by rehydration through graded alcohol .
microwave oven was used for antigen retrieval , which was performed in citrate based buffer , with ph 6.0 and then allowed to cool until room temperature .
endogenous peroxide was neutralized using peroxide block for 5 min to block the nonspecific antigen sites .
slides were washed in phosphate buffered saline ( pbs ) for 5 min in two changes .
later the slides were incubated with protein block for 5 min followed by washing in pbs for 5 min in two changes .
the slides were then incubated with liquid mouse monoclonal primary antibody ( p53 [ clone do7 ] , ki-67 [ mib-1 ] , bcl-2 [ clone 124 ] and bax [ 1:70,000 dilution ] ) ( sigma aldrich companies , usa ) for 1 h. slides were incubated with post primary block for 30 min , washed in three changes of pbs for 5 min each and excess pbs was removed .
the peroxidase activity was developed with a drop of freshly prepared 3 diaminobenzidine tetrahydrochloride working solution for 5 min to aid in visualization of the sections .
slides were rinsed in distilled water and then sections were counterstained with harris hematoxylin for 3 min followed by rinsing in tap water for 5 min .
histopathologically confirmed case of oscc was taken as positive control for the expression of p53 , ki-67 , bcl-2 , and bax markers . omitting
the use of primary antibody and carrying out the successive steps of ihc as usual , gave negatively stained slides for p53 , ki-67 , bcl-2 , and bax markers .
the immunoexpression of ki-67 , p53 , bcl-2 , and bax was assessed using light microscope ( olympus ch 20i - japan ) under 10 and 20 magnifications by one blinded pathologist .
criteria given by sousa et al . and dekker et al . were followed for identification of keratinocytes in the basal and parabasal layers .
cells were considered immunopositive if they presented a brown nuclear coloring , regardless of intensity for p53 and ki-67 ( p53 and ki-67 are nuclear markers ) .
cells were considered positive if they presented cytoplasmic staining for bcl-2 and bax ( bcl-2 and bax are cytoplasmic markers ) .
the basal keratinocytes positive for the antigen expression in the basal and parabasal layers of the epithelium were considered as the positive cells and those negative for the expression were considered as negative cells . in labeling index
positive staining areas for particular ihc marker or percent tumor cells staining positive as measured by ihc staining were counted by analyzing five high power fields ( 20 ) for each slide [ figures 1 and 2 - olp ; figures 3 and 4 - oed ] .
ki-67 , p53 , bcl-2 , and bax expression were classified according to the number of positively stained cells per 1000 counted cells .
the percentage of positive cells was scored according to the method of nakagawa et al .
immonohistochemical view ( 20 ) ( a ) p53 and ( b ) ki-67 . showing nuclear brown staining of tumor markers in basal and parabasal cells of oral lichen planus .
immonohistochemical view ( 20 ) ( a ) bcl-2 and ( b ) bax . showing cytoplasmic high intensity brown staining of tumor markers in basal and parabasal cells of oral lichen planus .
showing nuclear brown staining of tumor markers in basal and parabasal cells of oral epithelial dysplasia .
showing cytoplasmic high intensity brown staining of tumor markers in basal and parabasal cells of oral epithelial dysplasia . scoring system for staining intensity ( nakagawa et al . ) statistical analysis was performed using spss version 18.0 ( spss inc . ,
pair - wise comparison between the markers and the groups was done using mann - whitney u - test .
pair - wise comparison between the markers and the groups was done using mann - whitney u - test .
this study was undertaken to evaluate and compare the tumor suppressive , proliferative and apoptotic activity of cells in normal subjects , old and subjects with oed using immunohistochemical markers ; ki-67 , p53 , bcl-2 , and bax .
table 2 shows the comparison of p values between the three groups for p53 , ki-67 , bcl-2 , and bax .
group 1 was statistically significant with groups 2 and 3 for p53 , ki-67 and bcl-2 .
two - by - two comparison between the three studied groups for p53 , ki-67 , bcl-2 and bax table 3 shows the intensity of staining for all markers among three groups . in group 1 , only 20% have shown positivity for p53 , which were mildly stained . in relation to ki-67 ,
majority of the staining was of mild type ( 13.3% ) in both groups 2 and 3 cases .
intensity of staining for bcl-2 marker was predominantly moderate in group 2 ( 36.7% ) and 3 ( 66.7% ) cases .
severe staining was found to be more in group 3 ( 20% ) than group 2 ( 10% ) cases .
distribution of staining intensity for p53 , ki-67 , bcl-2 and bax in three groups according to the criteria of nakagawa et al .
table 4 shows the percentage positivity of all markers among the three groups . in group 1 , only 2 ( 20% )
have shown positivity for p53 and ki-67 protein , no subject for bcl-2 and 5 ( 50% ) for bax proteins . in group 3 , 23 ( 76.6% ) , 26 ( 86.58% ) , 7 ( 23.31% ) , and 22 ( 73.26% ) have shown positivity for respective markers .
the present study focused on evaluation of malignant potential with ihc in oed and olp by comparing the frequency of positively stained proteins ( p53 , ki-67 , bcl-2 , and bax ) depending on their intensity of staining .
the age range of patients diagnosed in group 2 was 30 - 52 years with mean age of 40.1 years with 46.6% males and 53.3% females , which was similar to sugerman et al . who have reported that females were more affected than men with olp ( 1.4:1 ) . in group 3
, the age ranged between 31 and 54 years with mean age of 41.1 years and males ( 53.3% ) were more affected when compared to females ( 46.6% ) .
our results were similar to brothwell et al . who have shown 38 males ( 59.4% ) and 26 females ( 40.6% ) with ages ranging from 20 to 86 years .
the normal p53 protein has a very short half - life ; therefore , can be hard to detect in normal tissues .
however , the protein can remain in the tissues longer due to mutations , or a defect in the degradation pathway or by binding to other proteins .
the physiological function of p53 protein is that of preventing accumulation of genetic damage in cells either by allowing for repair of the damage before cell division or by causing death of the cell .
the mutant p53 protein is normally not active , thus leading to the loss of the tumor suppressor function of the protein .
the alterations of p53 impair the ability of the cells to repair and undergo apoptosis in response to dna damage , which will lead to uncontrolled cell growth . in group 1 of this study ,
the p53 positivity was found to be 20% , which was quite lower than fakhrjou and toutounchi study ( 40% ) .
this variation may be due to small sample size and also because of excess cell damage in few benign conditions and it does not necessarily indicate mutations or malignant transformation .
the p53 positivity , in group 2 , was 69.9% in this study which is almost similar to taniguchi et al .
in contrast to our study , substantially higher p53 positivity was present in fakhrjou and toutounchi study ( 86.7% ) and acay et al .
they explained that p53 protein ( clone do-7 ) reacts only with its mutant form , thus revealing the presence of mutation in the protein and hence olp has potential malignant transformation . in sousa
study , the p53 positivity ( 41.7% ) was lesser compared to our study , which could be due to sample size .
in this study , the p53 positivity in group 3 ( 76.7% ) is almost similar to raju et al .
they explained that the tumor suppressor gene p53 plays a central role in controlling the progression of cell cycle from g1- phase to the s phase and alterations in this gene may provide cancer cells with a growth advantage leading to uncontrolled proliferation as well as increased p53 levels .
in contrast to our study , lower p53 positivity was present in sousa et al .
( 64.3% ) studies and this might be due to substantial differences in detection techniques as well as the varied habits practiced in different geographical regions and races .
the expression of p53 positivity in groups 2 and 3 , in the present study , is a relevant finding .
the p53 is a nuclear protein whose mutation is strongly associated to several cancer types .
it has been shown that alterations in the expression of p53 is essential for carcinogenesis and can indicate an important step in transformation of normal to neoplastic epithelium . according to stoll et al .
the loss of p53 function is found in at least half of oral cancer cases .
therefore , the similar expression of p53 in groups 2 and 3 can be an important indicator of malignant transformation potential of these lesions .
ki-67 is a cell cycle associated human nuclear protein present in perichromosomal region , the expression of which is strictly associated with cell proliferation and is widely used in pathology as a proliferation marker to measure the growth fraction of cells in human tumors .
the ki-67 antigen starts to be expressed in the s phase , progressively increasing through s and g2 phases and reaching a plateau at mitosis .
after cell division , the cells return to g1 with a stock of ki-67 antigen , whose level decreases rapidly during this phase .
ki-67 positivity was 20% in group 1 which is similar to fakhrjou and toutounchi study ( 20% ) who explained that ki-67 is a nuclear protein that can be detected in all phases of cell cycle except go phase and plays a pivotal role in maintaining cell proliferation . in group 2 ,
the ki-67 positivity was found to be 73.2% which is lower than fakhrjou and toutounchi ( 86.7% ) and acay et al .
the authors explained that this high positivity of ki-67 could be due to an increased cell proliferation , secondary to repeated break down of cycling cells leading to increased state of proliferation .
the ki-67 positivity in group 3 was found to be 86.6% in the present study with almost similar value observed in a study by raju et al .
the percentage positivity for ki-67 in group 2 is lower than that of group 3 and this difference is in the expected direction in that dysplastic lesions have a higher growth fraction . in this study , based on the ihc evaluation ,
both p53 and ki-67 were significantly more prevalent in specimens in groups 2 and 3 compared with group 1 .
these findings indicate that there might be a potential tendency for malignancy in groups 2 and 3 and both ki-67 and p53 might be used as relevant prognostic markers .
the bcl-2 proto - oncogene blocks a distal step in an evolutionary conserved pathway of apoptosis .
its abnormal expression , usually in terms of over expression in genetically modified cells such as tumor cells , contributes to the expansion of the damaged cell clone by preventing cell turnover due to programmed cell death , leading to cellular immortalization . by promoting cell survival
. moreover , increased bcl-2 expression in cancer cells possibly reflects tumor cell resistance to apoptosis and may have implications for their responsiveness to treatments .
the bcl-2 positivity in group 1 was ( 0% ) almost similar to leyva - huerta et al .
bcl-2 positivity was found to be 19.9% , which was similar to sousa et al .
study , the bcl-2 positivity was 10% , which was slightly lower than our study and suggested that the low rate of apoptosis in olp is a consequence of the antiapoptotic action exerted by bcl-2 .
study found no bcl-2 immunoexpression in olp group and the authors suggested that this protein does not seem to be involved in olp epithelial changes and the loss of bcl-2 antiapoptotic control is associated with a concomitant loss of other prosurvivor molecules or an increase in the proapoptotic molecules . in group 3 ,
bcl-2 positivity was found to be 23.3% in our study , which was slightly lower than sousa et al .
this might be due to subtle inherent genetic differences among different populations , gender and age of the population and anatomical location of the studied lesions .
in the various studies , it is observed that bcl-2 is present from initial stages of carcinogenesis up to appearance of metastasis in oral cancer .
therefore , the high immune expression of bcl-2 in groups 2 and 3 compared with group 1 may indicate premalignant potential of these two groups .
alterations in bcl-2 expression play a role in cellular differentiation and development of tumors and this oncoprotein has a role in early stages of tumor progression as it is up regulated in sequentially progressing oed .
it plays a tumor suppressor role in human malignancies and high bax expression is associated with favorable prognosis in several cancer types . according to zhan et al .
, bax expression is selectively induced in apoptosis proficient cells ; therefore , oscc cells with elevated bax expression could be those that are primed for apoptosis due to the inherent genetic instability associated with malignant transformation . in this study ,
the positivity rate for bax in group 1 ( 50% ) , group 2 ( 56.61% ) and group 3 ( 73.26% ) had no statistical significant difference and was almost similar to sousa et al .
though bax is a pro apoptotic protein , it is not used much as a marker to assess malignant transformation in groups 2 and 3 .
although bax and bcl-2 are strongly associated in apoptosis , no correlation between these proteins was observed in this study , which can be explained by the existence of different mechanisms of apoptosis regulation .
the expression of bax was lower in group 2 compared to group 3 possibly preventing the death of genetically damaged cells and consequently increasing the malignant transformation risk .
a potential advantage of the use of immunohistochemical markers is their application to routinely processed surgical specimens .
however , technical pitfalls of ihc technique should also be considered as a limitation , because differences in antigen retrieval procedure and diverse antibody clones are sources for various interpretations of protein expression .
therefore , there is a need for identification of any alterations that can indicate a possible malignant transformation .
groups 2 and 3 established a potential tendency for malignancy compared to group 1 and ki-67 , p53 and bcl-2 can be considered as reliable prognostic markers for malignancy .
though bax is a proapoptotic protein , it appears that it is not of much use as a marker to assess malignant transformation in these groups .
alterations in expression of these proteins play a role in cellular differentiation and development and progression of tumor as these proteins participate actively in oral carcinogenesis . | background : the issue of a possible malignant transformation in the lesions like epithelial dysplasia and oral lichen planus ( olp ) is a matter of serious controversy .
the purpose of this study was to suggest the malignant transformation rate in olp and oral epithelial dysplasia ( oed ) by using immunohistochemical expression of the tumor markers ki-67 , p53 , bcl-2 , and bax.materials and methods : this was a cross - sectional study carried out among 70 samples , including 10 samples from normal healthy mucosa categorized into group 1 , group 2 ( 30 olp ) , and group 3 ( 30 oed ) samples .
five sections ( 4 m thick ) were obtained and stained with monoclonal antibodies such as ki-67 , p53 , bcl-2 , and bax and analyzed for number of positive cells and also for intensity of staining .
statistical analysis was done using mann - whitney u - test ( p < 0.05).results : significant results were found only for expressions of ki-67 , p53 , bcl-2 markers in both study groups ( p < 0.05 ) . in these groups , the intensity of staining was mostly mild to moderate for all studied tumor markers . in this study ,
subjects with an average positive ihc expression of ki-67 , p53 , bcl-2 , and bax markers in normal mucosa was about 22.5% , which was significantly lower when compared with olp ( 54.9% ) and oed ( 64.9%).conclusion : the high propensity for malignant transformation in oed followed by olp suggests that a wide range of inherent and extrinsic factors contribute to the disease progression and malignant transformation . |
henoch - schnlein purpura ( hsp ) is a common systemic vasculitic disorder that occurs during hildhood and its course is often self - limiting ( 1 , 2 ) .
multiple studies have reported less frequent but poorer outcomes in adult hspn ( 1 , 2 ) and there have been a few data on elderly - onset patients ( 3 ) .
iga nephropathy is usually an indolent progressive adult disease , whereas hspn is mainly seen during childhood .
hspn more often shows nephritic - nephrotic syndrome and chronic renal failure compared to iga nephropathy ( 4 ) .
although the pathophysiologic mechanisms of hspn are not completely understood , thecomplement activation , vascular damage and crescent formation in glomerular injury in severe hspn appear to play an important role .
however , the serum complement levels are usually normal ( 5 ) and anti - neutrophil cytoplasmic antibodies ( anca ) is negative in most patients with hspn ( 2 , 3 , 6 ) .
we experienced a case of elderly - onset crescentic hspn with nephritic - nephrotic syndrome associated with hypocomplementemia and positive myeloperoxidase anca ( mpo - anca ) after a major operation and antibiotic use .
a 76-yr - old man was transferred to our hospital at 7th july 2011 with 1-month history of oliguria , gross hematuria , abdominal pain , pitting edema and skin eruption ( palpable purpura ) in both legs .
three months ago , he was admitted to another hospital with jaundice , but then diagnosed with early common bile duct cancer , and he underwent whipple 's operation .
antibiotics were used because of leakage in the suture from the surgery but there were no further surgical problems .
the antibiotics administered included ceftriaxone , amikacin , meropenem and piperacillin / tazobactam . however , serum creatinine increased from 1.0 to 6.3 mg / dl .
a physical examination revealed multiple nontender purpura mixed with erythema ranging from 2 to 4 mm in diameter over his edematous lower legs and the dorsal side of his feet and toes ( fig .
1 ) . most of the skin lesions were non - indurated , although palpable or indurated purpura mixed with erythema could be identified in some areas . on admission
, laboratory investigations showed a reduced serum hemoglobin level of 10.5 g / dl and an elevated serum c - reactive protein level of 5.93 mg / dl .
in addition to an elevated serum creatinine level of 6.4 mg / dl ( fig .
2 ) , an increased blood urea nitrogen level of 45.7 mg / dl , urine rbc many / hpf and 24 hr urine protein showed 8,141 mg / day .
c3 was low at 64.89 mg / dl , and anca was positive at mpo - anca 1:40 ( by enzyme - linked immunosorbent assay [ elisa ] and indirect inmunofluorescence [ if ] ) and serum fluorescent antinuclear antibody test , cryoglobulin and other serology findings were within the normal range .
ultrasonography showed diffuse parenchymal swelling , homogenous increase cortical echogenecity , ascites and bilateral pleural effusion .
renal biopsy showed crescentic glomerulonephritis on light microscopy ( lm ) , and mesangial and extracapillary ig a deposition on if .
circumferential fibrocellular and cellular crescents were noted in 5 out of a total of 8 glomeruli .
most glomeruli were diffuse endocapillary proliferation in size and cellularity due to endocapillary proliferation with mild lymphocytic and neutrophilic infiltration . if microscopy revealed conspicuous staining for ig a in the mesangium and segmentally subendothelial portion along the glomerular capillary walls ( fig .
the patient complained of abdomen discomfort and experienced pyrexia up to 38.5. we started prednisolon 60 mg without using antibiotics , and after that the fever improved . starting steroid therapy and hd , abdomen discomfort , purpura on legs , pretibial pitting edema and pleural effusion were improved .
however , his renal function was not improved and he has been under maintenance hd .
hsp is a typical disease of childhood , and shows a near 20-fold disparity in incidence between adults and children ( 1.3 vs 22.1 per 100,000 population ) .
adult - onset hsp may involve more severe hspn and organ injury , but there is limited data on elderly - onset hsp ( 3 ) .
the classic clinical symptoms include purpuric rash , abdominal pain , arthralgias , and hematuria , but the spectrum of hsp may vary from only minimal petechial rash to severe gastrointestinal , neurologic , pulmonary , and renal disease .
hspn is often triggered by antecedent infection , medication and other miscellaneous factors ( 8) . hspn and iga nephropathy are currently considered related diseases , as both are indistinguishable in the renal histologic and immunofluorescent findings , and both have been described in twins .
hspn more often shows extrarenal manifestation , nephritic - nephrotic syndrome and chronic renal failure compared to iga nephropathy ( 4 ) .
pathogenesis of hspn is not completely understood , but it has pathologically been regarded as a specific immune - mediated entity induced by environmental factors , particularly infections ( 9 ) .
the role of infection , particularly group a beta - hemolytic streptococcal , in the etiology of hsp has interested many investigators , but the role of bacterial antigens remains questionable in a majority of cases .
other causes are hypersensitivity reactions induced by numerous drugs , but no common drug has yet been identified . in a previous case report , prodromal illness and drug to hsp in three members of the same family were penicillin with sore throat and colon resection after diverticulitis with sulfonamide due to wound infection ( 10 ) .
pathophysiologic mechanisms of hspn including complement activation , vascular damage and crescent formation in glomerular injury appear to play an important role .
hypocomplementemia generally has not been seen in hspn , but excessive consumption and deposition of c3 in the kidneys may have been the cause of reduced c3 serum level in a severe hspn case ( 5 ) .
anca with specificity for myeloperoxidase ( mpo ) was noted in primary small vessel vasculitis ( 11 ) .
although the prognosis of patients with overlapping hspn with anca remains unclear , clinical correlations suggest that immune complex deposits in the kidney may potentiate the effect of anca in producing more severe glomerulonephritis than classic pauci - immune vasculitis or hspn . as hspn overlapping syndrome characterized by the synergistic effect of mpo - anca and
the iga immune complex may result in a fatal outcome , aggressive immunosuppressive therapy should be initiated as early as possible ( 12 ) .
we examined mpo - anca twice using an enzyme immunoassay ( elisa ) in our hospital and indirect if assay in another hospital .
it was found that mpo - anca was positive and a titer of 1:40 was relatively weak . however , large multicenter studies combining indirect if and elisa have demonstrated a specificity approaching 99% for wegener 's granulomatosis , microscopic polyangitis , or their renal - limited variant ( 13 ) .
the anca have an important role in pathogenicity of small vessel vasculitis , and intermittent anca positivity is an independent risk factor for relapse ( 11 - 13 ) .
therefore , mpo - anca may have some role in the pathogenesis of our crescentic hspn case . in a retrospective study , 2 cases with anca out of a total of 42 hsp
because the presence of crescents is a histologic feature of hspn that represents a prognostic factor and constitutes the basis for the international study group of kidney disease in childhood ( iskdc ) pathology classification , the study of crescent pathophysiology may provide useful information as a therapeutic strategy ( 14 ) . a cohort study ( 3 ) of 250 adults suffering from hsp was retrospectively analyzed for a median follow - up period of 14.8 yr . the risk of progression to renal insufficiency , which ranges from 5% to 15% in children , seems to be much higher in adults , at approximately 30% .
they reported to define long - term prognosis and risk factors for severe renal failure in adults with hspn .
renal function impairment and proteinuria level at presentation and , the degree of interstitial fibrosis , percentage of sclerotic glomeruli , and presence of glomeruli with fibrinoid necrosis on renal biopsy were associated with a poor renal prognosis .
our elderly patient underwent a kidney biopsy - proven , sudden developed hspn associated with nephritic - nephrotic syndrome with hypocomplementemia and p - anca after a major operation for the treatment of cancer .
after being initially treated with prednisolone , his purpura and edema on the legs improved , but renal function did not improve .
this case implies that the prognosis of elderly - onset crescentic hsp nephritis with positive mpo - anca and hypocomplentemia may be a poor outcome .
therefore , closed inspection would be helpful to monitor the progression of elderly - onset hsp , and the clinician should not delay renal biopsy to confirm the diagnosis at the time of clinical suspicion of renal involvement .
earlier diagnosis and prompt treatment of elderly - onset hspn will prevent the progress of end - stage renal failure . only few cases with elderly onset hspn
we describe elderly onset hspn with mpo - anca can be crescentic glomerulonephritis rapidly progressed to end stage renal failure . | henoch - schnlein purpura ( hsp ) is common in childhood and often self - limiting .
there have been limited studies on elderly - onset hsp nephritis ( hspn ) .
a 76-yr - old man was transferred to our hospital with a 1-month history of oliguria , abdominal pain , edema and palpable purpura in the legs .
three months ago , he was admitted to another hospital with jaundice , and consequently diagnosed with early common bile duct cancer .
the patient underwent a whipple 's operation .
antibiotics were administrated because of leakage in the suture from the surgery . however , he showed progressive renal failure with edema and purpura in the legs .
laboratory investigations showed serum creatinine 6.4 mg / dl , 24-hr urine protein 8,141 mg / day , myeloperoxidase anti - neutrophil cytoplasmic antibodies ( mpo - anca ) 1:40 and c3 below 64.89 mg / dl .
renal biopsy showed crescentic glomerulonephritis , as well as mesangial and extracapillary ig a deposition .
we started steroid therapy and hemodialysis , but he progressed to end - stage renal failure and he has been under maintenance hemodialysis .
we describe elderly onset hspn with mpo - anca can be crescentic glomerulonephritis rapidly progressed to end stage renal failure . |
breast cancer is the most common cancer among women and its incidence is increasing . however , its mortality decreases with better screening programs , public education , and medications .
disease - free survival and overall survival depend on multiple factors , including the stage of the disease , hormone receptor status , age , and prescribed chemotherapy agents [ 2 , 3 ] .
, younger premenopausal women develop recurrence sooner and recurrence after decades is not so common . here , we report 6 cases that remained disease - free for 13 - 32 years after the primary breast cancer .
the goal of this report is to show that breast cancer survivors are at risk for relapse even after years .
in addition , even high risk patients can have a long period of disease- free survival .
in this report , we reviewed 6 cases with a mean age of 40.1 ( 30 - 57 ) years at diagnosis ( table 1 ) .
in addition , 1 patient had ductal carcinoma in situ ( dcis ) and had received radiation . besides , 2 patients had breast cancer with negative axillary lymph nodes and 1 case had 6 positive nodes . except for 1 patient who had dcis ,
others had received cyclophosphamide , methotrexate , and 5-fluorouracil ( cmf ) ( 3 - 6 cycles ) .
the patient was a 62-year - old lady who was operated for a right breast carcinoma 32 years ago ( in 1979 ) . at that time , right side modified radical mastectomy ( mrm ) was done for her and then she received 6 cycles of combination chemotherapy ( cyclophosphamide , methotrexate , and 5- fluorouracil ) .
she had no regular follow up , but was well until february 2011 when she developed lymphadenopathy in the right supraclavicular fossa .
moreover , physical examination and mammography from the contralateral breast revealed no lesion and abdominal ultrasonography revealed liver metastases .
the patient was a 71-year - old lady who had breast cancer and underwent left side mrm and received 3 courses of combination chemotherapy by cyclophosphamide , methotrexate , and 5-flourouracil in 1986 .
she also received tamoxifen for 5 years and was well until january 2012 when she developed severe pelvic pain .
whole body bone scan was performed for her and showed multiple bone metastases in the first and fifth lumbar spines , left humerus , and greater trochanter of the right femur .
she received 20 gray in 5 fractions for the involved areas of lumbar spines and femur .
now , she is receiving zoledronic acid and has no evidence of distance metastasis except in bones .
the patient was a 70-year - old woman who was affected by left side breast cancer in october 1998 and left side mrm was done for her .
she received 6 cycles of chemotherapy by cmf and then radiotherapy to the chest wall and the regional lymph nodes .
her whole body bone scan showed multiple bone metastases at pelvic bones , multiple vertebrae , and skull .
the patient was a 56-year - old woman who was operated ( quadrantectomy and axillary lymph node dissection ) due to the right side infiltrating ductal carcinoma in january 1991 .
she had a 2 cm infiltrating ductal carcinoma ( idc ) and no lymph node involvement .
then , she received chemotherapy and radiotherapy and was under regular follow up . in november 2011 , she developed some nodules on the skin of the right breast and biopsy showed the recurrence of the primary tumor .
the patient was a 47-year - old woman who had right side dcis in july 1997 .
the patient was operated ( quadrantectomy without axillary nodal dissection ) and afterward she only received radiotherapy to the breast .
she did not have a regular follow up in spite of the history of breast cancer in her mother .
twelve years later in july 2009 , she was operated ( mrm ) due to local recurrence .
she had a 1.5 cm idc and 6 positive axillary lymph nodes from the total of 9 nodes .
then , 8 cycles of chemotherapy ( ac and taxane ) were performed for her and she received radiotherapy to the chest wall by superficial x- ray and the regional lymph nodes by co60 .
after that , letrozole was started for her estrogen receptor ( er ) , and progestrone receptor ( pr ) positive , and human epidermal growth factor receptor 2 ( her2 ) negative ( er and pr positive , her2 negative ) .
one and a half years later in march 2010 , she developed bone and lung metastasis and she is receiving chemotherapy now .
the last patient was a 61-year - old woman who underwent left side mrm due to a 4 cm breast cancer in march 1993 .
she was treated with 6 cycles of cmf and then irradiation to the chest wall and the regional nodes . in november 2007
, she developed abdominal pain and a mass was found in the epigastric area . in ultrasonography ,
multiple factors , such as race and ethnicity , are effective in breast cancer survival . for instance , one study showed better survival in non- hispanic whites compared to blacks and other groups .
this difference is present in cases with the same stage , er - pr status , and treatment [ 2 , 7 ] .
tumor size and lymph node status are also directly related to the prognosis of breast cancer . in a study by rosen et al . , the mean recurrence time was 4.9 years in the cases with 1 involved lymph node and 3.9 years in those with 2 or more involved lymph nodes .
their study also revealed the importance of the menstruation status . in that study , the perimenopausal cases had higher mortality in comparison to pre and post - menopausal women .
all the cases of the current study , except for 1 , were in the pre- menopausal status .
some authors suggested body weight as a negative prognostic factor which may influence the prognosis by interfering in the hormonal mechanism .
some studies have shown that younger age at presentation is accompanied by a higher risk of recurrence ; however , no difference has been reported in some other studies [ 6 , 10 ] . on the other hand ,
chen et al . performed a retrospective study and found that younger age was accompanied by better local control . in this study , the patients were not so old and the importance of age in late relapse should be studied in prospective studies .
the patients with negative tumoral tissue for er , pr , and her2 ( or triple negative ) comprise 15 - 20% of the breast cancer cases and are among the worst outcome groups .
this feature is associated with other high risk factors , such as young age and high grade .
the time of recurrence has been studied widely and most of the recurrences have been shown to occur in the first decade . the recurrence time and pattern
has been reported to have a double peak shaped curve ; the first peak occurring in the second year and the other one in the 6th to the 9th year post - treatment . in node
in other words , the recurrence rates were more constant and relatively similar in both premenopausal and postmenopausal women .
nevertheless , after 8 years of treatment , the recurrence rate in hormone receptor negative cases is less than the hormone positive ones . it should be mentioned that chemotherapy lowers the recurrence rate and the second peak appears later .
late failure ( more than 5 years ) is more prevalent in early stage breast cancer . in a study on patients by quadrantectomy ,
the rate of late recurrence was 53% for t1 lesions and 25% for t2 lesions .
the first cases with prolonged dfs were reported in 1887 and recurrence occurred 25 and 19 years after mastectomy . surely , they had not used chemotherapy and radiotherapy .
therefore , genetic or molecular studies may be needed to enlighten the dark horizon to us .
dcis by definition is a non - invasive disease ; however , invasive components may be missed in tissue sampling or even in post - operative pathologic samples .
these cases may have lymph node ( ln ) metastasis at presentation or may behave like an invasive disease [ 16 , 17 ] . in general , her2 receptor status and p53 mutation are widely checked in breast cancer cells , but their role in late relapse is not so clear . in the recent years , some biomarkers are used to realize which patient is at risk for recurrence or metastasis . in this regard , cytokeratin-19 may be the best factor which can be used to find the tumor cells in bone marrow or blood .
however , it is not accurate enough yet to be used as a test for decision making or as a prognostic index in breast cancer patients . the current guidelines ( nccn 2012 ) recommend annual mammogram for long time .
the question is whether this time interval is enough , more , or less than what is required .
the chemotherapy agent , surgery ( especially lymph node dissection ) , hormone therapy , and trastuzumab have also their own long- term side effects .
thus , more studies are needed in order to determine the effect of these factors on prolonged survival .
another ethical point is whether a young woman with breast cancer should be told that she will have a short survival or not .
our first case that was 30 years old on arrival delivered and grew up 5 children after diagnosis of breast carcinoma .
late relapse is not so common in breast cancer but can occur in any stage .
therefore , we suggest life- time follow up for every patient with breast cancer including dcis cases .
late relapse is not so common in breast cancer but can occur in any stage .
therefore , we suggest life- time follow up for every patient with breast cancer including dcis cases . | backgroundbreast cancer is the most common cancer and the leading cause of cancer death among the women worldwide .
the risk of local and distant recurrence is the highest during the first two years following the initial treatment
. very late relapse ( after 12 years ) is uncommon in breast cancer survivors.methodsherein , we report the characteristics and outcomes of 6 such cases of breast cancer.resultsthe mean age of the patients was 40.1 years ( range 30 - 57 ) and the mean disease free survival was 19.6 years.conclusionlate relapse is not so common in breast cancer but can occur in any stage .
therefore , we suggest life - time follow up for every patient with breast cancer . |
punjab ( india ) has 3 main wetlands , i.e. harike , kanjli and ropar wetlands which are included in ramsar list of international importance .
harike wetland ( 31 17 n latitude and 75 12 e longitude ) is the largest freshwater wetland ( in northern india ) of 4100 ha area with beas and sutlej as primary inflows .
it is a habitat for as many as 26 species of fishes which include catla , cirrhinus , channa , mystus , chitala chitala , cyprinus and ambassis ranga .
these wetlands are the major natural fisheries resource for food in whole of the punjab state .
the study indicates that large variety of fishes in these wetlands are infested with myxozoan parasites .
myxozoans include histozoic and coelozoic parasites infecting not only freshwater and marine fishes but have also been detected in molluscs , amphibians , reptiles , waterfowl and mammals ( moncada et al . , 2001 , eiras , 2005 ) .
as demonstrated firstly by wolf and markiw ( 1984 ) , it has been proven that myxozoan species require an alternate invertebrate host ( usually an annelid ) to complete the life cycle . among myxosporeans ,
( 2005 ) reported 744 valid species , while lom and dykova ( 2006 ) counted 792 including 7 amphibian species .
several other reports on myxobolus species are available from punjab and west bengal , india ( basu & haldar , 2004- basu et al . , 2009 ,
about 131 species of myxobolus have been recorded in india ( kaur and singh , 2012 ) and are mainly differentiated by morphological , morphometric characteristics of spores , besides host and organ or tissue specificity . presently , the molecular analyses have been an important tool in the study of these parasites and this has expanded their taxonomy to the phylogenetic analyses .
this has led to correct identification and differentiation of morphologically indistinguishable myxobolid species ( kent et al . , 2001 , eszterbauer , 2002 , molnar et al . , 2010 ,
the most revealing aspects of analyses based on ssu rdna sequences is the incongruence of phylogenetic trees with classification based 63on spore morphology alone ( bartosova et al . , 2009 ) .
so far , there are 14 sequences i.e caudatus
kc865607 ; myxobolus cuttacki 465682 ; m. orrissae
kf448527 ; myxobolus basuhaldari
km029974 , km029975 , km029976 ; m. kalavatiae
km029973 ; m. meerutensis
km029977;m .
bhadrensis
km029968 , km029969 , km029970 , km029971 , km029972 and m. catlae
km029967 ( mondal et al . , 2014 , szekely et al . , 2014 ,
, 2014 , abraham et al . , 2015 ) from india are available in the genbank . in this study
, we present morphological and molecular characterization of m. dermiscalis n. sp . infecting the scales of l. rohita collected from harike wetland , punjab , india along with 18s rdna based phylogenetic analysis with myxobolus group and other related texa . in future
, the molecular methods can be implied on the diagnostics of the economically important myxozoan parasites in this part of the world .
fresh specimens of indian major carp , rohu , l. rohita were collected from the fisher - man at harike wetland during the period june 2013 to july 2014 .
the infected scales were removed with the help of forceps in a petridish containing 0.9% saline .
the pseudocysts were visible with the naked eye and appeared as creamish white patches on the scale .
the plasmodium were teased on a clean slide to liberate spores and were examined under the microscope .
fresh spores were treated with 8% koh solution for the extrusion of polar filaments . for permanent preparation ,
the prevalence ( in percentage ) of m. dermiscalis infecting scales of l. rohita was calculated according to bush et al .
the intensity of infection was determined by following the index proposed by kaur and attri ( 2015 ) for infection on the scales . 0 = no infection ; 1 = one pseudocyst per scale in 10% of total scales ( indicating light infection ) ; 2 = two pseudocysts per scale in 1520% of total scales ( moderate infection ) ; 3 = three to four pseudocysts per scale in 50% of total scales ( heavy infection ) ; 4 = four to five pseudocysts per scale in 100% of scales ( severe infection ) .
the pseudocysts ( 50 in numbers ) present on ethanol fixed scales were ruptured with the help of a sharp needle in a watch glass having double distilled water .
the contents in the watch glass containing spores were collected in 1.5 ml micro centrifuge tubes .
polymerase chain reaction ( pcr ) was carried out according to the andree et al .
( 1999 ) at the final volume of 25 l using the primers mx5-mx3 which amplified the fragments of 1597 bp respectively of the 18s rdna gene .
the amplification reactions were conducted with 3075 ng of genomic dna , 12.5 l of 1 reaction buffer ( hi media ) , 1.0 l of each primer , 1.0 l of total dna and 10.5 l of double purified water .
amplification was done by initial denaturation at 95 c for 3 min , followed by 33 cycles of denaturation at 95 c for 30 s , annealing of primers at 58 c for 30 s , extension at 72 c for 1 s. the final extension was at 72 c for 10 s. the pcrproducts were analysed agarose gel electrophoresis , and size was estimated by comparison with the1 kb plus dna ladder ( fig . 1 ) .
extracted from spores using the dneasy blood & tissue kit ( qiagen ) following the manufacturer 's instructions .
the product was then quantified in a nanodrop ( thermocycler ) . in the present study
, the pcr amplified products were sequenced at the molecular diagnostics & research laboratories ( mdrl ) pvt .
1597 bp of 18s rdna sequences of m. dermiscalis n. sp . were deposited in genbank with accession number km092529 .
phylogenetic analysis involving 23 nucleotide sequences was performed using the bayesian , maximum likelihood ( ml ) , maximum parsimony ( mp ) and neighbour joining ( nj ) methods .
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1000 replicates ) .
the evolutionary distances were computed using the kimura 2-parameter method kimura ( 1980 ) and units of the number of base substitutions per site .
the best fit model for analysis for the current data was gtr + g with lowest bis ( bayesian information criterion ) was estimated by using model test tool in mega 6.06 .
round to irregular , white , 4 - 5pseudocysts per scale , histozoic , present within a cavity and measure 0.53.6 mm in diameter .
the spores measure 5.847.84 3.985.98 m , oval to spherical in frontal view having rounded anterior and posterior ends .
parietal folds two , present on the posterio - lateral margins of the shell valves .
polar capsules are two , equal , measure 3.985.98 1.853.85 m and are pyriform with distinct neck at the anterior end .
polar filaments form 56 coils and are arranged obliquely to the polar capsule axis , 7.2 m in length when extruded .
two capsulogenic nuclei are present beneath each of the polar capsule . sporoplasm agranular , homogenous occupying whole of the extracapsular space behind the polar capsules and contain one nucleus and a large iodinophilous vacuole
type specimen : paratype are spores stained with ziehl neelsen and iron - haematoxylin , deposited in the parasitology laboratory , department of zoology & environmental sciences , punjabi university , patiala ( india ) .
intensity of infection with parasite species ( index ) : 4 ( severe infection ) .
etymology : the specific epithet dermiscalis has been given on the basis of location of pseudocysts within the layers of dermal scales .
the edited nucleotide sequence was 1597 bp of m. dermiscalis n. sp . which was deposited in gen - bank under the accession number of km092529 .
blastn search ( www.ncbi.nlm.gov/blast ) with 21 myxobolus exhibited 9093% sequence homology and maximum homology of 98% recorded was recorded with myxobolus sp . ( km401439 unpubl ) infecting scales of a cyprinid , l. rohita in myanmar available in the genbank ( table 1 ) . in the phylogenetic tree based on the final edited alignment with neighbour joining analysis ( fig .
5 ) , m. dermiscalis was placed with the m. sp . with highest bootstrap value and confirmed the clustering pattern of maximum likelihood and maximum parsimony analysis ( 100% in ml , 100% mp ) .
the present species has been compared with m. squamae ( keysseltz , 1908 ) from scales of abramis brama ; m. mrigalae ( chakravarty , 1939 ) from scales of cirrhinus mrigala ; m. yogendrai ( tripathi , 1952 ) , m. rewensis ( srivastava , 1979 ) and m. vanivillasae ( seenappa and manohar , 1980 ) from scales of cirrhinus mrigala ; m. rohitae ( haldar et al . , 1981 ) from scales of l. rohita , labeo calbasu , labeo bata and puntius sarana ; m. indirae ( gupta and khera , 1988 ) from scale , tail , fin , cartilage and head of cirrhinus mrigala m. episquamae ( egusa et al . , 1990 ) from scales of mugil cephalus ; m. squamaphilus ( molnar , 1997 ) from scales of a. brama ; m. dermatis ( haldar et al . , 1983 ) from scales of l. rohita ; and m. saugati ( kaur and singh , 2011 ) from scales and gill lamellae of catla catla and l. rohita .
the present species , m. dermiscalis n. sp . was compared with all the above mentioned species and was found different in spore morphology and morphometrics .
the spores of the present species are characterized in having oval to spherical shape with rounded anterior end without intercapsular process and posterior end with two parietal folds .
polar capsules are two , equal , pyriform with distinct neck . in this respect ,
the present species differed from m. squamae , m. yogendrai , m. rewensis , m. vanivillasae , m. rohitae , m. indirae , m.episquamae , m. squamaphilus , m. dermatis , and m. saugati in lacking the intercapsular process .
furthermore , it differed from m. saugati , m. rohitae , m. dermatis and m. indirae in having two parietal folds placed posterio - laterally on the shell valves .
the present species is also different from m. mrigalae in which several triangular markings are present , in addition to unequal polar capsules . in view of the above differences in morphology and morphometrics , the present species under study
the universal primer sets mx5 and mx3 successfully amplified approximately 1600 bp fragments of the 18srdnagene from the present sample of myxobolus ( fig . 3 ) .
the edited nucleotide sequence was 1597 bp of m. dermiscalis n. sp . which was deposited in genbank under the accession number of km092529 .
infecting scales of a cyprinid , l. rohita in myanmar available in the genbank ( unpubl ) , which showed 98% homogeneity ( table 1 ) . in the phylogenetic tree based on the final edited alignment with neighbour joining analysis ( fig . 5 ) ,
. with highest bootstrap value and confirmed the clustering pattern of maximum likelihood and maximum parsimony analysis ( 100% in ml , 100% mp ) .
the present study indicated close similarity based on 18s rdna suggesting sister species , however , the morphological comparisons could not be made due to lack of published record .
minimum evolutionary divergence between the two species was estimated using bayesian information criterion ( bis scores ) using best model test tool in mega 6.06 ( fig .
4 ) . in order to estimate the pattern of nucleotide substitution involving 23 nucleotide sequences using gtr + g model in ( table 2 ) .
rates of different nucleotide substitution(r ) from one base ( transitional -in bold ) and to another base(transversional - in italics ) were assessed .
maximum transition was c g having 19.99 probability value followed by tc 14.38 , ag 13.21 , ga 12.62 .
as for trasversions at , gt 5.31 ; ta , ca 5.26 , tg , cg 247 5.51 , ac , gc 3.82 .
the nucleotide frequencies were 26.44%(a ) , 26.69%(t / u ) , 248(g ) 19.19%(c ) and 27.68%(g ) .
furthermore , the information generated confirmed the importance of features such as host , tissue tropism , geographical location of the host and correlation with the results of molecular data based on comparison of 18s rdna sequence ( kent et al .
, 2001 , seo et al . , 2012 , zhu et al . , 2012 , shin et al . , 2013 ) .
index of intensity of infection by m. dermiscalis n.sp was recorded as 4 indicated by the presence of four to five pseudocysts per scale in 100% of scale .
the prevalence of m. dermiscalis n. sp . infesting scales of l. rohita was 59.7% .
this study is the first report the molecular and phylogenetic characterization of scale infecting m. dermiscalis n. sp . from indian subcontinent . as the list of indian species of the genus myxobolus
have increased over the years ( basu et al . , 2009 , bandyopadhyay et al .
, 2006/2007 ; singh & kaur , 20122014 ) thus the correct identification in future studies must focus on molecular and phylogenetic characterization together with morphological characteristics , host , organ and tissue specificity .
the present study describe a myxobolid species commonly found infecting scales of an indian major carp , l. rohita in freshwater habitat .
the intensity of infection was severe as indicated by the scale pseudocyst index and highly symptomatic to the fish host .
further investigations are being carried out on the presence of gill infecting myxozoan parasites among major indian carps in polycultured conditions .
this is important to have assessment of the impact on these parasites on the carp fishes . beside morphological features ,
genetic data has been provided in the present manuscript which can form basis for development of diagnostics of this species . | in the present study , a new species myxobolus dermiscalis n. sp . infecting scales of labeo rohita , an indian major carp from harike wetland in punjab , india has been described on the basis of spore morphology and amplification of a part of 18s rdna gene .
the pseudocysts of m. dermiscalis n. sp . are milky white with irregular outline , 0.53.6 mm in diameter embedded within the dermal scale in the form of a cavity .
the spores 5.847.98 3.985.98 m in size , having two equal polar capsules 3.985.98 1.853.85 m in size .
the most differentiating feature from closely related species , myxobolus saugati ( kaur and singh , 2011 ) is the presence of two parietal folds at the posterior lateral margins of the shell valves .
the present species is regarded as host , organ and tissue specific in nature . the partial sequence of ssu gene of m. dermiscalis n. sp . clustered with other myxobolus species infecting cyprinids available in the genbank .
blast search revealed 98% homogeneity with myxobolus sp ( km401439 ) infecting scales of l. rohita in myanmar ( unpubl .
data ) .
the present myxobolid parasite has been recorded to cause serious , highly symptomatic disease of the scales , causing their loosening from the skin of l. rohita .
it rendered the host fish unsightly giving it cloudy appearance with white patches and mucoid body surface .
scale pseudocyst index ( spi ) has been provided to record the intensity of infection . |
in drug discovery projects , the three - dimensional ( 3d ) structure of the receptor is not always available . in such cases ,
pharmacophore model building , 3d quantitative structureactivity relationship ( qsar ) , comparative molecular field analysis ( comfa),(1 ) and ligand - based virtual screening all depend on a good algorithm to flexibly align small molecules . in light of the importance of the subject
on top of the methods mentioned in the comprehensive review of lemmen and lengauer,(2 ) new efforts have continued to appear .
similar to docking , the small molecule alignment problem can be divided into two parts .
second , a scoring function needs to be able to distinguish good alignment poses from other possibilities .
the current work is about obtaining a good scoring function for this purpose . in principle , a scoring function that can distinguish the correct alignment pose from incorrect ones is not necessarily the same as a scoring function that upon minimization gives poses that resemble the correct one as much as possible . in practice , a good scoring function probably performs well for both purposes . in this work ,
we are primarily interested in a scoring function for distinguishing good poses . in any case , we believe that the exact answer to the molecular alignment problem may not be simple .
for example , using only carbons in the binding pocket for superposition may give a slightly different answer from that using all the pocket atoms or using the pharmacophore elements involving the ligand and the pocket .
moreover , the ligand may have some freedom of movement inside a pocket . hence trying to nudge down the geometric difference between a proposed alignment hypothesis and the correct alignment all the way to zero
may not be necessary , or even meaningful , as long as the correct alignment mode is obtained . for scoring functions used in docking , one can use molecular mechanics force fields derived from first principles .
such functions need to be derived from some statistical analysis of a training set of known molecular alignments .
the protein data bank(17 ) ( pdb ) is an obvious source to obtain such a training set .
just in the five years since 2003 , the number of entries has more than doubled ( see http://www.pdb.org ) . moreover , many old entries have been revisited and cleaned up , and many low - resolution structures have been superseded by better ones . by
now , there are hundreds of systems of entries with identical proteins but different ligands .
we believe that now is a good time to make use of the growth in the pdb to revisit the derivation of a scoring function for molecular alignment .
for the problem of small molecule alignment , there are two main types of scoring functions .
the first type is atom based : when two molecules are being aligned , the score consists of a sum of terms that are based on intermolecular atom pairs ( i.e. , each pair has one atom coming from each molecule ) .
the second type of score is field based : the electrostatic or steric fields of the molecules or their surfaces are compared to arrive at a score
. this would be somewhat slower to compute since the fields or the molecular surfaces need to be calculated from the atomic coordinates .
hence it would be difficult to flexibly refine an ensemble of aligned molecules or to deal with a huge number of conformers .
indeed several of the field - based alignment methods rely on an independent conformational search engine to generate good conformers ( e.g. , shapelets,(6 ) brutus,(13 ) the molecular field extrema method,(11 ) and mimic(18 ) ) .
since our scoring function will be used to distinguish good binding poses among a huge number of possibilities , it needs to be calculated quickly .
an atom - based function would hence be more appealing than a molecular field - based function . when creating a hypothesis for an alignment of molecules , it is desirable for atoms ( from different molecules that are being aligned ) with similar properties to be near to each other . intermolecular atom pairs that are far away should have little influence on the goodness of an alignment .
this can be achieved by associating each intermolecular atom pair with similar properties with a gaussian scoring term : w exp(r ) , where r is the distance between the two atoms , is a parameter controlling the range of the interaction , and w is the weight of this term .
it is also desirable for the alignment hypothesis for atoms with incompatible properties ( e.g. , hydrophobic vs hydrophilic ) to not superpose .
this can be achieved using the same functional form but with a negative value for w. besides atoms , hydrogen - bond projected features can also be considered .
these features represent the expected positions of the hydrogen - bonding partner on the binding pocket based on the ligand geometry
. the overlap of hydrogen - bond projected features of the same type should be favorable for an alignment hypothesis .
again , the same gaussian score can work , with r being the distance between the intermolecular features .
while the gaussian function is widely used in molecular alignment methods ( e.g. , fap,(4 ) pharao,(5 ) moe,(16 ) sq,(19 ) and seal(20 ) ) , there are other functional forms that can provide a similar effect .
these include piecewise linear functions ( e.g. , flame),(12 ) and piecewise linear and quadratic functions ( e.g. , tfit).(21 ) it can be construed that indeed any bell - shaped function can work in a similar fashion . in their pioneering work 20 years ago , kearsley and smith(20 ) noticed that the lorentzian function ( 1/(1 + r ) ) yielded similar results to the gaussian but was significantly faster to compute .
however , the advance in computational technology to calculate exponents has eliminated this advantage of the lorentzian function .
for this work we chose the gaussian because it involves a minimal number of tunable parameters and has a simple , continuous shape .
another consideration is that function optimization procedures often require the first and second derivatives of the function . in the case of the gaussian ,
once the function has been calculated , its first and second derivatives can be obtained without much extra work .
a scoring function s could thus be constructed based on a sum of gaussian terms :
in the formula , tk is a term encouraging / discouraging the overlap of a particular pair of types ( ak , bk ) of atoms / features ; wk is the weight of the term ; i and j are atoms / features from the two molecules ; rij is the distance between the intermolecular atom / feature pair ; and is a parameter controlling the range of this type of interaction .
ligands binding in the same mode are expected to have substantial volume overlap . the first term in the scoring function , t0
the terms t1 and t2 encourage the overlap of pairs of intermolecular donors and acceptors .
after these attractive terms come the repulsive terms that discourage the overlap of intermolecular atom pairs of incompatible types .
first , hydrophobic and hydrophilic atoms ( atoms capable of hydrogen bonding ) are not expected to occupy the same part of the binding pocket , as reflected by t4 and t5 .
second , hydrogen - bond acceptors that are not also donors are expected to avoid positions of donors that are not also acceptors .
the last two terms , t8 and t9 , are based on hydrogen - bond projected features .
the scoring function consists of a summation of terms , tk s , each of which has the form :
each row of the table corresponds to one term , tk , and i and j denote atoms ( for t0t7 ) or features ( for t8 and t9 ) coming from the first and second molecule , respectively . these atoms / features have to be of certain types , ak and bk , as given in the second and third columns of the table , and rij is the distance between the atoms / features .
the names of the various constants for the different terms , wk and , are given in the fourth and fifth columns of the table .
all the atom - based terms have the same value for , namely a , while all hydrogen - bond projected feature - based terms have another value for , namely p . to keep the number of parameters low , the same value of , a ,
the projected feature terms used a different value of , p , allowing these terms to be more diffuse than the atom - based terms if necessary .
atom - based terms were followed by projected feature terms . for the atom - based terms ,
based on this order , the scoring function was built up term by term . at each stage , the one or two parameter(s ) of the newly added term was / were optimized .
a series of scoring functions were created using different values for the newly added parameter(s ) .
the parameter value(s ) corresponding to the best scoring function in the series would be picked .
after the scoring function was optimized , it was incorporated into the flexible alignment functionality in molecular operating environment ( moe).(22 ) molecular alignment results were compared with two recent publications .
proschak et al.(6 ) performed rigid - body alignments for eight thermolysin ligands using their shapelets method and compared their result with two previous alignment methods .
we performed validation runs on these thermolysin ligands to see how our scoring function fared .
chen et al.(23 ) studied the geometric accuracy of aligning small molecules using rocs(26 ) and flexs.(27 ) they used eight protein targets as test sets : hiv protease , cyclin - dependent kinase 2 ( cdk2 ) , estrogen receptor 1 ( esr1 ) , mitogen - activated protein kinase 14 ( p38 ) , thermolysin , human rhinovirus capsid , elastase , and trypsin .
six of these ( all but thermolysin and elastase ) consist exclusively of structures from the publicly accessible pdb.(17 ) we used these six systems for validation runs .
both rigid - body alignments and flexible alignments were compared . for training a scoring function for
the validation runs , all systems that contain any pdb entries involved in the test sets were naturally excluded .
ligands from identical or almost identical pockets ( see below for details ) from the pdb were used for training the scoring function . only pdb entries with a resolution of better than 2.5 were used .
ligands were defined as freestanding molecules with no more than 300 heavy atoms and 10 residues . in order to exclude waters and very small molecules ,
each ligand must have at least one pair of heavy atoms separated by three bonds .
ligands without carbon atoms or with exotic elements ( elements other than c , h , n , o , s , p , f , cl , br , and i ) were discarded .
atp analogs were excluded because their large population in the pdb would likely skew the data .
ligands without any rings were also excluded so as to remove the lipids without excluding any drug - like compounds .
we first obtained a list of protein domains from structural classification of proteins ( scop).(28 ) for each domain , all pdb entries containing that domain were superposed using the protein backbone of that domain .
overlapping ligands were clustered into groups so that each group would correspond to one pocket . for each pocket ,
since we started from scop domains , it was possible that pockets within a group could involve different residues or have different geometries . for our purpose of scoring function training
, it may not be very meaningful to study the alignment of ligands in different pockets .
a table was then constructed with each row corresponding to one protein and each column corresponding to one carbon cluster .
a cell of the table has the residue type as the entry if the protein contributes to that cluster .
the pocket similarity between a pair of proteins was defined to be the fraction of table columns that are identical ( i.e. , same residue type , or empty cell , for all cells in the column ) . after the pocket similarities between all protein pairs were determined , the proteins were clustered , using complete linkage and pocket similarity as the metric , with a cutoff of 0.8 . in other words ,
any two pockets in the same group had at least 80% of the table columns identical .
each original pocket group might thus be divided into multiple groups . for each resulting group ,
a fine superposition on the 3d structures was performed again using carbons common to all pockets , since the definition of the pocket might have changed due to the change in the content of the ligand set .
this whole procedure was repeated until there was no new division of the pocket groups . upon inspection
, some systems were found to be uninteresting in the sense that the aligned ligands only displayed one type of structure at most regions of the pocket .
studying this type of system would not give us more information than studying self - alignments .
. therefore the training ligands were restricted to those having no more than 12 rotatable bonds .
finally , to ensure diversity , a set of representative ligands was chosen for each system such that no pair of representatives had a tanimoto similarity of over 0.8 on their maccs fingerprints .
only 100 systems remained after these final filters , and 85 systems had no more than 20 representative ligands .
this is our comprehensive aligned small molecules data set . for training a scoring function for the validation runs , 11 of the 100 systems in the comprehensive data set were excluded because they contained entries that would be used in the runs .
this was more than the number of test systems ( 7 ) because some test systems ( e.g. , the 57 ligands in the cdk2 system ) had variations in the pocket residue composition or geometry and were hence subdivided into more than one pocket group when our comprehensive data set was constructed . to keep things simple ,
the training of the scoring function was based on trying to align a flexible source ligand onto a rigid target ligand .
dummy poses were generated by making small adaptations to the moe docking engine,(22 ) as described below .
n aligned ligands , all n n pairs of ligands were considered with the following requirement .
the fraction of heavy atoms in the target that lie within 2 of any heavy atom of the source must exceed 0.5 , and the fraction of heavy atoms in the source that lie within 2 of any heavy atom of the target must exceed 0.75 .
this excluded ligands with different binding modes and ensured that no source ligand was substantially larger than the target .
if this resulted in more than 5000 conformers , a random subset of the systematically generated conformers would be used . only conformers with severe atomic overlaps
this is because we envisioned that when the scoring function is used to distinguish poses with the correct binding mode , any poses that are fished out can be refined to improve the pose geometry and any atomic overlap within the molecule will be relieved . for each sourcetarget pair ,
each time , a random set of three heavy atoms on the target ligand was chosen , together with a random conformer of the source ligand .
triplets of heavy atoms on the source ligand , which formed a triangle similar to that formed by the three chosen target atoms , were used to align the source ligand onto the target ligand .
poses whose root - mean - square deviation ( rmsd ) for the heavy atoms was under 1.5 were considered duplicated , and only one copy was kept .
for each scoring function , the scores for all dummy poses were calculated . for each set of alignments involving a particular pair of source and target ligands
, we counted the number of good poses , n25 , among the top 25 scoring poses .
as for whether a pose is good or not , we used fuzzy logic
. poses with an rmsd ( heavy atoms only ) from the correct pose of under 2.0 were considered good .
poses with an rmsd between 2.0 and 3.0 were considered partial good poses , with the partiality decreasing linearly from 1 to 0 . for each system ( i.e. , same pocket ) , we averaged the number of good poses for all sourcetarget pairs in the system .
the receiver operating characteristic ( roc ) curve is often used to measure the success of scoring functions for virtual screening.(29 ) in short , the roc curve plots the fraction of true positives recovered ( out of all active compounds ) against the fraction of false positives obtained ( out of all inactive compounds ) , as the cutoff value varies .
it is generally considered that the bigger the area under the curve , the better is the scoring function .
we felt that , for our purpose , using the area under the roc curve was not as suitable as using
the number of good poses amongst the top 25 , since we generally can only afford to consider a limited number of top scoring poses .
hence all but the very beginning part of the roc curve would be of significance to us .
we think that human inspection can typically handle 10 or so poses . on the other hand , if a thorough refinement is desired as part of an automatic process , then it would be reasonable to intensively process up to about 100 poses per ligand .
hence here we used the top 25 poses . as a precaution , we repeated all the calculations and validation runs using the top 100 poses instead of the top 25 .
it turned out that only one out of the six optimized parameters for the scoring function would come out different , and the quality of the of validation runs remained very similar .
the details are given in section a of the supporting information . as mentioned previously ,
the scoring function was built up term by term . at each stage , only one ( or two ) parameter was introduced .
each table corresponds to one stage of optimization where one ( or two ) parameter was optimized .
the resulting numbers of good poses among the top 25 , n25 , corresponding to the various values of the parameter being optimized are given in the tables .
the optimal value of the parameter and its corresponding value of n25 are highlighted in bold .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of a .
we started by including only the volume overlap term ( t0 in table 1 ) in the scoring function .
in other words , to a first approximation , the bigger the volume overlap between two molecules , the better was the score . in order to make the scoring function quick to calculate ,
the only parameter to be optimized was . obviously a smaller value for would result in a more diffuse gaussian function .
consider the simple case to align one atom ( the source ) onto a diatomic molecule ( the target ) .
this is similar to trying to align methane , with one heavy atom , onto ethane , with two heavy atoms .
suppose the internuclear distance of the diatomic molecule is 1.5 ( for comparison , a cc bond in a benzene ring is about 1.40 , and a cc bond in an ethane is about 1.54 ) .
the shapes of the scoring function for three different values of are plotted in figure 2 . for large values of ,
the source atom will see a score with two distinct peaks located very close to the two target atoms .
it will have a strong tendency to align with either one of the target atoms .
for example , if is 2.0 ( the blue curve of figure 2 ) , then the distance between the peaks will be 1.46
. the value of the score at the trough midway between the peaks will be 64% of the height of the peaks .
when decreases to 1.2 ( the black curve of figure 2 ) , the peaks will move toward each other , away from the atomic positions .
the distance between the peaks will drop to 1.18 , and the trough to peak height ratio of the score will increase to 94%
. in other words , the score has already very much smoothed out the atomic positions .
the source atom can settle for any position between the two atoms with little change on the score .
how the scoring function is controlled by the parameter , for the case of a target molecule consisting of two atoms , located at positions 0.75 and 0.75 . for a source atom to be superimposed onto the target molecule ,
the scoring function is exp(r12 ) + exp(r22 ) , where r1 and r2 are the distances from the source atom to the two target atoms .
the scoring functions corresponding to values of 2.0 , 1.5 , and 1.2 are plotted in blue , red , and black , respectively . given a scoring scheme and an initial aligned pose between two rigid ligands , one can refine the alignment by optimizing the score .
when we did this with many starting poses , we found that we ended up with fewer resulting aligned poses when we used smaller values of ( unpublished results ) .
this is in line with our expectation that smaller values of smooth out the score landscape . according to previous works of gaussian function - based alignment scores , values of of between 0.1 and 0.5
, we tried out a total of 7 values for a , from 0.06 to 0.7 .
the corresponding numbers of good poses among the top 25 are tabulated in table 2a .
it can be seen that for the values of a tested , a value of 0.5 was the best , with an average of 4.83 good poses among the top 25 . after establishing a value for a
, the relative weights of the atom - based terms were optimized one by one .
we first looked at the weight wda for the hydrogen - bonding atoms ( donor and acceptor ) similarity terms ( t1 and t2 in table 1 ) . for this
we tried out 7 values for wda , from 0.5 to 32 , in logarithmic intervals .
the corresponding numbers of good poses among the top 25 are tabulated in table 2b .
a value of 4 turned out to be the most favorable , with an average of 6.10 good poses among the top 25 .
the new terms are t1 and t2 , which correspond to the attraction between hydrogen - bond donor / acceptor atoms .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of wda .
after this , we turned to the similarity term between hydrophobic atoms ( t3 in table 1 ) . besides trying to establish the optimal weight whh for this term , we also tried out two schemes of defining hydrophobic atoms .
one used the conventional hydrophobic atom assignment in moe.(22 ) the other scheme was based on how far topologically an atom was from any hydrogen - bonding atom .
atoms that were two or more bonds away from any hydrogen - bond acceptors or donors were considered hydrophobic .
tables 2c and 2d give the numbers of good poses among the top 25 for various values of whh using the two schemes .
it can be seen that the two schemes gave very similar results , with the optimal weighting factor whh to be around 1.0 for both schemes .
the scoring function at this stage is ( t0 + t1 + t2 + t3 ) .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of whh .
the scoring function at this stage is ( t0 + t1 + t2 + t3 ) .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of whh .
next we considered the repulsion term between hydrogen - bonding atoms and hydrophobic atoms ( t4 and t5 in table 1 ) .
here we see that the first scheme of defining hydrophobicity was slightly better and that the optimal weight for the repulsion term , wr1 , was around 1.0 .
the scoring function at this stage is ( t0 + t1 + t2 + t3 + t4 + t5 ) .
the new terms t4 and t5 correspond to the repulsion between hydrophobic and hydrogen - bonding atoms .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of wr1 .
the scoring function at this stage is ( t0 + t1 + t2 + t3 + t4 + t5 ) .
the new terms t4 and t5 correspond to the repulsion between hydrophobic and hydrogen - bonding atoms .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of wr1 .
after this we investigated a repulsion term between hydrogen - bond donor and acceptor atoms ( t6 and t7 in table 1 ) .
perhaps surprisingly , it was found that this term did not seem to improve the scoring function ( see table 2 g ) .
the scoring function at this stage is ( t0 + t1 + t2 + t3 + t4 + t5 + t6 + t7 ) .
the new terms t6 and t7 correspond to the repulsion between hydrogen - bond donor atoms that are not acceptors and hydrogen - bond acceptor atoms that are not donors . refer to table 1 for the details of these terms .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of wr2 .
finally the projected acceptor and donor feature points were considered ( t8 and t9 in table 1 ) .
these points correspond to the expected positions of donors and acceptors on the binding pocket . in this sense
so we tried out different values of as well as different weights , wpf , for this term .
it can be seen that an p value of 0.125 coupled with a weight of 2.0 was a good combination .
otherwise an p value of 0.0625 coupled with a weight of 1.0 was also respectable .
the scoring function at this stage is ( t0 + t1 + t2 + t3 + t4 + t5 + t8 + t9 ) .
the new terms t8 and t9 correspond to the attraction between hydrogen - bond donor / acceptor projected features . refer to table 1 for the details of these terms .
this table gives the average number of good poses , n25 , within the top 25 scoring poses for various values of wpf and p .
to summarize , it was found that a good scoring function is of the form given by the terms of table 1 , except terms t6 and t7 , with the values of the various coefficients given in table 3 .
the optimal scoring function consists of the terms t0t5 , t8 , and t9 , as given in table 1 , with the various parameters as given by this table . for each of the parameters considered
however , there was a considerable number of ligandpocket systems , and in each system , there was a considerable number ( at least 25 , but often much more ) of pairs of sourcetarget ligands .
more importantly , the fact that there are smooth trends in the numbers in tables 2a2h , as the parameters were varied , serves to confirm that the numbers are accurate enough for our purpose of locating the optimal values for the parameters .
moe s flexible alignment ( flexalign ) functionality(22 ) was used for the validation runs . for rigid - body alignments , conformers from the pdb
flexalign generated random poses of the source molecule superposed onto the target molecule and optimized the alignment score s , while keeping the molecules rigid .
for the flexible alignments , for each source molecule , one randomized conformation was used as input .
flexalign generated a random conformer from this input , randomly superposed it onto the target molecule , and then optimized this pose under the influence of the alignment score between the source and target molecules .
the target molecule remained fixed throughout the process . in order to avoid unrealistic conformations ,
the internal energy u of the source molecule must also be considered alongside the alignment score s. for the internal energy , we used the mmff94 force field with a distance - dependent dielectric .
pose optimization was carried out by minimizing the effective energy , e , which was defined by
alignment solutions were also ranked according to their e values .
a value of 4 kcal / mol was used for k. this value seemed to give a good balance between the internal energy u and the alignment score s. the difference in the force field energy u between the conformation of the source molecule in the top scoring ( i.e. , the one with the lowest e value ) solution and its corresponding local energy minimum was generally small .
the average of this difference ( over the n n cross - alignments for each system ) for the cdk2 , hiv , p38 , esr1 , trypsin , and rhinovirus systems were , respectively , 0.32 , 0.76 , 0.25 , 0.33 , 0.25 , 0.25 kcal / mol per heavy atom .
in contrast , the average of the difference in the internal energy u between the ligand conformer in the crystal structure and its corresponding local energy minimum is over 1 kcal / mol per heavy atom for all the six systems .
we also investigated whether the value of the alignment score s or the effective energy e gives an indication of the goodness of the alignment .
our results were first compared with those in table 4 of proschak et al.(6 ) this involved pairwise rigid - body alignments on a system of eight thermolysin ligands .
for each ligand pair , one ligand was used as the target , while the other was used as the source .
an alignment was considered successful if the top scoring pose had a heavy atom rmsd of under 2 from the correct answer .
our results , together with those obtained by proshak et al.,(6 ) are tabulated in table 4 . according to proshak
when surfcomp(24 ) was used with the electrostatic potential , the success rate was 75% .
in contrast , our scoring function yielded a success rate of 93% ; there were only 2 failed cases out of 28 alignments .
our scoring function returned the correct answer as the second best scoring solution ( figure 3 , bottom ) . in any case
, our best scoring solution looks highly plausible with heavy overall volumetric overlap as well as the overlap of the aromatic ring and two carbonyl groups ( figure 3 , top ) .
carbons of our solutions ( top and bottom are best and second best scoring , respectively ) are in cyan .
the eight thermolysin ligands have pdb i d s 1thl , 1tlp , 1tmn , 3tmn , 4tmn , 5tln , 5tmn , and 6tmn . for each pair of nonidentical ligands ,
one ligand was chosen as the template , and the other ligand was aligned onto the template .
if the top scoring solution yielded an rmsd of under 2 with the crystallographic pose for the heavy atoms , then it was considered successful .
this table gives the success rates for the 28 cases , as reported on table 4 of proschak et al.(6 ) tests were also run on six systems mentioned in chen et al.,(23 ) where rocs(26 ) and flexs(27 ) were used .
an alignment was considered correct if the top scoring solution had a heavy atom rmsd of under 2 with the crystallographic overlay .
except for the rhinovirus system , which will be examined in more details below , and for the flexible alignment of the cdk2 system , our results are significantly better than those obtained by chen et al . using flexs or rocs . for rigid - body alignments ,
our fraction of correct results was at least 40% and reached 80% or more in two systems .
naturally , flexible alignment is more difficult than rigid - body alignment , and so the percentages of correct results are lower for all systems except rhinovirus ( see analysis below ) .
for each of the six systems , pairwise cross - alignments were carried out for all n n ligand pairs , where n is the number of ligands in the system .
if the top scoring result yielded an rmsd of under 2 for the heavy atoms , then it was considered correct .
this table gives the percentage of correct results for the n n ligand pairs in the system , as reported on table 2a of chen et al.(23 ) for the cdk2 system , our results for the rigid - body alignments but not for the flexible alignments are significantly better than those of chen et al .
cdk2 is a large system ( 57 ligands ) with a complex pharmacophore model.(31 ) if the six ligands 1di8 , 1p5e , 1pkd , 1pxi , 1pxj , and 1wcc were excluded , then our results would improve from 39.6% to 47.6% correct for the rigid - body alignments and from 22.4% to 26.6% correct for the flexible alignments . of the 57 ligands in the system , only 7 ligands have more than 33 or less than 17 heavy atoms , and 1p5e , 1pkd
, 1pxi , 1pxj , and 1wcc are among them , with 13 , 36 , 14 , 14 , and 8 heavy atoms , respectively .
since the pharmacophore model is complex,(31 ) it seems that volumetric overlap is an important factor for alignment .
hence ligands that are much smaller or much larger than average would be difficult to align . moreover , an important pharmacophore element is the set of two hydrogen - bond donors and one acceptor interacting with the hinge region of the protein .
these correspond to pharmacophore features d1 , d2 , and a1 mentioned in zou et al.(31 ) and are shown in figure 4 . out of the 57 ligands , only 4 do not have at least 2 out of these 3 features , and ligands 1di8 , 1p5e , and 1wcc are among them .
important pharmacophore features for the cdk2 ligands include two hydrogen - bond donors ( magenta sphere ) and one acceptor ( cyan sphere ) .
the fact that they are relatively comparable in size ( all but three have between 39 and 61 atoms ) , do not have a homogeneous ( spherical ) shape , and have rich feature sets might have helped the rigid - body alignment result , because there are not that many possibilities for the molecules to align well .
one possibility for our superior results is that we have trained our scoring function using a significantly larger data set from the pdb than the version of flexs or rocs that were used . of all six test systems ,
the drop in performance in going from the rigid - body to the flexible mode is most pronounced for the hiv system .
of the 28 hiv ligands , 5 have 10 rotatable bonds . 7 have between 12 and 16 rotatable bonds .
for the 13 ligands of the p38 system , our scoring function produced an overall correct rate of 43% and 30% for the rigid - body and flexible alignments , respectively ( table 5 ) .
it is worth noting that one of the ligands , 1wbo , is significantly smaller than the others ( figure 5 ) .
it can be seen that the ligand 1di9 ( magenta in figure 6 ) binds in a mode different from the others .
the four ligands 1m7q , 1ouk , 1ouy , and 1ove ( yellow in figure 6 ) also bind in a mode somewhat different from the others . for the remaining 7 ligands ,
our scoring function actually got 73% of the pairwise cross - alignments correct in the rigid - body mode , and 53% correct in the flexible mode . and for the 16 pairwise cross - alignments of the four ligands 1m7q , 1ouk , 1ouy , and 1ove , our scoring function got them all , 100% , correct in the rigid - body mode and 94% ( all but one of the 16 ) correct in the flexible mode .
ligands within each group have comparable sizes and can be considered to bind in a similar mode , as can be seen in figure 7 . for the pairwise cross - alignments of all 13 ligands , our scoring function got 59% and 41% correct in the rigid - body and flexible modes , respectively .
however , for the group of 6 smaller ligands , we got 94% of the 36 rigid - body cross - alignments correct and 72% of the flexible alignments correct . for the group of 7 larger ligands , we got 92% of the 49 rigid - body cross - alignments correct and 65% of the flexible alignments correct .
the 6 smaller ligands ( 1a52 , 1gwq , 1l2i , 1x7e , 1x7r , and 3erd ) are in cyan . the 7 larger ligands ( 1r5k , 1sj0 , 1uom , 1xp1 , 1xp9 , 1xqc , and 2bj4 ) are in magenta . the crystallographic alignment of the 7 trypsin ligands is given in figure 8 .
ligand 1tni ( cyan in figure 8) assumes a binding mode different from the others . for the remaining 5 ligands ,
our scoring function got all 25 cross - alignments correct in both the flexible and rigid - body modes .
figure 9 gives our result for flexibly aligning 1tnj onto the much larger ligand 1pph .
our second best scoring solution ( figure 9 , bottom ) yielded the correct alignment mode .
as for our best scoring solution ( figure 9 , top ) , it involves a perfect atom to atom matching for all atoms of 1tnj .
the aromatic ring is mapped to an aromatic ring of 1pph , all aliphatic carbons are mapped to aliphatic carbons on 1pph , and the nitrogen atom is mapped to a nitrogen on 1pph .
our third best scoring solution ( figure 10 , bottom ) roughly yielded the correct binding mode .
our best scoring solution ( figure 10 , top ) involves a matching of atoms similar to that of the best scoring solution of aligning 1tnj onto 1pph ( figure 9 , top ) .
our second best scoring solution ( figure 10 , middle ) also involves a good matching of atoms , with the phenyl ring of 1tnl aligned to a six - membered ring of 1pph .
the remaining ligands are 1tnh , 1tnj , 1tnk , 1tnl , 3ptb . flexibly aligning 1tnj onto 1pph ( carbons in green ) .
carbons of our solutions ( top and bottom : best and second best scoring ) are in cyan . flexibly aligning 1tnl onto 1pph ( carbons in green ) .
carbons of our solutions ( from top to bottom : first , second , and third best scoring ) are in cyan . for the 8 rhinovirus ligands , our scoring function got 50% of the pairwise cross - alignments correct in both the rigid - body and flexible modes .
as shown in figure 11 , all eight ligands have a long shape and are almost symmetric .
there is a heterocyclic ring on either end of each ligand , connected by a long , flexible linker chain .
as noted in the original publication for the x - ray structures,(32 ) the ligands have two binding modes , one being the inverse of the other , as shown in figure 11 .
it is easy for the alignment algorithm to superpose the two rings on either end but difficult to distinguish between the two binding modes .
hence all the cross - alignments within a binding mode were obtained correctly , but the inverse binding mode was obtained for each pair across the different binding modes .
the carbons are in magenta for one mode ( 2rm2 , 2rr1 , 2rs1 , and 2rs3 ) and in cyan for the other mode ( 2r04 , 2r06 , 2r07 , and 2rs5 ) .
when the scoring function was trained for the validation runs , systems involved in the test sets were excluded . after the validation runs , the training process was repeated to make use of all data , including systems involved in the test sets .
it turned out that using all the data did not lead to any change in the scoring function .
the updated tables 2a2h are given in tables ah of section b of the supporting information .
although the numbers of good poses among the top 25 ( n25 ) have changed slightly , the positions for the optimal values for all the parameters have not .
based on the pdb(17 ) and scop,(28 ) a comprehensive set of aligned ligands binding in the same pocket has been compiled .
we used only high - resolution structures and considered only pockets that are highly similar and have diversified , drug - like ligands . for each pair of ligands binding in the same pocket ,
one ligand was used as the target , and dummy aligned poses of the other ligand were generated . by studying the correlation between the score of these dummy poses and their similarities with the correct answer , a small molecule alignment scoring function was built up term by term .
eventually we obtained the scoring function composed of terms given by table 1 , except terms t6 and t7 , with parameters as given by table 3 .
we verified our scoring function by comparing results of rigid - body and flexible alignments performed using the flexible alignment functionality in moe(22 ) with those reported in the literature .
we found that our results are superior to those of five methods reported in two recent publications .
we are happy to share our comprehensive aligned small molecules data set with other scientists so that they can use it to optimize their own scoring functions . | a comprehensive data set of aligned ligands with highly similar binding pockets from the protein data bank has been built .
based on this data set , a scoring function for recognizing good alignment poses for small molecules has been developed .
this function is based on atoms and hydrogen - bond projected features .
the concept is simply that atoms and features of a similar type ( hydrogen - bond acceptors / donors and hydrophobic ) tend to occupy the same space in a binding pocket and atoms of incompatible types often tend to avoid the same space .
comparison with some recently published results of small molecule alignments shows that the current scoring function can lead to performance better than those of several existing methods . |
esthesioneuroblastoma also called olfactory neuroblastoma is a rare malignant neuroectodermal tumor of the sinonasal tract region , first described by berger et al . in 1924 . according to the world health organization
, olfactory neuroblastoma is defined as a malignant neuroectodermal tumor , that is , assumed to originate from olfactory receptor cells present high in the nasal cavity .
this justifies the most common site of the tumor being cribriform plate , middle superior nasal structures and anterior skull base .
these tumors show varied morphology with uncertain histogenesis which results in two extremes , i.e. an indolent growth with more than 20-year survival or an aggressive tumor with limited survival and distant metastasis .
most of the pathologists encounter difficulties in the diagnosis of these malignancies because of their complex anatomic location , non - specific symptoms , variable morphology and unfamiliarity which leads to misdiagnosis as benign tumors . a case of aggressive olfactory neuroblastoma , which recurred thrice following complete surgical excision ,
a 24-year - old female patient reported to the outpatient department of our hospital complaining of swelling associated with pain on the left side of the face since 1 month .
initially the swelling was about peanut size , which gradually increased to present size , and was associated with pricking and localized pain .
her dental history revealed surgical excision of a tumor in 2007 and subsequent recurrent lesion at the same site in 2013 which was again operated . at present , extra - oral examination revealed a solitary diffuse swelling in the left infraorbital region of the face measuring approximately 4 cm 4 cm , extending mediolaterally from the medial canthus of the eye to frontozygomatic region and superioinferiorly from the lower eyelid to 1 cm above the left corner of mouth obliterating the nasolabial fold [ figure 1 ] .
intraoral examination showed deficient maxilla and the absence of teeth in relation to 1428 region [ figure 2 ] .
extraoral photograph revealing infraorbital swelling on the left side of the face intraoral examination depicting deficient maxilla and the absence of teeth in relation to 1428 region computerized tomography revealed a comminuted fracture of the lateral wall of the left orbit with an asymmetric outline of the globe .
the posterior wall of left orbit was also incomplete with little soft tissue masses seen protruding into its posterio - lateral aspect .
part of soft tissue was seen extending toward the anterior wall of left maxillary sinus which was completely effaced .
two metal plates were seen , supporting the anterior lateral wall of the left maxillary antrum as artifacts .
these plates were presumed to be placed in the previous surgeries [ figure 3 ] .
computerized tomography revealing comminuted fracture on lateral wall and floor of the left orbit with the tumor mass extending into the orbit on reviewing the dental history , clinical and radiological features , a provisional diagnosis of recurrent ameloblastoma of the left infraorbital region was given .
the tumor was surgically excised with normal tissue margins surrounding the lesion and the excised specimen was sent for histopathological examination .
microscopic examination revealed a nasociliary epithelium with adjacent tumor area composed of small round cells arranged in lobular pattern separated by fibrovascular stroma .
these tumor cells are arranged in pseudorosettes and glandular patterns , and showed round to oval hyperchromatic nucleus with few presenting salt and pepper chromatin pattern .
the adjacent bone tissue is infiltrated by these tumor cells along with new bone formation ( osteoid ) .
the stroma is fibro - cellular and -vascular with focal areas of neurofibrillary matrix , giant cells and mitoses [ figure 4 ] .
photomicrograph showing ( a ) small round cells arranged in sheets and lobules ( h&e stain , 40 ) , ( b ) rosettes in neurofibrillary matrix ( arrow ) and new bone formation ( arrowhead ) ( h&e stain , 100 ) , ( c ) homer wright rosettes ( arrow ) with central neurofibrillary matrix ( h&e stain , 400 ) , ( d ) fibro - cellular and -vascular stroma ( h&e stain , 200 ) based on clinical , radiological and histological findings , a final diagnosis of olfactory neuroblastoma was established . for the confirmation of the diagnosis , immunohistochemistry with a panel of markers
was performed [ table 1 and figure 5 ] , establishing the diagnosis as olfactory neuroblastoma . with the suspicion about the former treated pathologies , past records of the patient were reevaluated and subjected to histopathological and immunohistochemical examination .
histopathology revealed the characteristic features of olfactory neuroblastoma and was confirmed immunohistochemically with the marker neuron - specific enolase ( nse ) which showed strong positivity [ figure 6 ] .
immunohistochemical markers performed photomicrograph showing immunohistochemical staining for ( a ) neuron - specific enolase : strongly positive ( ihc stain , 200 ) , ( b ) chromogranin : positive ( ihc stain , 100 ) , ( c ) s-100 : negative ( ihc stain , 40 ) , ( d ) proliferating cell nuclear antigen : positive ( ihc stain , 40 ) , ( e ) ki-67 : positive ( labeling index : < 10% ) ( ihc stain , 100 ) [ inset : showing two positive cells , ( ihc stain , 400 ) ] , ( f ) pancytokeratins : focally positive ( ihc stain , 200 ) , ( g ) epithelial membrane antigen : negative ( ihc stain , 100 ) , ( h ) caldesmon : negative ( ihc stain , 100 ) , ( i ) cytokeratin-7 : negative ( ihc stain , 100 ) .
nse : neuron - specific enolase , pcna : proliferating cell nuclear antigen , ema : epithelial membrane antigen photomicrograph showing immunohistochemical staining of marker neuron - specific enolase ( nse ) showing strong positivity in previously treated pathologies .
( a ) section of tumor mass resected in 2007 ( ihc stain , 100 ) , ( b ) section of tumor mass resected in 2013 ( ihc stain , 100 )
although there is no much evidence regarding the origin of olfactory neuroblastoma , few studies reported the possible source of this tumor to be specialized neuroectodermal olfactory cells . even in the present case , there was an evidence of olfactory epithelium extending along the tumor mass .
other specific sites of origin for this tumor include jacobson 's vomeronasal organ , sphenopalatine ganglion , ectodermal olfactory placode , ganglion of loci ( nervus terminalis ) and autonomic ganglia of the nasal mucosa .
most commonly reported in all age groups with a bimodal peak in second and sixth decade with no gender predilection . in the present case , the female patient first developed tumor at an age of 16 with recurrences noticed at 22 and 24 years .
tumors in advanced stages usually present orbital symptoms such as proptosis and excessive lacrimation , which were also noticed in the current case report . computed tomography ( ct )
the ct features of olfactory neuroblastoma are nonspecific and so can not be differentiated from other tumors such as undifferentiated lymphoma , rhabdomyosarcoma , squamous cell carcinoma , extramedullary plasmacytoma and amelanotic melanoma .
the aggressive nature of this tumor is determined by kadish staging system , which is based on the clinical spread of the tumor [ table 2 ] .
moritaet al . modified kadish staging system by incorporating group d for tumors with regional ( cervical lymph node ) or distant metastases .
an extensive research of previous cases of olfactory neuroblastomas revealed their presence in kadish stage c. the present case was also categorized under stage c as the tumor mass was shown to be extending into the orbit .
however , kadish system could not give a definite comparison between the prognosis of surgically operable and inoperable tumors because tumors in the neck , orbit , intracranial portion and distant metastasis were all included in the same stage .
hence , histopathological grading is also mandatory to predict the treatment outcome and survival rate of this tumor .
kadish clinical staging system histopathologically , hyams grading system is used to differentiate low - grade ( grade 1 and 2 ) from high - grade tumors ( grade 3 and 4 ) .
grading is based on few defining features of olfactory neuroblastoma such as lobular architecture , rosettes , neurofibrillary matrix , pleomorphism , mitoses , necrosis and presence of calcifications [ table 3 ] .
two types of rosettes are seen , of which pseudorosettes ( homer wright rosettes ) are the more common .
they are characterized by a delicate neurofibrillary and edematous stroma that forms the center of a palisaded arrangement of tumor cells .
true rosettes ( flexner wintersteiner rosettes ) have a tight , gland - like annular arrangement . in the present case ,
most of the tumor cells are arranged in pseudorosette pattern in a fibro - cellular and vascular connective tissue stroma representing a stromal reaction to the proliferating tumor islands .
hyams histopathological grading system histopathological evaluation of our case revealed the tumor in hyams grade 2 , which is considered as a low - grade tumor .
however , according to kadish system , our case can be categorized as high - grade malignancy . despite its shortcomings ,
the other classification systems for olfactory neuroblastoma are according to biller et al . , and dulguerov and calcaterra .
, among all classification systems , only kadish system revealed a statistically significant discrimination among stages for relapse - free survival .
the differential diagnosis included primary intraosseous carcinoma , ameloblastic carcinoma and salivary gland carcinoma . to differentiate whether it was derived from the salivary gland epithelium , a panel of immunohistochemical markers such as epithelial membrane antigen ( ema ) , cytokeratin-7 ( ck-7 ) and caldesmon was used which showed a negative staining pattern .
hence , the salivary gland tumors were ruled out . as histopathology showed nasociliary epithelium which shows olfactory neural cells , markers specific for neuroectodermal cells , such as nse and chromogranin ,
were used which showed a strong positive reaction with nse and weak positivity with chromogranin . to determine the proliferation rate , immunohistochemical staining with ki 67 and proliferating cell nuclear antigen ( pcna )
was done , which showed the labeling index of ki 67 to be < 10% and strong positivity with pcna .
other markers include synaptophysin , neurofilament protein , class iii beta - tubulin , microtubule - associated protein , chromogranin , glial fibrillary acidic protein and leu-7 .
the present case showed focal positivity for cks which signifies that there are focal areas of epithelial differentiation which is a common feature of olfactory neuroblastoma .
caldesmon is a marker for neoplastic myoepithelial cells and ck-7 is usually positive for salivary gland neoplasms .
s-100 positivity is mostly seen in the periphery , which represents the schwann cells or sustentacular cells usually present along the periphery of neoplastic lobules . in our case , s-100 staining was negative which is a common feature seen in high - grade tumors .
ki-67 positivity showed < 10% labeling index , which implies that the tumor is well differentiated and associated with long - term survival , whereas poorly differentiated ( hyams grade 3 and 4 ) are associated with a high proliferation index .
a combined treatment of surgery and radiation therapy ( rt ) resulted in a better recurrence -free rate ( 60100% ) compared with surgery alone ( 1456% ) .
lund et al . considered the above combination as the gold standard treatment . in the present case , as the tumor was extended into the orbit ( kadish stage c ) and as the tumor recurred thrice , aggressive surgical excision with adjuvant radiotherapy is the mainstay of treatment .
elkon included the presence of metastasis and local extension of the tumor ( e.g. ethmoidal , nasopharynx , orbital ) as negative prognostic factors .
a proper diagnosis should be made through clinical , radiographic and histopathological findings and the tumor should be staged and graded before proceeding to surgery . aggressive surgical resection and rt is the mainstay of treatment and patients must be followed up carefully as local or regional recurrences are most commonly associated .
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has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
| esthesioneuroblastoma is a rare malignant tumor of the sinonasal tract .
oral and maxillofacial pathologists rarely encounter this tumor in their daily practice . because of their complex anatomic location , non - specific symptoms , varied histomorphology and unfamiliarity , most of the times
, the tumor is diagnosed as benign tumor and thereby conservative treatment results in multiple recurrences .
a recurrent case of esthesioneuroblastoma in a 24-year - old female patient describing the clinical , histopathological and immunohistochemical features along with differential diagnosis is discussed . |
hepatocellular carcinoma ( hcc ) is the sixth most common malignancy worldwide and the third most common cause of global cancer related mortality [ 1 , 2 ] .
hcc burden disproportionately impacts developing countries and males ; as of 2008 , 85% of cases occurred in africa and asia , with worldwide male : female sex ratio of 2.4 .
risk factors for the development of hcc include chronic liver inflammation from hepatitis b and c infection , autoimmune hepatitis , excessive alcohol use , nonalcoholic steatohepatitis , primary biliary cirrhosis , environmental carcinogens such as aflatoxin b , and genetic metabolic disease ( such as hemochromatosis and alpha-1 antitrypsin deficiency ) .
prognostic and therapeutic options are dependent upon the severity of underlying liver disease , and median overall survival ( os ) for metastatic or locally advanced disease is estimated at 58 months .
hcc is relatively refractory to cytotoxic chemotherapy , likely due to overexpression of multidrug - resistant genes , protein products such as heat shock 70 and p - glycoprotein , and p53 mutations .
presently , systemic therapeutic options in the locally advanced or metastatic setting are limited to sorafenib , an oral multikinase inhibitor targeting raf kinase , vascular endothelial growth factor ( vegf ) , and platelet - derived growth factor ( pdgf ) receptor tyrosine kinase signaling . although the transition from normal hepatocyte to hcc is not fully understood , hepatocarcinogenesis is a complex multistep process driven by accumulation of heterogeneous molecular alterations from initial hepatocyte injury to metastatic invasion .
dysplastic nodules subsequently progress to early hcc through cumulative genetic alterations , while advanced hcc often involves intrahepatic metastasis and portal vein invasion .
molecular alterations implicated in hcc development include mutations in oncogenes and tumor suppressor genes ( p53 and p16 ) , epigenetic alterations , chromosomal changes , and aberrant activation of signaling cascades necessary for proliferation , angiogenesis , invasion and metastasis , and survival .
pathogenesis of early and advanced hcc may be modulated through different mechanisms ; for example , p53 mutations , p16 gene silencing , and aberrant akt signaling are more frequently observed in advanced hcc [ 46 ] .
the molecular pathogenesis of hcc is multifactorial and is reliant upon dysregulation of multiple pathways including wnt / b - catenin , mitogen - activated protein kinase ( mapk ) , phosphatidylinositol-3 ( pi3k)/akt / mammalian target of rapamycin ( mtor ) , vegf , pdgf , insulin - like growth factor ( igf ) , epidermal growth factor ( egf ) , tgf - beta , and hepatocyte growth factor [ 6 , 7 ] .
the hepatocyte growth factor ( hgf ) and its transmembrane tyrosine kinase receptor , cellular met ( cmet ) promote cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions .
hgf - cmet signaling is critical for normal processes such as embryogenesis , organogenesis , and postnatal tissue repair after acute injury .
hgf - cmet axis activation is also implicated in cellular invasion and metastases through induction of increased proliferation ( mitogenesis ) , migration and mobility ( motogenesis ) , three - dimensional epithelial cell organization ( morphogenesis ) , and angiogenesis .
hgf was first discovered in 1984 as a mitogenic protein for rat hepatocytes in vitro .
hgf was subsequently found to be indistinguishable from scatter factor , a fibroblast - derived motility factor promoting epithelial cell dispersal and three - dimensional branching tubulogenesis .
hgf is secreted primarily by mesenchymal cells ( or by stellate and endothelial cells in the liver ) as an inactive single - chain precursor ( pro - hgf ) which is bound to heparin proteoglycans within the extracellular matrix .
hgf transcription is upregulated by inflammatory modulators such as tumor necrosis factor alpha , il-1 , il-6 , tgf - beta , and vegf [ 11 , 12 ] .
circulating pro - hgf undergoes proteolytic conversion via extracellular proteases including hgf activator ( hgfa ) , urokinase - type plasminogen activator , factors xii and xi , matriptase , and hepsin into an active two - polypeptide chain heterodimeric linked by a disulfide bond .
hgfa is a serine protease which is secreted primarily by the liver and circulates as pro - hgfa ; pro - hgfa is activated by thrombin in response to tissue injury and malignant transformation [ 13 , 14 ] .
the active form of hgf includes an -chain containing four kringle domains ( k1 to k4 ) and an amino - terminal loop domain ( n ) , and a -chain ( c - terminal ) containing a serine protease homology ( sph ) domain .
hgf is a ligand for the met receptor , also known as cellular met or cmet .
the met protooncogene was first isolated in 1984 from a human osteosarcoma - derived cell line driven by a chromosomal rearrangement tpr - met , resulting from fusion of translocated promoter region located on chromosome 1q25 and met sequence located on chromosome 7q31 .
the tpr - met rearrangement encodes for a prototype of the cmet receptor tyrosine kinase family .
the cmet receptor is expressed predominantly on the surface of endothelial and epithelial cells of many organs , including the liver , kidney , prostate , pancreas , kidney , muscle , and bone marrow . like hgf , cmet is synthesized as an inactive single - chain precursor and undergoes proteolytic cleavage into a disulfide linked heterodimer consisting of an extracellular -chain and transmembrane -chain .
the extracellular domain includes an amino - terminal semaphorin domain , terminal cysteine - rich psi domain , and four ipt repeat immunoglobulin domains .
the cytoplasmic portion includes a juxtamembrane region including two phosphorylation sites , a tyrosine kinase ( tk ) domain and a carboxyl terminal region for substrate docking . under normal conditions , hgf interacts with cmet via a paracrine signaling loop and triggers cmet kinase activation within the cytoplasmic portion ( see figure 1 ) .
hgf contains two cmet binding sites including a high affinity constitutively active site on the -chain ( n - terminal and first kringle domain , or n - k1 ) and a low affinity site on the -chain ( c - terminal ) [ 8 , 19 ] .
the exact mechanism of cmet receptor activation and the contribution of both binding sites to receptor activation by full length hgf are not yet established , although it is evident that both - and -chain sites have distinct functions . while -chain n - k1 portion activates cmet and induces met dimerization , -chain residues bind cmet once the receptor is already occupied by hgf n - k4 and subsequently induce phosphorylation and downstream signaling .
hgf binding induces autophosphorylation of tyrosine residues y1234 and y1235 within the cmet tk domain activation loop .
phosphorylation also occurs at two sites within the carboxyl terminal region ( y1349 and y1356 ) , forming a multifunctional high affinity binding docking site that recruits a range of intracellular adaptors containing src homology-2 domains [ 2124 ] .
recruited adaptor proteins and kinase substrates include growth factor receptor - bound protein 2 ( grb2 ) , grb2-associated binder ( gab1 ) , phospholipase c- , stat3 , shc , src , shp2 , pi3k , and ship1 [ 23 , 24 ] .
these and other adaptor proteins provide scaffolding for a larger apparatus of network proteins , ultimately promoting activation of multiple signaling pathways . of these , grb2 and gab1 are critical effectors that interact directly with the receptor ; grb2 binding to the cmet docking site through y1356 results in downstream signaling via the ras / mapk pathway , while gab1 phosphorylation by met kinase activates the pi3k pathway .
other signaling proteins that are activated by cmet include p38 , jnk , and nuclear factor kb .
alterations in transcription induce cell cycle progression , antiapoptosis , increased cell motility , angiogenesis , and survival .
the hgf - cmet pathway serves as a hub for multiple heterogeneous signaling networks and is also modulated by the activation of other receptor tk families such as the epidermal growth factor receptor ( egfr ) , human epidermal growth factor receptor 2 , insulin - like growth factor 1 receptor , raf kinase , and vegf .
the hgf - cmet pathway plays an essential role in mammalian development in terms of morphogenesis , mitogenesis , and motogenesis , and angiogenesis hgf - cmet signaling is likely to be critical in many aspects of adult homeostasis including cardiac and hepatic tissue injury repair [ 26 , 27 ] , skin wound repair , and skin immunity regulation . targeted disruption of the hgf or met genes results in embryonically lethal knockouts with impaired organogenesis of the liver and placenta .
preclinical models demonstrate that hgf functions as a hepatotrophic factor enhancing hepatic regeneration and suppressing hepatocyte apoptosis [ 31 , 32 ] ; expression of hgf is increased in response to liver injury , while neutralization of endogenous hgf or met knockout facilitates liver damage and fibrotic changes with delayed repair . under normal physiologic conditions ,
hgf - induced cmet activation is strictly controlled by paracrine ligand delivery followed by ligand activation at target cell surfaces and ligand - activated receptor internationalization / degradation . despite multiple checkpoints ,
hgf - cmet axis dysregulation occurs in a variety of solid tumors and hematopoietic derived malignancies and plays a key role in malignant transformation by promoting tumor cell migration , epithelial to mesenchymal transition , invasion , proliferation , and angiogenesis .
dysregulated cmet signaling upregulates protease production ( plasminogen dependent and independent ) and increased cell dissociation via extracellular matrix degradation , facilitating tumor invasiveness and metastasis .
mechanisms of pathogenic activation include aberrant paracrine or autocrine ligand production , overexpression of hgf and cmet , upregulation of genes encoding proteases for hgf / cmet processing , constitutive kinase activation independent of cmet gene amplification , and cmet gene mutations leading to ligand - independent kinase activity [ 3436 ] .
the hgf - cmet axis is implicated in hepatocarcinogenesis through multiple mechanisms , many of which are still being elucidated .
overexpression of hgf and cmet [ 3741 ] is observed in 33% and 2048% of human hcc samples , respectively .
the prognostic utility of cmet and hgf overexpression is uncertain ; while some studies show no correlation between cmet overexpression and tumor size or invasive behavior [ 38 , 42 ] or hgf levels and survival , others demonstrate an inverse relationship with survival .
specifically , cmet overexpression was found to correlate with poorly differentiated hcc and increased intrahepatic metastases along with decreased five - year survival .
after hepatectomy , cmet overexpression in hcc tissue has been correlated with early tumor recurrence , metastasis , and shorter 5-year survival [ 41 , 45 , 46 ] .
a cmet - regulated gene expression signature was found to define a subset of human hcc with poor prognosis and aggressive phenotype and correlated with increased vascular invasion , increased microvessel density , and decreased mean survival time .
higher hgf levels negatively correlate with survival per biomarker analysis of the sharp trial and prior data and positively correlate with tumor size ; however given that hgf is secreted as an inactive precursor , overexpression alone is unlikely to guarantee pathway dysregulation .
hgf has also been investigated as a potential biomarker for hcc development , but may also be a specific marker for liver cirrhosis .
while gains in 7q have been reported in hcc , gene amplification has not been reported as a significant source of increased cmet .
crosstalk between cmet and egfr [ 55 , 56 ] and cmet and vegf signaling pathways is also implicated in promoting tumor survival .
cmet - csrc has been shown to mediate egfr phosphorylation and cell survival in the presence of egfr inhibitors .
cmet is both an independent angiogenic factor and interacts with angiogenic survival signals promoted through vegf . by upregulating hypoxia - inducible factor , hypoxia results in increased hgf expression in tumor and surrounding normal interstitial cells and
hgf - cmet signaling induces upregulation of tumoral vegf expression and endothelial vegfr2 expression and downregulation of endogenous inhibitors of angiogenesis [ 58 , 59 ] .
dual vegf and cmet axis activity demonstrates increased capillary formation in vivo , tubulogenesis in vitro , and tumoral microvessel density .
given the predominant role of dysregulated hgf - cmet signaling in hepatocarcinogenesis , pharmacologic cmet inhibition is a promising therapeutic strategy .
targets for inhibition of the cmet signal transduction pathway include ligand - receptor interaction , cmet kinase activity , and cmet and adaptor protein interaction .
hgf - cmet axis inhibitors can be broadly classified into biologic antagonists , c - met adaptor protein inhibitors , small - molecule downstream pathway inhibitors , and small synthetic met tyrosine kinase inhibitors ( tki ) . of these , tkis are the most common and the only ones to have completed phase 2 testing in hcc as of march 2013 .
table 1 shows the selected hgf - cmet inhibitors in active clinical trials for either hcc or advanced solid malignancies ( including hcc ) as of march 2013 .
a comprehensive listing of hgf - cmet inhibitors in active clinical trials for all malignancies is maintained by the bottaro nci lab and is available at https://ccrod.cancer.gov/confluence/display/ccrhgf/home .
biologic antagonists inhibit cell surface interactions such as ligand - receptor binding or receptor clustering , preventing activation of downstream signaling .
these include hgf competitive analogs , met decoy receptor , and anti - hgf - cmet monoclonal antibodies .
hgf competitive analogs compete with ligand for receptor binding without causing cmet dimerization and activation and include nk2 [ 60 , 61 ] , nk4 [ 6264 ] , and uncleavable hgf ; preliminary safety and drug development data in humans are pending .
nk2 may be the least promising hgf competitive analog due to being a potent mitogen and promoting metastases . met decoy receptors
are soluble forms of the cmet extracellular domain which compete with hgf and inhibit cmet dimerization ; in vitro and in vivo mice models demonstrate suppression of hgf - induced tumor cell migration and metastasis [ 68 , 69 ] .
monoclonal antibodies targeting hgf and the extracellular domain of cmet are currently being explored in clinical trials ( see table 1 ) , but data are not yet available for hcc . given the importance of adaptor proteins in propagating downstream cmet signaling , cmet adaptor inhibitors offer unique potential for cmet specific inhibition .
as described above , cmet signaling is initiated through autophosphorylation of cytoplasmic tyrosines that form docking sites for adaptor proteins .
grb2 and gab1 are critical effectors that interact directly with the cmet receptor , ultimately recruiting a larger apparatus of network proteins necessary for cmet signaling .
gab1 coupling with cmet requires gab1 binding to the sh3 domain of grb2 , and cmet binding to the sh2 domain of grb2 [ 72 , 73 ] .
c90 is a selective grb2 antagonist with demonstrable inhibition of gastric cancer cell motility and matrix invasion in vitro and impaired metastatic spread of human prostate cancer cells in vivo ; data in human studies have not been reported to date [ 74 , 75 ] .
small - molecule downstream pathway inhibitors directed towards inhibition of stat3 phosphorylation showed preliminary tolerability in a phase i trial of advanced solid tumors , but data are not yet available for hcc .
synthetic small met tkis inhibit downstream signal transduction by preventing phosphorylation , either via competitive binding of the intracellular adenosine triphosphate ( atp ) site in cmet 's tk domain , or noncompetitive binding of a cmet region outside of the atp binding site .
while some of the tkis in development are cmet specific , others target multiple pathways including vegf , pdgfr , fms - related tyrosine kinase 3 ( flt3 ) , v - kit feline sarcoma viral oncogene homolog protein ( kit ) , and anaplastic lymphoma kinase ( alk ) . preclinical studies and clinical trials show tolerability and efficacy of cmet tkis across a variety of solid malignancies . as of march 2013 ,
promising results in the phase 2 randomized setting for hcc are available for two cmet inhibitors : tivantinib and cabozantinib .
tivantinib ( arq 197 ) is an oral low - molecular - weight tki which is non - atp competitive .
safety and tolerability without drug - related worsening of hepatic function were previously reported in a phase ib trial of 20 cirrhotic patients ( child - pugh a and b ) with hcc , with 2 or less prior systemic chemotherapy regimens .
rimassa and colleagues reported results at asco 2012 of a phase ii trial assigning 107 patients with unresectable hcc with ecog ps 0 - 1 and child - pugh a in a 2 : 1 randomization to either second - line tivantinib or placebo with crossover allowed .
although no difference in median os occurred , the primary endpoint of time to progression ( ttp ) was met and favored tivantinib versus placebo ( 6.6 versus 6.2 months , hr = 0.90 , p = 0.63 ; 6.9 versus 6 weeks , hr = 0.64 , p = 0.04 , resp . ) .
patients with high met expression ( defined as 50% or more cells in the tumor specimen with 2 + or 3 + staining intensity ) versus low met expression receiving tivantinib demonstrated a significant improvement in both os and ttp
( 7.2 versus 3.8 months , hr 0.38 p = 0.01 ; 11.7 versus 6.8 weeks , hr 0.43 , p = 0.03 , resp . ) .
no detrimental effect was reported in patients with low met expression , and common adverse events were neutropenia , asthenia , poor appetite , and anemia .
as of november 2012 , a phase iii trial for tivantinib and hcc patients is in development .
cabozantinib ( xl 184 ) is an unselective oral multikinase tki targeting cmet , kit , rearranged during transfection ( ret ) , vegfr1 and 2 and 3 , flt3 , axl receptor tyrosine kinase ( axl ) , and tie family angiopoietin 1 receptor .
cabozantinib has shown efficacy in the phase 3 setting for medullary thyroid carcinoma with progression - free survival ( pfs ) improvement and phase 2 setting for advanced solid tumors ( including hcc ) .
verslype and colleagues reported preliminary results of a phase 2 randomized discontinuation study of cabozantinib in 41 patients with hcc , child - pugh score a , and one prior line of systemic treatment .
all patients initiated cabozantinib for a 12-week lead in time frame ; patients with partial response continued on study drug while patients with progression of disease discontinued .
32 patients with stable disease were blindly randomized 1 : 1 to continue cabozantinib or receive placebo .
the primary endpoint was overall response rate ( rr ) during the lead in time phase and pfs for patients entering the randomization phase .
independent of prior sorafenib use , median os was 15.1 months , median pfs was 4.4 months , and median time on study was 6 months .
common grade 3 adverse effects were hand - foot syndrome , diarrhea , and thrombocytopenia . met expression was not evaluated prospectively , and given the broad activity of cabozantinib , it is unclear how much activity is attributable to met inhibition alone .
in fact , the inhibition of both vegf and met concurrently may be particularly effective .
vegf inhibition leads to increased met signaling , either from resultant hypoxia or direct interactions between vegfr2 and met [ 81 , 82 ] .
preliminary evidence of efficacy against hcc was seen in a phase i combination study of tivantinib with sorafenib including a complete response and another prolonged partial response lasting greater than one year .
inc280 is a selective met tki in phase i testing for early hcc , currently accruing .
foretinib ( gsk 1363089 ) is a small - molecule tki targeting both met and vegf in phase i / ii testing for advanced hcc .
based on preclinical trials and phase 2 data for tivantinib and cabozantinib , inhibition of cmet signaling is a promising therapeutic strategy in hcc .
although it is unclear which genetic and molecular abnormalities implicated in hgf - cmet dysregulation are predictive of sensitivity to cmet targeted therapy , ongoing trials must address the challenge of identifying patients most likely to achieve maximal benefit and minimal toxicity .
two current strategies for patient selection based on tumor biomarkers are quantification of tumor cmet content via immunohistochemical ( ihc ) and immunoassay tissue analysis and assessment of met sequence status .
pharmacodynamic serum markers are under evaluation in ongoing trials as a strategy to assess clinical response .
tumoral cmet overexpression as measured by commercially available ihc kits appears to correlate with efficacy of cmet inhibitors in hcc and other solid tumors , although more prospective data are necessary for validation .
identification of cmet phosphorylation status is a potentially powerful target for targeted antibodies ; two site immunoassays of flash - frozen tissue samples yielding precise measurements of met content and phosphorylation activation are currently under development .
another promising stratification strategy is assessment of met sequence status including met mutations , met amplification , and chromosome 7 polysomy .
preclinical studies of cmet targeted agents demonstrate variable efficacy based on met mutation location ; for example , pf-2341066/4217903 is a selective inhibitor with increased activity against certain cmet atp binding site mutations in comparison to met kinase domain activation loop mutations . in vitro studies of su11274 , a small - molecule tk competitive atp binding site inhibitor , show selective inhibition of two out of four identified met mutations .
amplification of cmet is associated with increased clinical response to foretinib ( xl 880 ) in phase 2 gastric cancer data , and cmet copy number correlates with increased clinical response to tivantinib in addition to erlotinib in advanced nsclc .
ancillary pharmacodynamic and pharmacokinetic marker studies in a variety of solid tumors show variable correlation with clinical response .
preclinical data with crizotinib in gastric cancer cell lines demonstrate variable biomarker modulation of cmet inhibition with cmet dependent gastric cancer cell lines versus cmet - independent lines [ 88 , 89 ] .
clinical data shows changes in plasma concentrations of hgf , vegf , soluble met , soluble vegfr2 , pigf , and epo during treatment with various tkis [ 90 , 91 ] . the predictive utility of these biomarkers in patients with cmet - dependent hcc is unclear ; further analyses and prospective validation are necessary .
hgf - cmet is an intriguing target for the development of companion diagnostic tools as an adjunct tool for patient selection and stratification for cmet therapy . in vitro and in vivo animal studies
suggest that radiolabelled dyes containing cmet binding peptide successfully targets cmet receptors with higher imaged based tumor uptake .
a variant of su11274 with radiomethylation modification is being utilized as a pet visualization agent to quantify cmet receptor in xenograft models .
inhibition of cmet is a promising therapeutic strategy in hcc . given the heterogeneous mechanisms underlying cmet dysregulation , there is an urgent and unmet need for the development of predictive biomarkers to identify which subsets of cmet - dependent hcc tumors are most likely to benefit from specific classes of inhibitors .
as more agents move into phase 2 and 3 trials for hcc , one important consideration is the emergence of acquired and primary resistance mechanisms from de novo or preexisting mutations .
these may be overcome by rational combination therapy directed against multiple pathways , different levels of ligand - receptor - tki interaction , and the presence of cmet addiction .
innovative clinical trial designs ( such as the discontinuation cabozantinib design ) with incorporation of enriched patient cohorts , biomarker analyses , pharmacodynamic markers , and companion diagnostics are essential in moving forward . | under normal physiological conditions , the hepatocyte growth factor ( hgf ) and its receptor , the met transmembrane tyrosine kinase ( cmet ) , are involved in embryogenesis , morphogenesis , and wound healing .
the hgf - cmet axis promotes cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions .
hepatocellular cancer ( hcc ) is the third most common cause of worldwide cancer - related mortality ; advanced disease is associated with a paucity of therapeutic options and a five - year survival rate of only 10% .
dysregulation of the hgf - cmet pathway is implicated in hcc carcinogenesis and progression through activation of multiple signaling pathways ; therefore , cmet inhibition is a promising therapeutic strategy for hcc treatment .
the authors review hgf - cmet structure and function in normal tissue and in hcc , cmet inhibition in hcc , and future strategies for biomarker identification . |
depression , a severe psychiatric disorder [ 1 , 2 ] , affects up to 20% of the population in the us within their lifetime and is more prevalent in women than men [ 36 ] .
although depression has been studied for decades , its cellular and molecular mechanisms still remain largely unknown .
as many as 3040% of patients with major depressive disorder have treatment - resistant depression which does not respond to currently available antidepressant therapies .
it is therefore important to identify the mechanisms underlying depression in order to develop effective therapeutic strategies .
chronic stress , especially psychosocial stressors in humans , is one well - known risk factor for the development of depression [ 6 , 913 ] .
enhancement of neuronal plasticity is essential for adaptive intracellular changes during the normal stress response , which promotes dendritic growth , new synapse formation , and facilitates neuronal protein synthesis in the face of an acute challenge .
in addition , a successful stress response requires continuity of the response to ensure normal brain function and promote survival [ 9 , 14 , 15 ] . on the one hand ,
brief or moderate stressors actually enhance neural function in most cases , while severe or chronic stressors are detrimental and can disrupt the ability of the brain to maintain its normal stress response , eventually leading to depression [ 1518 ] .
furthermore , it has been shown that significant but brief stressful events ( acute stress ) result in the differentiation of stem cells into new nerve cells that improve the mental performance of rats .
on the other hand , chronic stress increases the levels of the stress hormone glucocorticoid and suppresses the production of new neurons in the hippocampus .
this response results in decreased dendritic spine density and synapse number and impaired memory [ 17 , 2024 ] .
the relationship between stress and psychiatric diseases has been well established for 20 years in the clinic [ 25 , 26 ] .
chronic stress paradigms in rodents , the classical animal model of depression , recapitulate many of the core behavioral features of depression and respond to antidepressant treatments [ 10 , 23 , 27 ] . however , the precise nature of relationships among the effects of chronic stress , the dysregulation of spine / synapse plasticity , and the molecular mechanisms of depression remain poorly understood .
this minireview summarizes our current understanding , obtained from animal models of chronic stress , of remodeling of dendritic spines in five regions of the brain during depression .
dendritic spines are tiny membranous protrusions from the dendritic shaft of various types of neurons .
they typically receive excitatory input from axons , although sometimes both inhibitory and excitatory connections are present on the same spine . over 90% of all excitatory synapses that occur in the cns
are localized to dendritic spines , which are cellular substrates of brain connectivity and the major sites of information processing in the brain [ 61 , 62 ] .
it is widely accepted that the regulation of dendritic spine number , size , and shape is of importance to the plasticity of synapses , as well as learning and memory [ 63 , 64 ] .
the morphology of spines is highly variable and commonly categorized into three types : thin , mushroom , and stubby ( figure 1 ) [ 65 , 66 ] .
large mushroom spines are memory spines carrying more biochemical signals [ 67 , 68 ] and a number of human disease states are associated with alterations with spine morphology and/or spine density .
spines are thin if the length is greater than the neck diameter and the diameters of the head and neck are similar ( figure 1 ) .
spines are classified as mushrooms if the diameter of the head is greater than the diameter of the neck .
spines are defined when they are no longer than three m . the length of dendritic filopodia is normally > 3 m and < 10 m .
the normal dendritic spine density ranges from 0.2 to 3.5 spines per 1 m of dendrite depending on the neuron type , age , and position along the dendrite as well as the method of counting .
thin and stubby spines , as well as dendritic filopodia , are prevalent during development .
the spine neck is an important structure for a mushroom spine to perform its normal function because the spine neck prevents ca exchange between the spine head and dendrite shaft .
this is important for the regulation of synaptic transmission and may be neuroprotective , preventing excitotoxicity to the dendrite and neuron by restricting excessive influxes of ca within the synaptic region [ 69 , 73 ] .
different spine types may serve different functions and changes in the ratio of these spines may have a greater effect on neuronal excitability and function .
it is generally accepted that thin spines are learning spines , whereas large , mature , and less motile mushroom spines harboring larger and stronger synapses are memory spines that are responsible for the maintenance of neuronal networks and long - term memory .
large mushroom spines with large heads are stable and are likely to contain smooth endoplasmic reticulum , a spine apparatus , polyribosomes , and endosomal compartments in which posttranslational modification of proteins , local protein synthesis , local recycling of receptors , and membrane management occur , respectively .
large mushroom spines that contain abundant ampa receptors are not restricted to pairing with presynaptic axonal terminals containing more synaptic vesicles .
they can also associate with presynaptic astroglial terminals , which enhance synapse formation , stabilization , and synapse elimination .
mushroom spines with small heads are motile and unstable and contribute to weak or silent synaptic connections . dendritic spine pathology is associated with many psychiatric diseases [ 71 , 7678 ] .
the formation , growth , and elimination of the dendritic spines are precisely controlled , which requires the reorganization of the neural network in response to acute stress or learning processes .
these processes are commonly dysregulated or disrupted in chronically stressed animals [ 46 , 79 ] . therefore
it is well established that depression is closely associated with selective structural changes , altered cellular resilience , and neuronal atrophy .
moreover , depression is associated with reduction in astrocytes and reduced / or increased volume of some brain regions that affect mood and cognition , which involve structural and molecular remodeling of dendritic spines in the hippocampus , prefrontal cortex , amygdala , and nucleus accumbens [ 7 , 23 , 49 , 62 , 8083 ] .
antidepressants have reversed some of these structural changes observed in animal models of depression [ 13 , 83 , 84 ] .
these studies have generated the hypothesis that alterations of the dendritic spines and the plasticity at excitatory synapses contribute to symptoms of depression [ 5 , 8588 ] .
animal models are essential tools for studying and understanding specific symptoms of human psychiatric disorders , though none of the current models fully recapitulate stress - related psychiatric disorders described in humans .
most of the current knowledge about the mechanism underlying depression has come from animal models .
several chronic stress models have been used to model depression - like behaviors in rodents such as chronic restraint stress ( crs ) , chronic unpredictable stress ( cus ) , and chronic social defeat stress ( csds ) .
behavioral tests of anhedonia ( sucrose preference ) or despair ( forced swim test and tail suspension test ) have been widely used to determine depression - like behaviors induced by these three models .
depression - like behaviors induced by these models can often be reversed by chronic antidepressant treatments [ 27 , 86 ] .
it is , however , worth noting that there are some rats or mice that do not respond to traditional antidepressants , which is similar to treatment - resistant depression in human subjects . here
crs has been used widely to study the morphological , hormonal , and behavioral alteration in several brain regions in rodents , such as the hippocampus , prefrontal cortex , amygdala , and nucleus accumbens because it is inexpensive and relatively easy to implement ( tables 14 ) . to study dendritic morphology and spine formation
, this method typically involves restraining an animal for 16 h each day in a restraint device ( bag or tube ) for a period of 1421 days or more .
a disadvantage of the crs model is the habituation of rats or mice to repeated exposure to homotypic restraint stressors ; the response of plasma corticosterone , the major glucocorticoids in rodents , to the final stressor is diminished in animals that had been stressed for 14 days [ 153156 ] .
the pattern of hypothalamic corticotrophin - releasing hormone ( crh ) heteronuclear rna and mrna responses to crs is similar to the response of corticosterone , decreasing with increasing frequency of exposure to the repeated restraint stressor .
animals habituate over time and finally show no increase in hypothalamic - pituitary - adrenal ( hpa ) axis activation and no increase in expression of hypothalamic crh [ 30 , 153 , 156 ] .
the duration of crs may differentially affect learning / memory and ca3 dendritic atrophy with shorter periods of crs ( 713 days ) serving an adaptive function to enhance learning and memory .
on the other hand , longer crs duration ( 21 days or more ) causes maladaptive changes such as dendrite atrophy , spine loss , and impaired memory [ 15 , 157 , 158 ] .
crs - induced habitation of hpa axis contrasts with the hyperactivity of the hpa axis accompanied by increased crh levels [ 43 , 159 ] and the hypersecretion of cortisol [ 160 , 161 ] in depressed patients , showing that activation of hpa axis is a hallmark of major depression [ 162 , 163 ] .
depending on duration and intensity of chronic stress , some studies report that exposure of animals to crs induces depression - like behaviors such as anhedonia ( decreased sucrose preference ) [ 164169 ] , which is a core symptom of human depression [ 10 , 27 ] .
the duration and intensity of crs as well as animal strains may determine whether crs can be used as a valid animal model of depression to produce anhedonic - like behavior .
the original , three - week chronic unpredictable severe stress ( cus ) model with diverse severe and unpredictable stressors ( electric shocks , immobilization , cold swimming , isolation housing , and other strong stimuli ) was developed by katz and coworkers [ 171 , 172 ] . in order to accurately recapitulate the human condition , willner and colleagues replaced severe stressors in katz 's model with mild stressors .
additionally , willner and colleagues augmented the cums model with a variety of mild and unpredictable stressors ( e.g. , overnight illumination ; presence of novel objects ; periods of food and/or water deprivation ; cage tilt ; change of cage mate ) . in willner 's model ,
exposure of animals to 713 mild stressors up to 3 months produced a longer lasting depression - like behavior , anhedonia [ 173175 ] . the cus model used in duman 's group
was modified from willner 's model . in duman 's model , animals were exposed to 10 [ 108 , 176 ] or 12 unpredictable stressors , 2 times per day , for up to 35 days , which produced depression - like behaviors . the duration of cus is 21 days for the experiments using cus alone or 35 days for the experiment using cus together with antidepressant treatments [ 106 , 108 , 176 ] .
it is worth noting that cus model used by duman 's group is different from the cums protocol , not only in the duration and number of stressors / day , but also at the level of stressor intensity ( rotation on a shaker 1 hour , cold 4c 1 hour , lights off for 3 hours , lights on overnight , strobe light overnight , aversive odor overnight , 45 tilted cages overnight , food and water deprivation overnight , crowded housing overnight , and isolation housing overnight ) [ 108 , 176 ] .
the modified cums model used in our laboratory consists of daily exposure of animals to 8 chronic unpredictable mild stressors , one stressor per day , for 21 days .
the same stressor is not applied in two consecutive days [ 24 , 177 ] .
the different abbreviations of chronic unpredictable mild stress ( cus , cms , or cums ) were used in several modified versions by different laboratories .
we use cums as a common denotation in this review . in comparison with the crs model ,
cums overcomes stress habituation of the hpa axis occurring during stress , in which the response of plasma corticosterone to the final stressor is still sustained in animals which had been stressed for 15 to 35 days [ 27 , 30 , 106 , 155 ] .
depression - like behaviors and deficits in synaptic plasticity are gradually developed during cums [ 24 , 173 ] .
the cums model recapitulates many of the core behavioral characteristics of human depression that are reversible by chronic treatments with traditional antidepressant agents [ 10 , 27 ] and is more relevant to human disease .
therefore , the cums model has been widely used as an animal ( specifically rat ) model of depression .
our results show that , during cums , rats require three weeks to develop depression - like behaviors accompanied by both functional changes in ca3-ca1 synapses and decreased spine density in the dendrites of ca1 and ca3 pyramidal neurons [ 24 , 177 ] .
this is in line with willner 's cums paradigm , in which animals were exposed to initial unpredictable stress for three weeks to develop depression - like behaviors prior to the onset of antidepressant treatments . because of its advantage of the gradual development of depression - like behaviors during cums [ 24 , 175 ] , this model is useful in studying depression - like behaviors such as anhedonia [ 27 , 86 , 174 , 178 ] .
in addition , this cums model is useful for inducing depression - like behaviors in female mice because chronic social defeat stress protocol can not successfully induce depression - like behaviors in c57bl/6j female mice .
a recent report shows that c57bl/6 mice , one of the most widely used mouse strains , are resistant to the commonly used cums protocol due to the variety of genetically modified lines .
a recently revised , eight - week cums protocol has been developed and used to induce depression - like behaviors in c57bl/6 mice .
interestingly , male and female rodents are differentially affected by cums , depending on the behavioral and neurobiological markers that are being measured .
csds is one of the most frequently used rodent models for depression and has been used to induce depression - like behaviors in mice such as social avoidance and anhedonia [ 86 , 144 , 182185 ] . during each defeat period , an intruder , a male c57bl/6j mouse ,
is allowed to interact for 10 minutes with an aggressive and large cd1 mouse during which the intruder is rapidly investigated , attacked , and defeated by the resident cd-1 mouse .
the experimental c57bl/6j mice are exposed to a different resident aggressor for 10 minutes each day for 10 consecutive days [ 183 , 184 , 186188 ] .
on the one hand , after completing the social defeats , 30% of animals do not show depression - like behaviors known as resilient , a positive adaptation in the face of stress , threat , or severe adversity [ 189 , 190 ] . on the other hand ,
a disadvantage of this model is that it is limited in studying only male mice because female c57bl/6j mice are not easily defeated by cd-1 mice .
this model has been widely used to induce depression - like behaviors and study the molecular mechanisms underlying depression [ 139 , 141 , 142 , 146 , 149 , 191 , 192 ] .
this model is also used to induce depression - like behaviors in rats [ 192 , 193 ] .
the hippocampus plays an important role in learning and memory and is particularly sensitive to stress and glucocorticoids [ 194 , 195 ] .
rodent hippocampus contains high levels of glucocorticoid receptors ( grs ) and mineralocorticoid - like receptors ( mrs ) .
the affinity of mr for corticosterone is 6- to 10-fold higher than that of gr , but it is gr that is activated after stress and is involved in its feedback action on stress - induced neural plasticity .
chronic stress decreases gr expression or its numbers and finally alters the balance of gr / mr in the male hippocampus [ 197 , 198 ] , which is thought to be a protective mechanism against the damaging effects of chronic stress .
chronic exposure of male rats to glucocorticoids induces depression - like behaviors and causes the synaptic deficits in the hippocampus .
a recent report shows that grs , acting via mr , decrease resilience to stress via downregulation of mglu2 receptors in mice during cums .
chronic stress and glucocorticoids impair hippocampal function , which in turn contributes to the hpa axis dysregulation [ 195 , 198 ] .
the blunting of the feedback mechanism is believed to underlie sustained high levels of glucocorticoids in some depressed patients .
people with depression have a significantly smaller hippocampus than healthy individuals [ 200205 ] , which may result from a decrease in dendritic arbors and spine density in hippocampal neurons .
many structural and functional studies show that dendritic retraction or atrophy , characterized by both reduction in total dendritic length and a simplification of dendritic arbors , is found in the dendrites of ca3 pyramidal neurons but not the dendrites of ca1 pyramidal neurons in response to cums or crs [ 37 , 38 , 41 , 42 ] ( table 1 )
the different sensitivity of ca1 and ca3 to chronic stress may result from the differences between these two regions in afferents / efferents , the levels of grs , nmda receptors , 5-ht receptors , and gaba inhibitory tones [ 207211 ] .
gr levels are higher in the ca1 region than the ca3 region , where the receptors are activated by stress hormone corticosteroids [ 209 , 212 ] .
in addition , it has been repeatedly shown that apical dendrites of ca3 pyramidal neurons are more susceptible to the effects of sustained crs than ca3 basal dendrites .
dendritic retraction in apical but not basal dendrites of ca3 pyramidal neurons is found after cums , chronic psychosocial stress [ 56 , 57 ] , and crs [ 28 , 30 , 31 , 33 , 37 , 38 , 41 , 42 , 44 , 49 , 56 , 213 , 214 ] .
crs - induced depression - like behaviors and ca3 dendritic atrophy are not permanent but recovered to control levels after certain stress - free period following the end of crs procedure [ 33 , 49 , 158 , 213 , 215 ] .
importantly , ca3 dendritic retraction induced by crs requires corticosterone secretion and intact nmdar function .
treatments of chronically stressed rats with either the steroid synthesis blocker cyanoketone or competitive nmda receptor antagonist ( cgp 43487 ) blocked crs - induced dendritic retraction .
similar to cums , rats usually require three weeks to develop depression - like behaviors and ca3 apical dendritic atrophy because only 21 days , but not 7 to 13 days of crs , induces reversible impairments of spatial memory performance and ca3 apical dendritic atrophy [ 157 , 158 ] .
in addition , atrophy of apical dendrites , but not basal dendrites of ca3 pyramidal neurons , is found after chronic exposure to elevated glucocorticoid levels , which mimics chronic stress .
chronic stress - induced hippocampal ca3 dendritic retraction and elevated glucocorticoid release contribute to impaired spatial memory .
chronic stress - induced alterations of spine density in ca3 pyramidal neurons depend on stressor types , animal species , sex , and the duration of stress .
crs causes either a decrease [ 30 , 33 , 36 , 42 , 44 ] , an increase [ 29 , 34 ] , or no change in the spine density in the dendrites of male rat ca3 pyramidal neurons .
crs - induced loss of synapses in male rat ca3 apical dendrites can be recovered following water maze training [ 34 , 36 ] .
one report shows that crs causes a decrease in dendritic spine density , especially in thin and mushroom spines in mouse ca1 pyramidal neurons , but does not affect total spine density in mouse ca3 pyramidal neurons , due to increased stubby spine density and decreased thin and mushroom spine density .
the degree of stress - induced spine loss in ca3 pyramidal neurons correlates significantly with the memory defects and loss of ltp in mice . in comparison with crs , both 21-day cums and 30-day cums decrease spine density in male rat ca3 pyramidal neurons [ 24 , 49 ] , whereas 14-day cums increases spine density in male mouse ca3 pyramidal neurons , which is consistent with our report that two - week cums enhances ltp induction in ca3-ca1 synapses in male rat hippocampus .
psychosocial stress ( 1 h / day for 28 days ) does not affect spine density in ca3 pyramidal neurons of male tree shrews .
ca1 is a hippocampal region crucial for long - term memory . in comparison with ca3 pyramidal neurons ,
chronic stress - induced changes in spine density in ca1 pyramidal neurons are less characterized .
acute stress ( 30 , 1 sec , 1 ma , 60 hz shocks to the tail ) increases spine density in the apical dendrites of male hippocampal ca1 pyramidal neurons but decreases it in the same area of female hippocampus . these increases and decreases in spine density are dependent on nmda receptor activation .
similar to acute stress , the same crs regimen causes a decrease in spine density in the apical dendrites of hippocampal ca1 pyramidal neurons in male rat and male mouse [ 40 , 43 , 46 , 48 ] but causes an increase in spine density in the same region in female rats [ 37 , 38 , 42 ] .
one recent study shows that crs decreases spine density in basal dendrites , while it increases apical dendritic arbors in the ca1 pyramidal neurons of the ventral hippocampus in female but not in male rats .
in contrast to female rats , female mice show a decrease in spine density in ca1 pyramidal after exposure to same 21-day crs .
additionally , an ultrastructural study of ca1 synapses shows that 21-day crs causes an increase in the size of the postsynaptic density in male rat ca1 .
similar to crs , cums also causes a decrease in spine density in the dendrites of ca1 pyramidal neurons in male rat .
stress - induced increase in spine density in the apical dendrites of ca1 pyramidal neurons in female rat and same stress - induced decrease in spine density in the same area in male rat are completely prevented by nmda receptor antagonist cpp [ 35 , 219 ] , but exposure of nmda receptor antagonist cpp to the stress procedure does not affect corticosterone levels or the corticosterone response to stress , suggesting a key role of nmda receptor activation in stress - induced increases or decreases in spine density .
similar to sex - dependent alterations of dendritic spines induced by both acute stress and crs in hippocampal ca1 pyramidal neurons , there is a sex difference in crs - induced changes in hippocampal - dependent spatial learning and memory .
crs impairs spatial learning and memory in males but not in females [ 38 , 197 ] .
furthermore , recent studies suggest that cums - induced glutamatergic dysfunction in excitatory temporoammonic- ( ta- ) ca1 synapses of the hippocampus serves as an underlying cause of depression [ 53 , 220 ] .
this suggests that restoring spine loss or excitatory synaptic dysfunction in the hippocampus could be a novel therapeutic target for depression .
similar to cums , chronic exposure of male rats to corticosterone for 3 - 4 weeks induces depression - like behaviors and causes a decrease in ampar - mediated excitation at temporoammonic - ca1 synapses accompanied by decreased expression of glur1 protein .
blocking cums - induced increase of corticosterone during cums with the corticosterone synthesis inhibitor metyrapone prevents stress - induced depression - like behaviors .
similar to male rats , exposure of male mice to 35-day corticosterone treatments shows anxiety / depression - like behaviors , accompanied by a reduction in spine density , mainly in thin and stubby spines but not in mushroom spines in ca1 pyramidal neurons .
chronic corticosterone - induced decreases in spine density in the hippocampal ca1 pyramidal neurons and depression - like behaviors recover to normal levels concomitantly after 25-day treatment with fluoxetine .
these studies suggest that corticosterone secreted during stress plays a key role in chronic stress - induced depression - like behaviors , dysfunction of excitatory synapses , and alteration of dendritic spines in the hippocampus ; rescuing chronic stress - induced loss of dendritic spines and/or synaptic dysfunction may rescue depression - like behaviors . the medial pfc ( mpfc ) , an information processing center ,
is often divided into the anterior cingulate , prelimbic ( pl ) , and infralimbic ( il ) subregions .
the mpfc plays a critical role in the integration of cognitive and emotionally relevant information , modulation of subcortical systems , and attention [ 222225 ] .
the mpfc expressing high levels of glucocorticoid receptors is a target site for glucocorticoids and plays an important role in the regulation of the response of hpa axis to stress and antidepressant response [ 225 , 227 , 228 ] .
it is widely reported that the mpfc volume is decreased in a subset of depressed patients [ 201 , 205 , 229233 ] . however
, a recent report shows that the decreased volume of the mpfc is found in male but not in female depressed patients .
the decreased volume of the mpfc in depressed patients [ 201 , 205 , 229 , 230 ] is in line with decreased expression of synaptic - function - related genes and loss of synapses in the mpfc of subjects with major depression disorder .
in addition , glial cell loss , reductions in the density and size of neurons in the postmortem mpfc of subjects with major depression , may contribute to pathology of depression [ 235 , 236 ] .
animal studies show that the retraction of apical dendrites of pyramidal neurons in the mpfc induced by chronic stress is accompanied by alterations in fear conditioning and extinction .
crs - induced dendritic retraction and spine loss in the hippocampal and mpfc neurons are accompanied by cognitive impairments , which are mediated by each respective structural alteration [ 92 , 109 , 217 ] .
it is well documented that crs results in a retraction of the distal part of apical dendritic arbors of layers ii / iii pyramidal cells [ 76 , 89 , 90 , 92 ] and a decrease in spine density on those neurons [ 91 , 93 , 96 , 237 ] in the mpfc of male rats , which is similar to that found in hippocampal ca3 region [ 41 , 42 , 44 ] .
the pattern of crs - induced dendritic reorganization is similar to that seen after daily corticosterone injections [ 238 , 239 ] .
crs also alters spine morphology with an overall decrease in mean dendritic spine volume and surface area , a reduction in large mushroom spine density , and an increase in small thin spine density in the mpfc of male rats .
one conflicting study , however , reports that crs - induced decrease in spine density in the male rat mpfc is characterized by a decrease in thin and stubby spine density without affecting mushroom spine density .
crs causes a reduction of length and branch number in the apical dendrites of the neurons in the mpfc of young ( 3 months ) and aged ( 20 months ) male rats .
surprisingly , crs - induced retraction of apical dendrites , however , is reversed with recovery in young ( 3 months ) but not aged ( 20 months ) animals . in young rats ,
crs results in dendritic spine loss and alters the patterns of spine morphology . in contrast , crs does not affect spine density and spine shape in aged animals , showing that dendritic spines become progressively less plastic in the aging brain .
interestingly , chronic immobilization stress does not affect spine density in a subpopulation of il neurons in the mpfc that project to the basolateral amygdala ( bla ) in male rats , suggesting this pathway may be particularly resilient against the effects of stress .
randomly selected neurons in the il of the mpfc , however , show dendritic retraction after crs .
since most layer ii / iii neurons project intracortically , the majority of randomly selected pyramidal neurons may be local cortical neurons with no projections to the bla .
an independent study reports that il neurons , but not pl neurons , in the mpfc are highly sensitive to a brief exposure to stress and the same form of stress impairs fear extinction in mice .
however , these il neurons are putative local cortical neurons without projections to the bla .
a conflicting report shows that crs causes dendritic retraction in pl neurons of rat mpfc , while this dendritic retraction is prevented by the d1r antagonist sch23390 , and the same d1r antagonist causes dendritic retraction in the pl neurons of the mpfc in unstressed rats .
however , the effects of crs on dendrites in the il neurons of mpfc are not studied in this report .
these results show that dopaminergic transmission in the pl neurons of the mpfc during stress may contribute directly to the crs - induced retraction of apical dendrites , while dopamine transmission in the absence of stress is important in maintaining normal dendritic morphology .
recent reports show that acute foot - shock stress not only produces an increase in the number of excitatory synapses and docked vesicles in the mpfc , but also induces rapid and sustained increases in spine density accompanied by atrophy of apical dendrites in the pl neurons of the mpfc .
importantly , these synaptic changes induced by acute stress are prevented by chronic antidepressant desipramine treatments [ 240 , 241 ] .
optogenetic activation of the mpfc exerts potent antidepressant - like effects , showing that the activity of the mpfc may play a key role in the development of depression - like behaviors and antidepressant responses .
similar to hippocampus , alteration of stress - mediated dendritic arbors in the mpfc is sex dependent .
crs causes retraction of apical dendrite arbors in the mpfc in male , while it increases apical dendrite arbors in the female mpfc in which crs - induced dendritic plasticity is estrogen dependent .
rat mpfc is sexually dimorphic , which is characterized by a bigger and more complex apical dendritic tree in the pl neurons of the mpfc in healthy male rats than that in healthy female rats [ 243 , 244 ] .
the ofc , a part of the pfc in the frontal lobes in the brain , is involved in cognitive functions , decision - making , and emotional processing .
the studies from neuroimaging and neuropathology show that the ofc is involved in pathophysiology of major depression .
decreases in cortical thickness , neuronal size , neuronal density , and glia densities in the ii
the decrease in neuronal sizes in layer 3 of the ofc from depressed subjects is confirmed by another postmortem study .
neuroimaging and functional studies also show that patients with major depression have reduced ofc volume and reduced density of pyramidal neurons in layers v and iii of the ofc . in contrast , animal studies show that 3-week cums increases both the volume of layers ii / iii in the lateral orbital subregion and the volume of layer ii in the ventral orbital subregion of the ofc , which is accompanied by an increase in the length of apical dendrites in the ventral orbital subregion of the ofc .
interestingly , crs causes a 43% increase in the dendritic arbors in the ofc neurons , an effect opposite to what is observed in the mpfc neurons where the same crs causes 20% retraction of apical dendritic arbors in layer ii / iii pyramidal neurons of the mpfc .
further studies are needed to explore the discrepancy between the data from imaging analysis or postmortem studies and the findings from animal models .
our recent study showed that 3-week cums caused a decrease in spine density in the ofc pyramidal neurons , which was accompanied by both depression - like behaviors and decreased expression of kalirin-7 and psd95 in the ofc ( chang xu , shu - chen an , and xin - ming ma , unpublished ) .
kalirin-7 plays an essential role in maintaining dendritic spine density , size , and synaptic functions [ 250 , 251 ] .
similar to cums , chronic exposure of male mice to corticosterone for 20 days that recapitulates blood corticosterone levels found after crs exposure in mice also decreases spine density in the ofc neurons , which fails to recover after one week of washout period .
this suggests that chronic stress - induced decrease in spine density is not reversible in the ofc neurons .
the amygdala , a structure within the subcortical limbic system , is involved in the processing of emotion and motivation such as fear and anger .
the amygdala is also responsible for determining what memories are stored and where they are stored .
imaging studies show an increase [ 253255 ] or decrease [ 256 , 257 ] or no change in amygdala volume or increased activity of amygdala [ 201 , 259 , 260 ] in patients with major depression .
a conflicting mri study reports a trend towards smaller left amygdala volumes in depressed patients compared with healthy controls .
a postmortem study shows that depressed subjects have a larger lateral nucleus and a greater number of total bla neurovascular cells than controls .
there are no differences in the number or density of neurons or glia between depressed and control subjects .
to our knowledge , it is not clear whether cell size in bla is altered in depressed patients .
animal studies show that chronic stress generally results in an increase in spine density and enhanced dendritic arborization in the amygdala ( table 3 ) .
this is in contrast to the hippocampus and pfc ( tables 1 and 2 ) .
acute immobilization also causes an increase in spine density without any effects on dendritic arbors in bla spiny neurons , showing that these neurons are very sensitive to stress .
chronic stress causes an increase in dendritic arborization and spine density in the bla spiny neurons of male rats [ 122 , 123 , 125129 ] and male mice [ 114 , 118 , 120 , 124 ] .
in contrast , crs causes a decrease in spine density in spiny neurons in the medial amygdale , which are gabaergic neurons .
crs - induced increase in dendritic arbors and spine density in the bla pyramidal neurons and crs - induced depression - like behavior in wild - type mice are absent in fatty acid amide hydrolase ( faah ) deficient mice suggesting a key role of faah in maintaining normal amygdala function in the face of chronic stress .
chronic immobilization stress - induced dendritic hypertrophy in the bla spiny neurons is not reversible .
this is distinct from hippocampal ca3 and mpfc atrophy , which is reversible within the same period of stress - free recovery .
a single dose of corticosterone induces dendritic hypertrophy in the bla accompanied by enhanced anxiety .
chronic exposure of mice to corticosterone for 20 days mimicking chronic stress increases dendritic length and spine density in the bla .
chronic exposure of rats to corticosterone for 2 weeks causes an increase in the levels of memory - related genes including arc / arg3.1 and egr-1 and enhances the consolidation of fear memory processes in the lateral amygdala .
in addition , tianeptine , an antidepressant , exerts the opposite roles in chronic stress - induced synaptic changes in the amygdala and hippocampus .
animal studies indicate that the neuronal circuitry of the pfc - nac - ventral tegmental area ( vta ) underlies drug reward responses and contributes to relapse to cocaine seeking [ 263 , 264 ] .
excitatory axonal terminals from glutamatergic neurons of the pfc form the synapse onto nac medium spiny neurons ( msns ) , which also receive dopaminergic ( da ) inputs from the vta .
the vta receives gabaergic inputs from the nac and glutamatergic inputs from the pfc [ 265 , 266 ] .
in addition , the nac also receives glutamatergic inputs from ventral hippocampus and basolateral amygdala .
the nac serves as a hub of the brain 's reward pathways and plays a central role in mood and emotion regulation .
depressive symptoms , such as anhedonia , and depression severity are correlated with reduced nac volume and reduced nac responses to rewards in depressed patients [ 205 , 269 ] .
an optogenetic study shows that inhibition of the vta - nac projection induces resilience , whereas inhibition of the vta - mpfc projection enhances susceptibility , highlighting a key role of pfc - nac - vta circuitry in the development of depression .
therefore , dysregulation of pfc - nac - vta reward circuitry may contribute to the pathophysiology of depression [ 13 , 146 , 271 ] .
similar to the effect of cocaine abuse , chronic stress may alter dendritic spines and synaptic plasticity in the pfc - nac - vta circuitry . a recent study , however , reports that chronic social defeat stress- ( csds- ) mediated increase in glutamatergic transmission at the intralaminar thalamus- ( ilt- ) nac but not pfc - nac circuitry mediates stress - induced postsynaptic plasticity on the msns and depression - like behaviors in susceptible mice .
the msns of dorsal striatum receive not only glutamatergic inputs from the cerebral cortex and the thalamus , but also da innervation from the midbrain .
these msns account for > 95% of the neurons in the striatum [ 273 , 274 ] .
the dorsal striatum and the nac are not distinguishable in their populations and expression of da receptors ( drs , d1r and d2r ) . approximately half of the striatal msns express the d1r [ 274 , 275 ] ; other half msns express the d2r [ 276 , 277 ] .
the degree of d1r / d2r colocalization remains controversial , ranging from 10% to 30% [ 275 , 278 , 279 ] .
d1r signaling enhances dendritic excitability and glutamatergic signaling in striatonigral msns , while d2r signaling exerts the opposite effect in striatopallidal msns ( indirect pathway ) [ 280282 ] .
crs causes a decrease in ampar / nmdar ratio in the d1r - msn of the nac compared to nonstressed control , while it does not affect ampar / nmdar ratios in d2r - msns of the nac .
this crs - induced decrease in the ratio of ampar / nmdar in the d1r - msn is accompanied by depression - like behaviors , showing a role of nac d1r - msns , at least in part , in the development of depression .
one report shows that enhanced activity in d1r - msns causes resilient behaviors , while inhibition of these d1r - msns induces depression - like behaviors after csds .
another report shows that csds specifically results in an increase in synaptic strength represented by the increased amplitude of uepscs ( unitary excitatory postsynaptic currents ) in large mushroom spines on d1r - msns but decreases synaptic strength on d2r - msns mushroom spines in the nac of resilient mice .
csds does not affect the uepsc amplitude in small thin spines on both d1r- and d2r - nac msns in resilient mice .
csds , however , does not alter synaptic strength in mushroom and thin spines on d1r- or d2r - msns in the nac in susceptible mice .
these data show that the nac d1r - msn of susceptible mice may be resistant to adaptation and play a critical role in the development of chronic stress - induced depression - like behaviors .
in addition , the inhibitor of kappab kinase ( ik ) in the nac is also a critical regulator of depression - like behavior , and the ik - nuclear factor kappab ( nfb ) plays a key role in the regulation of synaptic signaling and neuronal morphology in vitro and in vivo .
csds - induced increase in ik activity in the nac enhances social avoidance behavior and promotes the formation of thin spines .
inhibition of ik signaling results in a reversal of csds - induced social avoidance behaviors , suggesting that csds - induced depression - like behaviors are associated with ik - mediated increase in thin spine density in the nac .
interestingly , csds - induced increases in stubby spine density and the frequency of mepscs in the nac in susceptible mice are accompanied by an increase in the levels of ik in the nac .
these results show that csds - induced increases in stubby spine density and ik expression in the nac are correlated with depression - like behaviors .
csds - mediated downregulation of rac1 through an epigenetic mechanism contributes to depression - like behaviors and enhanced formation of stubby spines in the nac msns of susceptible mice .
furthermore , deltafosb , a transcription factor , plays an essential role in the mechanism of resilience in mice , supported by evidence that csds - mediated induction of deltafosb in the nac is not only necessary and sufficient for resilience in mice , but also required for the antidepressant fluoxetine to reverse depression - like behaviors induced by csds .
nr2b in the nac plays a key role in the modulation of csds - induced depression - like behaviors and synaptic plasticity .
csds - induced reduction in nr2b surface expression in the mouse nac neurons is restored by fluoxetine treatment .
behaviorally , restoration of nr2b loss prevents the behavioral sensitization of mice to chronic stress .
overexpression of dna methyltransferase ( dnmt3a ) increases dendritic spine density in the nac msns .
csds - induced depression - like behaviors are accompanied by an increase in the dnmt3a levels in the nac , suggesting that csds - induced depression - like behaviors are positively correlated with increased spine density in the nac neurons .
these studies highlight an important role of the nac in chronic stress - induced depression - like behaviors .
it is possible that stress may differently affect dendritic spines in the d1r - msns and d2r - msns of the nac .
more studies are required for a better understanding of the roles of d1r - msns and d2r - msns in chronic stress - induced depression - like behaviors and the underlying mechanisms . reduced nac volume in depressed patients [ 205 , 269 ] is not in line with the findings from animal models in which stress generally results in an increase in spine density in the nac msns .
csds causes an increase in spine density and the frequency of mepscs in the mouse nac msns .
in addition , the shell of the nac is thought to be a part of the extended amygdala .
chronic stress increases spine density in the neurons of the bla and the shell of nac even though these two neuron types are naturally different .
the downstream mechanisms of chronic stress - induced spine formation in these two distinct neuron types are not clear . taken together , these data show that altered spine density and synaptic plasticity in the nac msns are correlated with depression - like behaviors induced by chronic stress , which may be a target for developing the novel treatment strategies for depression .
the molecular mechanisms underlying spine loss and dendritic retraction induced by chronic stress in the hippocampus and pfc as well as enhanced spine formation found in the amygdala and nac in chronically stressed animals are not well understood .
expression of several synapse - related genes is decreased in the postmortem pfc of subjects with major depressive disorder .
one of these genes is gata1 ( gata - binding factor 1 ) , a transcriptional repressor that plays a key role in the formation of dendritic spines and dendrite arbor maintenance .
furthermore , a nuclear pore complex protein , nucleoporin p62 ( nup62 ) , and tyrosine phosphorylation of nup62 play a critical role in crs - induced dendritic retraction of hippocampal ca3 pyramidal neurons .
many synaptic proteins including kalirin-7 , spinophilin , homer1 , cofilin , rac-1 , cadherin , p - akt , p - gsk-3 , p - erk1/2 , pkc , ncam , psa - ncam , snap-25 , snap-29 , vamp1/2 , syntaxin 1a , synaptophysin , synapsin 1 , vglut2 , glur1 , glur2 , nr1 , nr2a , nr2b , psd95 , camkii , melanocortin 4 receptors , crh receptor 1 , and p190rhogap play an important role in the regulation of the spine formation and/or synaptic plasticity ; expression of these synaptic proteins in the brain is altered by chronic stress , and these proteins may play a key role in chronic stress - induced both depression - like behaviors and spine alterations ( table 14 ) [ 24 , 40 , 44 , 46 , 53 , 100 , 102 , 113 , 130 , 141 , 142 , 144 , 286292 ] .
in addition , chronic stress - induced alterations of several signal transduction pathways including camp - pka - creb , camp - erk1/2-creb , camp - pka , ras - erk , pi3k - akt , tnf-nfb , gsk-3 , mtor , and creb may be also associated with chronic stress - induced spine loss or increase in certain brain areas [ 7 , 22 , 293 ] .
a recent report shows that the homer1/mglur5 complex is involved in the development of csds - induced depression - like behaviors , suggesting a role of this complex in chronic stress - mediated spine plasticity .
presynaptic mglu2 receptors play a key role in cums - induced depression - like behaviors in male susceptible mice .
the rapid antidepressant - like properties of ketamine , an nmda receptor antagonist , result from increased synaptic signaling proteins and increased number and function of new spine synapses via activating the mammalian target of rapamycin ( mtor ) pathway in the rat mpfc and hippocampus [ 295298 ] .
s6k1 , a key mediator of activity - dependent synaptic protein synthesis , is the downstream of mtorc1 and plays a critical role in cums - induced depression - like behaviors .
postmortem studies show that the levels of nr2a , nr2b , mglur5 , psd-95 , and mtor as well as the levels of s6k , eif4b , and p - eif4b , the core downstream signaling targets of mtor , are decreased in the pfc of depressed patients .
these studies suggest that mtor signaling is a promising target for the development of novel antidepressant drugs [ 297 , 301 , 302 ] . taken together
, understanding chronic stress- and/or depression - induced alterations in dendritic spines , synapse plasticity , synaptic proteins , and their upstream / downstream signaling pathways may pave the path for developing efficiency therapeutic strategies for depression .
the search for the mechanisms through which chronic stress alters dendritic spines or synapse numbers in different brain regions should be a major future direction . | depression , a severe psychiatric disorder , has been studied for decades , but the underlying mechanisms still remain largely unknown . depression is closely associated with alterations in dendritic spine morphology and spine density . therefore , understanding dendritic spines is vital for uncovering the mechanisms underlying depression .
several chronic stress models , including chronic restraint stress ( crs ) , chronic unpredictable mild stress ( cums ) , and chronic social defeat stress ( csds ) , have been used to recapitulate depression - like behaviors in rodents and study the underlying mechanisms . in comparison with crs ,
cums overcomes the stress habituation and has been widely used to model depression - like behaviors .
csds is one of the most frequently used models for depression , but it is limited to the study of male mice .
generally , chronic stress causes dendritic atrophy and spine loss in the neurons of the hippocampus and prefrontal cortex .
meanwhile , neurons of the amygdala and nucleus accumbens exhibit an increase in spine density .
these alterations induced by chronic stress are often accompanied by depression - like behaviors .
however , the underlying mechanisms are poorly understood .
this review summarizes our current understanding of the chronic stress - induced remodeling of dendritic spines in the hippocampus , prefrontal cortex , orbitofrontal cortex , amygdala , and nucleus accumbens and also discusses the putative underlying mechanisms . |
the calcifying epithelial odontogenic tumor ( ceot ) is also known as pindborg tumor . the dutch pathologist , dr .
jens jorgen pindborg , described and designated ceot as a distinct pathologic entity in 1955 .
it is a benign , locally invasive neoplasm that constitutes about 1% of all odontogenic neoplasms .
ceot usually presents in patients aged between 30 and 50 years and both sexes are equally affected .
molar and premolar regions of the posterior mandible are the most common sites of intraosseous occurrence .
ten percent of ceots occur extraosseously , with a predilection to the anterior gingival region .
more than half ( 53% ) of these tumors are associated with an unerupted teeth , most commonly , mandibular molars .
clear cell , noncalcifying , bone and cementum forming and langerhans cell variants have been reported .
pigmented variants of odontogenic tumors are extremely rare and only 47 cases have been reported , of which 20 cases were reported in calcifying cystic odontogenic tumors ( ccot ) and one case was described as unclassifiable . however , no definitive case of pigmented ceot has been reported in literature so far .
the histogenesis of pindborg tumor has not been established conclusively ; however , most authors accept that intraosseous tumors arise from the stratum intermedium of the enamel organ whereas extraosseous tumors originate from the epithelial rests of the dental lamina or from the basal cells of gingiva .
tumors which arise in the maxilla tend to be more locally aggressive than those which arise in the mandible .
a 28-year - old female patient visited the dental outpatient department of our institute with a complaint of swelling in the left maxillary region for the past 6 months .
intraoral examination revealed a 3.5 cm 3 cm swelling , extending from the left molar to the left incisor region .
radiograph revealed a mixed pattern revealing a radiolucent mass with scattered radiopaque areas associated with an unerupted tooth fragment .
computerized tomography ( ct ) scan revealed a heterogeneous soft - tissue mass , epicentering the alveolar margin of the left maxilla around the root of the first molar tooth and measuring 3.6 cm 3.3 cm 3.2 cm , causing expansion with resultant thinning of the bony wall and a thin rim of calcification in the periphery [ figure 1a and b ] .
an eccentrically located small , unerupted tooth fragment measuring 8 mm 6 mm 6 mm was also identified [ figure 1a ] .
paraffin - embedded tissues were cut into 4-mm sections , stained with hematoxylin and eosin ( h and e ) , congo red and masson - fontana .
immunohistochemistry with monoclonal mouse anti - s100 antibody ( dako carpenteria , ca , usa ) and monoclonal mouse anti - melanoma ( hmb45 ) antibody ( biogenex , san ramon , ca , usa ) was performed .
( a ) axial section reveals a heterogeneous soft - tissue density lesion ( black arrows ) , filling the left maxillary antrum .
( b ) axial section shows the lesion ( black arrows ) in the left maxillary antrum , causing expansion with resultant thinning of the bony wall and thin rim of calcification in the periphery microscopic examination of h and e - stained sections demonstrated a tumor composed of epithelial cells arranged in sheets , nests , plexiform and occasional pseudoglandular pattern .
these epithelial cells were polyhedral with abundant dark staining eosinophilic cytoplasm , large vesicular nuclei with smooth nuclear margins and fine chromatin , separated by loose connective tissue stroma [ figure 2a ] .
epithelial cells were admixed with cells containing abundant brownish black pigment [ figure 2b ] .
homogeneous eosinophilic material , which is pathognomonic of ceot , was found in the intercellular areas [ figure 2c and f ] .
numerous foci of calcification and calcific spherules forming liesegang rings [ figure 2d and g ] , ossification [ figure 2e ] and acanthomatous areas were also observed .
homogeneous eosinophilic material stained positive with congo red and therefore is an amyloid - like material [ figure 3a ] .
melanin bleach using potassium permanganate and masson - fontana staining [ figure 3b ] demonstrated that the pigmented cells contained melanin .
immunohistochemistry revealed strong positivity for s100 [ figure 3c ] and hmb45 [ figure 3d ] in all pigment - containing cells .
( a ) polyhedral epithelial cells in sheets , plexiform and occasional pseudoglandular arrangement having abundant dark staining eosinophilic cytoplasm are separated by loose connective tissue stroma ( h&e , 100 ) .
( b ) abundant brownish black pigment containing cells admixed among tumor cells ( h&e , 200 ) . ( c ) tumor cells with intercellular areas showing homogeneous eosinophilic material undergoing calcification ( h&e , 100 ) . ( d ) foci of calcific spherules forming liesegang rings ( h&e , 40 ) .
( f ) polyhedral cells with acellular material undergoing calcification ( h&e , 100 ) globular calcifications with onion skinning ( liesegang rings ) ( h&e , 40 ) special stains and immunohistochemistry . (
a ) masson - fontanna stain showing strong positivity in the pigments , demonstrating the presence of melanin ( masson - fontanna stain , 100 ) .
( b ) congo red stain showing positivity in the homogeneous eosinophilic amyloid - like material ( congo red stain , 100 ) .
( c ) anti - s100 antibody showing positivity in round and spindled pigment - containing cells ( ihc stain , 200 ) .
( d ) anti - hmb45 antibodies showing positivity in pigment - containing cells ( ihc stain , 200 )
microscopic examination of h and e - stained sections demonstrated a tumor composed of epithelial cells arranged in sheets , nests , plexiform and occasional pseudoglandular pattern .
these epithelial cells were polyhedral with abundant dark staining eosinophilic cytoplasm , large vesicular nuclei with smooth nuclear margins and fine chromatin , separated by loose connective tissue stroma [ figure 2a ] .
epithelial cells were admixed with cells containing abundant brownish black pigment [ figure 2b ] .
homogeneous eosinophilic material , which is pathognomonic of ceot , was found in the intercellular areas [ figure 2c and f ] .
numerous foci of calcification and calcific spherules forming liesegang rings [ figure 2d and g ] , ossification [ figure 2e ] and acanthomatous areas were also observed .
homogeneous eosinophilic material stained positive with congo red and therefore is an amyloid - like material [ figure 3a ] .
melanin bleach using potassium permanganate and masson - fontana staining [ figure 3b ] demonstrated that the pigmented cells contained melanin .
immunohistochemistry revealed strong positivity for s100 [ figure 3c ] and hmb45 [ figure 3d ] in all pigment - containing cells .
( a ) polyhedral epithelial cells in sheets , plexiform and occasional pseudoglandular arrangement having abundant dark staining eosinophilic cytoplasm are separated by loose connective tissue stroma ( h&e , 100 ) .
( b ) abundant brownish black pigment containing cells admixed among tumor cells ( h&e , 200 ) . ( c ) tumor cells with intercellular areas showing homogeneous eosinophilic material undergoing calcification ( h&e , 100 ) . ( d ) foci of calcific spherules forming liesegang rings ( h&e , 40 ) .
( f ) polyhedral cells with acellular material undergoing calcification ( h&e , 100 ) globular calcifications with onion skinning ( liesegang rings ) ( h&e , 40 ) special stains and immunohistochemistry . (
a ) masson - fontanna stain showing strong positivity in the pigments , demonstrating the presence of melanin ( masson - fontanna stain , 100 ) .
( b ) congo red stain showing positivity in the homogeneous eosinophilic amyloid - like material ( congo red stain , 100 ) .
( c ) anti - s100 antibody showing positivity in round and spindled pigment - containing cells ( ihc stain , 200 ) .
( d ) anti - hmb45 antibodies showing positivity in pigment - containing cells ( ihc stain , 200 )
classically , ceot is comprised of polygonal epithelial cells with eosinophilic cytoplasm , well - defined cellular borders and vesicular nuclei with conspicuous nucleoli .
mild to moderate pleomorphism can be found , but atypical mitotic figures are usually absent .
this amyloid - like material , proposed to be derived from degradation of keratin filaments secreted by the epithelial cells , stains positive with congo red and is characteristic of pindborg tumor .
another important distinguishing feature of ceot is the presence of calcifications ranging from small to large aggregates or in the form calcific spherules forming concentric laminations known as liesegang rings .
five histopathologic patterns have been described in pindborg tumor : sheets and islands of polyhedral cells with well - defined cell outlines and intercellular bridges ; a cribriform pattern with the presence of eosinophilic substance resembling amyloid within the spaces , which undergoes calcification to form liesegang rings ; abundant polyhedral neoplastic cells admixed with many multinucleated giant cells ; polyhedral cells in nests resembling salivary gland tumors ; and predominantly pseudoglandular pattern composed of clear cells . in general , ceots are benign with an indolent biological behavior and a local recurrence rate of 10%15% .
it is important to recognize the variants of pindborg tumor because of their varied clinical course , aggressiveness , recurrence potential and malignant behavior .
clear cell variant is characterized by the presence of abundant clear cells in the pseudoglandular pattern .
the origin of clear cells is not clear and may arise from degenerated epithelial cells with glycogen , langerhans cells or odontogenic epithelium .
the absence of calcification in the noncalcifying variant is a sign of poor tumor differentiation and is associated with greater chances of recurrence .
bone and cementum forming variant shows extensive mineralized areas , either cellular resembling bone or acellular areas resembling cementum and composed of collagen .
collagen is most likely deposited by stimulated stromal cells of the tumor on any calcified nidus .
langerhans cell variant is distinguished by its occurrence in the asian population , involvement of anterior part of the maxilla , few nests and islands of clear tumor cells exhibiting positivity with s100 and cd1a , lack of calcification , excess of amyloid - like substance and presence of chronic inflammatory cells .
langerhans cells are claimed to play a major role in antigen presentation and regression of the tumor .
some authors suggest that the lesional odontogenic tissue has the potential to undergo neuroectodermal differentiation under certain situations and could give rise to melanocytes .
others propose that the process of odontogenesis is multifarious and occurs as a result of close and mutual interactions involving oral epithelium and ectomesenchyme , which in turn is derived from cranial neural crest .
hence , melanocytes which are also derived from neural crest might be present in odontogenic lesions .
a few other authors identified the presence of melanocytes in outer enamel epithelium and dental lamina in fetuses during early gestation ( 1218 weeks ) and hence stated that melanocytes could be seen in odontogenic lesions .
the same authors observed that the occurrence of pigmented lesions and melanocytes is common in pigmented races and suggested a strong racial predilection for these lesions . an additional speculation for the presence of melanocytes in odontogenic lesions includes the migration of melanocytes through the mesenchyme . in dog fetuses ,
the melanocytes were seen surrounding mesenchymal tissue in the dental anlage . it is observed that most of the pigmented odontogenic lesions have a prominent mesenchymal component either as in the form of dentin or in the form of calcification suggesting that the presence of calcification or dentin may activate the melanocytes previously present there , to form pigment .
takeda et al . demonstrated two types of pigment - containing cells : s100-positive spindle or round cells and hmb45-positive dentritic and spindle cells derived from melanocytes . while pigmented variants of ccot have been reported , it is not known if pigmented variants of ceot occur . here , we report the first case of pigmented variant of ceot in the jaw .
the tumor was found in a young asian female in the left maxilla and was associated with an unerupted tooth .
the tumor was extensively calcified , and it is possible that ossification could have triggered melanocytes to produce melanin .
the patient was asymptomatic for 18 months after surgery , indicating that the pigmented variant could be similar to conventional ceot in progression . however , prospective studies on a larger patient population are required to ascertain the prognosis of patients diagnosed with pigmented variant of pindborg tumor described in this case report .
since this variant possesses unique histologic and uncommon clinical features , it may possibly create problems for differential diagnosis .
therefore , it is important to recognize the occurrence of a pigmented variant of ceot .
further investigation is required to unravel the biological behavior of this variant , precise origin of melanocytes and the pathological implication of their presence in the odontogenic tumors .
| the calcifying epithelial odontogenic tumor ( ceot ) , also known as the pindborg tumor , is a benign locally invasive neoplasm .
common variants of ceot include noncalcifying , langerhans cell , bone and cementum forming and clear cell , which have a prognostic significance .
pigmented variants are known to occur in other odontogenic tumors . however , a definitive pigmented variant of ceot has not been reported in literature so far . here , we report the first case of pigmented pindborg tumor arising from the maxilla in a young female . the pigment was demonstrated as melanin by staining and confirmed by immunohistochemistry .
the pigmented variant of ceot did not recur within 18 months postsurgery .
our report indicates that it is essential to recognize the pigmented variant .
we discuss the common variants of ceot and potential histogenesis of the pigmented variant .
further studies are required to reveal the histogenesis of melanocytes and their pathological significance in the odontogenic tumors . |
type 1 diabetes mellitus ( dm1 ) affects nearly three million americans.1 it is a challenging disease to manage , often requiring complex medical regimens and significant effort from both the clinician and the patient .
the first manifestation of beta cell loss is loss of first - phase insulin secretion ( peak levels of insulin in the minutes following a glucose challenge ) and is then followed by widespread destruction of beta cells and persistent occurrence of hyperglycemia.2 this destruction is mediated by a host of immune factors , including beta - cell autoantigens , dendritic cells , macrophages , b lymphocytes , and t lymphocytes.3 the resulting insulin deficiency causes a state of glucose dysregulation , requiring exogenous insulin administration and careful glucose monitoring . despite optimal therapy , however , a subset of patients still experience unmanageable and unpredictable fluctuations in their blood glucose.4 the two most promising techniques for restoring endogenous insulin production have been whole pancreas transplantation and islet - cell transplantation .
islet transplant is significantly less invasive than pancreatic transplant and has been shown to significantly reduce the burden of exogenous insulin requirement .
islet transplantation has also been shown to slow or even halt the progression of serious diabetic sequelae such as cardiovascular complications , retinopathy , nephropathy , and neuropathy.5,6 in islet transplantation , pancreatic tissue from a donor is processed and islet cells are isolated .
a single healthy human pancreas can contain 415 million islets.7 isolation and purification is done using a combination of enzymatic tissue digestion as well as mechanical agitation .
the yield and purity of islets remains highly variable with some studies showing an islet equivalents count ranging from 100,000 to 300,000 per pancreas ( islet equivalent : a standardized value of islet volume).8 the isolated cells are then injected into the hepatic portal system of the recipient with the aim of producing a viable donor islet - cell population in the liver . to establish an islet population capable of producing enough insulin for clinical effect , a large number of these cells ( > 5,600 islet equivalents / kg ) must be transplanted.9 post - transplant , the c - peptide level is used as a marker for graft survival as its production often outlasts insulin independence and overall clinical effect of the transplant .
oftentimes patients require multiple infusions from multiple donors over time to achieve sufficient endogenous insulin production .
a drawback of this method is the chance of recipient sensitization , as they are exposed to such a large number of donor antigens.9 thus , the ideal method is one infusion of islets from one single donor .
additionally , limiting the number of required islet transfusions helps mitigate the cost , improves access to transplantation , and limits the burden of this procedure over time .
for example , characteristics such as glucose lability , preexisting insulin resistance , obesity , sensitization , and the presence of beta - cell autoantibodies affect the success and appropriateness of this intervention .
given the limits of islet availability and the significant burden that comes with receiving a transplant , selecting appropriate candidates for islet transplantation is essential . here , a number of important aspects and considerations in patient selection for islet transplantation are reviewed .
the knowledge that pancreatic dysfunction causes a syndrome similar to dm1 dates back to 1889 when von mering and minkowski discovered that pancreatectomy lead to hyperglycemia , ketoacidosis , and death.10 despite significant improvements in the understanding of dm1 throughout the 20th century , it was not until 1977 that the development of a clinically successful cellular replacement therapy emerged . using the newly discovered azathioprine and corticosteroids as immunosuppression , najarian et al described the first successful islet cell transplant.11 three years later , largiadr et al demonstrated the first case of insulin independence after islet allotransplantation.12 overall , however , outcomes from islet transplantation remained significantly worse than those of solid organ transplantation until the description of the so - called edmonton protocol in 2000.13 this landmark protocol provided a standardized procedure by which islet transplantation could be safely and effectively performed and could dramatically improve outcomes.13 in particular ,
the two most notable changes introduced by the edmonton protocol were the use of multiple islet transplantations to increase the mass of cells provided , as well as shifting immunosuppression away from steroids and using low - dose anti - interleukin 2 and sirolimus to minimize beta - cell toxicity . since the edmonton protocol , significant work has been done to improve islet transplantation.14 despite this , islet transplant remains relatively rare , with only 44 islet transplant centers currently active in the world.15 as islet transplantation becomes more common and more centers begin adopting it as a therapy for type 1 diabetics , the need for defining optimal recipient characteristics becomes ever more important .
therefore , appropriate selection of candidates is paramount . the following paragraphs address common considerations for evaluating which patients would benefit most from single donor islet transplantation .
the goal of islet and pancreas transplantation is to eliminate the requirement for exogenous insulin in type 1 diabetic patients .
currently 82% of patients receiving pancreas transplant are insulin independent at 1 year and 58% at 5 years.16 islet transplantation has been less successful at providing durable insulin independence , with only 66% of patients remaining off insulin at 1 year , and 44% at 3 years.17 interestingly , in a number of recent studies , patients who received islet transplants but failed to remain insulin independent had significantly reduced incidence of hypoglycemic events post - transplant.18 this was most recently demonstrated in a phase 3 trial by the clinical islet transplantation consortium.19 this trial demonstrated that patients had a 93% probability of being free from hypoglycemic events at 2 years post - transplant .
though islet transplant is less successful than pancreas transplantation at maintaining insulin independence , these findings suggest a role for islet transplant in patients with labile diabetes . in other words ,
this therapy could best serve those patients whose greatest impediment to a normal life is not insulin administration , but unpredictable and often debilitating hypoglycemic episodes and hypoglycemic unawareness .
the volume of infused islets correlates to the duration of insulin independence post - transplant.20 therefore , a barrier to islet transplantation has been the need for recurrent islet infusions due to decreasing graft function over time.21,22 however , a french study of 23 patients showed preserved hypoglycemic protection despite decreasing allograft function as measured by the -score ( a validated score for describing overall graft function).23,24 in other words , transplanted patients were protected against hypoglycemic events despite decreasing allograft function over time .
subsequent trials have been consistent with this finding . in a study of 12 islet recipients , for example ,
rickels et al reported dramatic and durable reduction in glycemic variability despite the fact that many of the patients did not remain insulin independent over time.25 one explanation for this finding is that in many patients with progressive graft - failure , some latent c - peptide production persists .
this production of c - peptide indicates that beta cells , though small in number , continue to survive and protect against hypoglycemia by stimulating glucagon production in nearby alpha cells via a paracrine pathway.26 it would seem , therefore , that single donor islet transplantation can be used instead of the traditional multiple infusions for patients whose primary goal is reduction of glycemic variability and hypoglycemic unawareness .
this would ultimately provide significant lifestyle benefit for brittle diabetics and expand the donor pool for those patients who would benefit from this therapy .
though these results are quite promising , it must be noted that the majority of the aforementioned trials have been very small .
further study with large prospective trials will reveal whether the initial findings are in fact applicable to the general population of type 1 diabetic patients .
two immune mechanisms are responsible for graft loss in islet transplant patients : 1 ) destruction of transplanted islet cells by the same mechanism that destroyed the native insulin - producing cells autoantibodies ; 2 ) graft loss due to rejection from alloimmunity .
both cellular and humoral mechanisms have been implicated in graft destruction of transplanted islet cells .
cellular immunity has been difficult to study given the inherent difficulty of sampling engrafted islets in the recipient liver . as such , humoral immunity and in particular antibody assays
though autoantibodies lead to the initial destruction of the islets in dm1 , studies have found that pre - transplant autoantibody titers are only weakly associated with graft survival and overall outcomes.27 although the presence of these autoantibodies pre - transplant are poorly predictive of graft loss , activation of these autoimmune pathways post - transplant is associated with graft loss and eventual failure.28 a study by piemonti et al , for example , showed that a statistically significant rise in anti - glutamic acid decarboxylase or anti - islet antigen 2 autoantibodies ( antibodies primarily responsible for beta cell loss in dm1 ) resulted in a significantly increased risk of graft failure when compared to those without a rise in those antibodies with a relative risk of graft failure of 5.3.27 furthermore , those patients who experienced an increase in autoantibody titer had a shorter median time to graft failure when compared to those who did not experience autoantibody increases . although autoimmunity is a significant aspect of dm1 , islet transplantation , like most other types of transplant , is also challenged by the issue of alloimmunity or the body s reaction against foreign antigens . in addition to studying autoantibodies ,
piemonti et al examined how donor - specific alloantibodies ( dsa ) affected graft failure in islet recipients .
the relative risk of developing graft failure in those patients with a rise in dsa , despite adequate immunosuppression , was 5.12 when compared to those without a rise in dsa .
risk factors for increased dsa included multiple islet infusions , human leukocyte antigen ( hla ) class i or ii mismatch , or pre - transplant dsa . additionally , dsa was higher in regimens where anti - thymocyte globulin was used for induction and mycophenolate mofetil was used for maintenance immunosuppression .
the groups in which dsa remained low were those in which induction was done using an anti - cd25 antibody , and where sirolimus or tacrolimus were used for maintenance.27 it is clear that immunity plays a significant role in islet cell transplant outcomes .
dm1 is unique in that autoantibodies play a role in the initial disease pathogenesis and also seem to affect the success of islet transplantation .
it is surprising that preformed autoantibodies are only weakly associated with outcomes and that significant negative effects were noted when those antibodies increased post - transplant .
preformed autoantibody titers , therefore , are difficult to use for predicting who will and will not have a successful outcome . currently , hla matching is not performed in islet transplantation.29 however , hla mismatch was associated with increased dsa and graft failure . a strategy for outcome improvement could include matching donor and recipients with hla ii match and hla i mismatch
this would theoretically prevent autoimmune destruction through hla i and limit the development of dsa and panel reactive antibody reactivity to class ii antigens .
immunity remains a significant challenge to islet transplant outcomes , and further study will be required to characterize optimal patient donor matching characteristics and develop improved immunosuppression that limits toxicity to engrafted islets .
the goal of islet and pancreas transplantation is to eliminate the requirement for exogenous insulin in type 1 diabetic patients .
currently 82% of patients receiving pancreas transplant are insulin independent at 1 year and 58% at 5 years.16 islet transplantation has been less successful at providing durable insulin independence , with only 66% of patients remaining off insulin at 1 year , and 44% at 3 years.17 interestingly , in a number of recent studies , patients who received islet transplants but failed to remain insulin independent had significantly reduced incidence of hypoglycemic events post - transplant.18 this was most recently demonstrated in a phase 3 trial by the clinical islet transplantation consortium.19 this trial demonstrated that patients had a 93% probability of being free from hypoglycemic events at 2 years post - transplant .
though islet transplant is less successful than pancreas transplantation at maintaining insulin independence , these findings suggest a role for islet transplant in patients with labile diabetes . in other words ,
this therapy could best serve those patients whose greatest impediment to a normal life is not insulin administration , but unpredictable and often debilitating hypoglycemic episodes and hypoglycemic unawareness .
the volume of infused islets correlates to the duration of insulin independence post - transplant.20 therefore , a barrier to islet transplantation has been the need for recurrent islet infusions due to decreasing graft function over time.21,22 however , a french study of 23 patients showed preserved hypoglycemic protection despite decreasing allograft function as measured by the -score ( a validated score for describing overall graft function).23,24 in other words , transplanted patients were protected against hypoglycemic events despite decreasing allograft function over time .
subsequent trials have been consistent with this finding . in a study of 12 islet recipients , for example , rickels
et al reported dramatic and durable reduction in glycemic variability despite the fact that many of the patients did not remain insulin independent over time.25 one explanation for this finding is that in many patients with progressive graft - failure , some latent c - peptide production persists .
this production of c - peptide indicates that beta cells , though small in number , continue to survive and protect against hypoglycemia by stimulating glucagon production in nearby alpha cells via a paracrine pathway.26 it would seem , therefore , that single donor islet transplantation can be used instead of the traditional multiple infusions for patients whose primary goal is reduction of glycemic variability and hypoglycemic unawareness .
this would ultimately provide significant lifestyle benefit for brittle diabetics and expand the donor pool for those patients who would benefit from this therapy .
though these results are quite promising , it must be noted that the majority of the aforementioned trials have been very small .
further study with large prospective trials will reveal whether the initial findings are in fact applicable to the general population of type 1 diabetic patients .
two immune mechanisms are responsible for graft loss in islet transplant patients : 1 ) destruction of transplanted islet cells by the same mechanism that destroyed the native insulin - producing cells autoantibodies ; 2 ) graft loss due to rejection from alloimmunity .
both cellular and humoral mechanisms have been implicated in graft destruction of transplanted islet cells .
cellular immunity has been difficult to study given the inherent difficulty of sampling engrafted islets in the recipient liver . as such , humoral immunity and in particular antibody assays
though autoantibodies lead to the initial destruction of the islets in dm1 , studies have found that pre - transplant autoantibody titers are only weakly associated with graft survival and overall outcomes.27 although the presence of these autoantibodies pre - transplant are poorly predictive of graft loss , activation of these autoimmune pathways post - transplant is associated with graft loss and eventual failure.28 a study by piemonti et al , for example , showed that a statistically significant rise in anti - glutamic acid decarboxylase or anti - islet antigen 2 autoantibodies ( antibodies primarily responsible for beta cell loss in dm1 ) resulted in a significantly increased risk of graft failure when compared to those without a rise in those antibodies with a relative risk of graft failure of 5.3.27 furthermore , those patients who experienced an increase in autoantibody titer had a shorter median time to graft failure when compared to those who did not experience autoantibody increases . although autoimmunity is a significant aspect of dm1 , islet transplantation , like most other types of transplant , is also challenged by the issue of alloimmunity or the body s reaction against foreign antigens . in addition to studying autoantibodies ,
piemonti et al examined how donor - specific alloantibodies ( dsa ) affected graft failure in islet recipients .
the relative risk of developing graft failure in those patients with a rise in dsa , despite adequate immunosuppression , was 5.12 when compared to those without a rise in dsa .
risk factors for increased dsa included multiple islet infusions , human leukocyte antigen ( hla ) class i or ii mismatch , or pre - transplant dsa . additionally , dsa was higher in regimens where anti - thymocyte globulin was used for induction and mycophenolate mofetil was used for maintenance immunosuppression .
the groups in which dsa remained low were those in which induction was done using an anti - cd25 antibody , and where sirolimus or tacrolimus were used for maintenance.27 it is clear that immunity plays a significant role in islet cell transplant outcomes .
dm1 is unique in that autoantibodies play a role in the initial disease pathogenesis and also seem to affect the success of islet transplantation .
it is surprising that preformed autoantibodies are only weakly associated with outcomes and that significant negative effects were noted when those antibodies increased post - transplant .
preformed autoantibody titers , therefore , are difficult to use for predicting who will and will not have a successful outcome .
currently , hla matching is not performed in islet transplantation.29 however , hla mismatch was associated with increased dsa and graft failure . a strategy for outcome improvement could include matching donor and recipients with hla ii match and hla i mismatch .
this would theoretically prevent autoimmune destruction through hla i and limit the development of dsa and panel reactive antibody reactivity to class ii antigens .
immunity remains a significant challenge to islet transplant outcomes , and further study will be required to characterize optimal patient donor matching characteristics and develop improved immunosuppression that limits toxicity to engrafted islets .
one of the particular risks of this therapy comes from the portal vein infusion process .
a number of studies have shown that portal vein hypertension , thrombosis , or infarction of the liver can all occur as a result of islet infusion .
rates of thrombosis range from 0% to 3.9% , and the risk of bleeding is even higher , ranging from 0% to 6.5%.3033 at least one group performing islet transplants has been preferentially cannulating only the left branch of the portal vein to facilitate future resection should serious complications of the infusion arise.34 given that islet infusion poses a risk to hepatic vasculature , some have proposed that there is an upper limit to the number of safe islet infusions that should be done.34 this risk must be balanced with the goals of transplantation , one of which being insulin independence , which is better achieved with multiple islet infusions . in addition to causing vascular insult , islet infusion may also lead to hepatic steatosis .
first found in animal models , it has only been recently shown that this occurs in humans after transplantation.35 various groups have started assessing for steatosis in their postoperative cohorts using a variety of imaging modalities , most notably ultrasound and mri.36,37 in these studies , rates of fatty liver post - transplant have ranged from 17% to 24% . there was no correlation between hba1c or body mass index and steatosis post - transplant in either study .
fat deposition tends to be transient and can vary widely throughout a patient s course .
interestingly , some patients with fat deposition tend to have better graft function , as defined by superior beta scores compared to their non - steatotic counterparts .
graft function tends to diverge late ( 40 + months ) , those with steatosis experiencing faster rates of graft exhaustion and increased islet loss.36 this finding is not universal , however , as at least one group has found contradictory data.38 steatosis of hepatic parenchyma is a cornerstone of nonalcoholic fatty liver disease ( nafld ) .
the disease is more often associated with diabetes mellitus type 2 but is seen in those with type 1 at higher incidences than the general population .
this association has been correlated to increased risk of heart disease and chronic kidney disease in these type 1 diabetics.39 the ramifications of this finding may be twofold with regard to islet transplantation .
1 ) although it is well understood that diabetes in and of itself causes heart disease and kidney dysfunction,40 islet grafts that cause steatosis may in turn also lead to increased rates of heart and kidney disease .
2 ) that steatosis caused by islet infusion is associated with shorter graft - survival times .
although steatosis in islet transplant is only just beginning to be studied , it may hold that preexisting steatosis such as that caused by nafld may also speed graft loss .
therefore , it could be that those with preexisting heart disease , kidney disease , or nafld are far less likely to benefit from islet transplant despite the fact that the progression of these diabetic complications are slowed by islet transplantation . though a speculation at this time , this hypothesis warrants further study as the findings may directly impact proper patient selection
though great strides have been made in islet transplantation , the treatment often falls short in its goal of producing durable , life - long insulin independence .
achieving long - term success with single donor islet transplantation has been difficult due to the smaller number of islets transplanted .
indeed , it has been shown that overall duration of graft survival is predicted by initial graft success.41 long - term insulin independence has largely been achieved by infusing a large number of islets from multiple donors . despite its shortcomings in terms of transplanted islet mass and insulin independence ,
single - donor islet transplant still provides significant benefit for a specific patient population : brittle diabetics .
hypoglycemic events are a source of great morbidity and mortality ; these episodes can largely be avoided with islet transplant even if insulin independence is never achieved , thus providing security and improved quality of life to recipients.4 additionally , rejection remains one of the greatest barriers to long - term graft survival .
data are beginning to emerge with regard to the role of recipients auto- and alloimmune responses to grafts .
so far , these factors have been difficult to apply to patient selection . with further study , knowledge of the immune processes specific to islet transplantation will allow superior patient donor matching and decrease the need for postoperative immunosuppression .
some current work has focused on reducing the exposure of transplanted islets to the recipient immune microenvironment by methods such as local drug release and cellular encapsulation.4244 stem cell strategies for type 1 diabetes have also been considered , as obtaining sufficient beta cells for transplantation remains a significant barrier to success .
results have been mixed , but stem - cell therapy remains a promising hope for cure.45 the majority of studies examining islet transplantation have been small and generally lacking in power . as this therapy continues to improve and become more common , larger studies will be able to better characterize the important factors related to patient selection .
what can be concluded now , however , is that islet transplantation remains an optimal therapy for patients with brittle diabetes and difficult - to predict hypoglycemic events . | type 1 diabetes mellitus is an autoimmune disorder of the endocrine pancreas that currently affects millions of people in the united states .
although the disease can be managed with exogenous insulin administration , the ultimate cure for the condition lies in restoring a patient s ability to produce their own insulin .
islet cell allotransplantation provides a means of endogenous insulin production . though far from perfected ,
islet transplants are now a proven treatment for type 1 diabetics .
however , proper patient selection is critical for achieving optimal outcomes . given the shortage of transplantable organs , selecting appropriate candidates for whom the procedure will be of greatest benefit is essential .
although many of those who receive islets do not retain insulin independence , grafts do play a significant role in preventing hypoglycemic episodes that can be quite detrimental to quality of life and potentially fatal .
additionally , islet transplant requires lifelong immunosuppression .
antibodies , both preformed and following islet infusion , may play important roles in graft outcomes .
finally , no procedure is without inherent risk and islet transfusions can have serious consequences for recipients livers in the form of both vascular and metabolic complications .
therefore , patient - specific factors that should be taken into account before islet transplantation include aims of therapy , sensitization , and potential increased risk for hepatic and portal - venous sequelae . |
the serine / threonine kinase akt ( pkb / c - akt ) promotes cell growth ( i.e. , accumulation of cell mass ) predominantly through activation of complex 1 of the mammalian target of rapamycin ( mtorc1 ) ( sarbassov et al . ,
mtorc1 is regulated by both nutrients and growth factors and plays a conserved role in cell growth control ( wullschleger et al . , 2006 ) .
glucose uptake and induction of glycolysis by akt have been studied in the context of the metabolic effects of insulin signaling on cellular and organismal levels ( whiteman et al . , 2002 ) .
however , the contribution of anabolic pathways other than protein biosynthesis to cell growth have only been considered recently ( plas and thompson , 2005 ) .
akt regulates cell metabolism through several downstream targets ( manning and cantley , 2007 ) .
akt stimulates glucose uptake via induction of translocation of the glucose transporter 4 ( glut4 ) to the plasma membrane and by increasing expression of glut1 ( kohn et al . , 1996 ; welsh et al . ,
akt promotes conversion of glucose into pyruvate through the glycolytic pathway by increasing the expression of glycolytic enzymes ( semenza et al . , 1996 ) and
by phosphorylating and activating hexokinase 2 ( hk2 ) ( robey and hay , 2006 ) .
pyruvate can either be converted into lactate , or it can enter the tricarboxylic acid cycle ( tca cycle ) , where it is fully oxidized to generate atp . in liver and adipose tissue , glucose is converted via pyruvate and citrate into lipids .
phosphorylation of tsc2 by akt ( inoki et al . , 2002 ; potter et al . ,
2002 ) results in activation of the rheb gtpase while phosphorylation of the pras40 induces the dissociation of this inhibitory component from the mtorc1 complex ( oshiro et al .
, 2007 ; sancak et al . , 2007 ; vander haar et al . , 2007 ) .
there are several lines of evidence that the akt / mtorc1 axis is an essential regulator of cell growth in vivo .
akt1/akt2 double - knockout mice exhibit severe growth deficiency ( peng et al . , 2003 ) and
mice deficient in s6-kinase 1 ( s6k1 ) show a significant reduction in size accompanied by hypoinsulinaemia and glucose intolerance ( pende et al . , 2004 ) .
the family of sterol regulatory element binding proteins ( srebp ) consists of three closely related members : srebp1a , srebp1c , and srebp2 ( eberle et al . , 2004 ) .
they have been identified as mediators of the effect of sterols on expression of enzymes involved in lipid and cholesterol homeostasis ( yokoyama et al . , 1993 ) .
srebps belong to the family of basic helix - loop - helix - leucine zipper ( bhlh - zip ) transcription factors .
they are synthesized as inactive precursors and localize to the endoplasmic reticulum ( er ) , where they bind to the sterol cleavage activating protein ( scap ) . the srebp / scap complex binds to copii proteins and translocates to the golgi where a two - step proteolytic cleavage releases the n - terminal half of the srebp protein and allows entry into the nucleus ( rawson , 2003 ) .
srebps bind to sterol regulatory element ( sre ) and e box sequences found in the promoter regions of genes involved in cholesterol and fatty acid biosynthesis .
studies in knockout and transgenic mice have shown that srebp1 preferentially regulates genes involved in fatty acid biosynthesis while srebp2 mainly regulates genes of the cholesterol pathway ( horton et al . , 2003 ) .
two key enzymes that are required to divert glycolytic carbon flux into lipid biosynthesis are atp - citrate lyase ( acly ) and fatty acid synthase ( fasn ) .
acly converts cytosolic citrate into acetyl - coa and oxaloacetate , thereby supplying the essential metabolite for lipid biosynthesis .
akt phosphorylates acly ( berwick et al . , 2002 ) , and acly activity is required for cell growth and tumorigenesis ( bauer et al . , 2005 ; hatzivassiliou et al . ,
fasn catalyzes the condensation of acetyl - coa and malonyl - coa to generate long - chain fatty acids .
we have previously shown that activation of akt induces expression of a number of lipogenic genes , including acly and fasn ( porstmann et al . , 2005 ) .
we show here that activation of srebp and akt - dependent induction of lipid biosynthesis requires the activity of mtorc1 .
furthermore , activation of srebp contributes to akt - dependent cell growth in mammalian cells in vitro and in drosophila melanogaster in vivo .
our data demonstrate the importance of lipogenesis for cell growth in two distinct model systems .
akt is involved in the regulation of cell growth in both mammalian and invertebrate systems by activation of protein biosynthesis through the mtor / s6k pathway ( sarbassov et al . ,
2005 ) . to investigate the involvement of de novo lipid biosynthesis in the regulation of cell growth by akt ,
we used immortalized human retinal pigment epithelial cells ( rpe ) expressing a conditional allele of the akt1 kinase ( myrakt - er ) ( porstmann et al . , 2005 ) .
cells were cultured in medium supplemented with 1% lipoprotein deficient serum ( lpds ) to reduce activation of endogenous akt and limit the availability of exogenous lipids .
activation of myrakt - er by addition of 4-hydroxytamoxifen ( 4-oht ) caused a 20%30% increase in median cell volume ( mcv ) ( figures 1a and 1b ) .
simultaneous treatment with the specific mtorc1 inhibitor rapamycin abolished the akt - dependent increase in cell volume .
rapamycin did not affect cell size in the absence of akt activation since the experiment was performed under serum starvation conditions .
however , rapamycin markedly reduced the akt - dependent increase in cellular protein content ( figure s1 ) .
importantly , we did not observe significant changes in cell - cycle distribution upon akt activation under the conditions used here ( data not shown ) .
we next used nuclear magnetic resonance spectroscopy ( nmr ) to measure changes in medium metabolite concentration before and after myrakt - er activation .
akt activation induced an almost 2-fold increase in glucose uptake and lactate production in rpe cells ( figure 1c ) consistent with a role for akt in activation of glucose uptake and glycolysis .
akt - dependent glucose uptake and lactate production were completely blocked in the presence of rapamycin ( figure 1c ) . likewise , we observed a reduction in akt - dependent amino acid uptake and cellular amino acid content by rapamycin treatment ( figure s2 ) .
analysis of incorporation of radioactive glucose , pyruvate , or acetate into cellular lipids showed that akt activation leads to a substantial increase in glucose - dependent lipogenesis .
crucially , this was completely blocked by rapamycin treatment ( figure 1d ) , indicating that activation of de novo lipid synthesis by akt requires mtorc1 function .
we have previously shown that akt induces accumulation of intracellular lipids including phosphoglycerides ( porstmann et al . , 2005 ) .
figure 1e shows that inhibition of mtorc1 by rapamycin prevents akt - dependent accumulation of unsaturated ( ufa ) and saturated fatty acids ( fa ) as well as phosphatidylcholine ( pc ) and phosphatidylglycerol ( pg ) levels ( figure 1e ) .
we next asked whether induction of lipid biosynthesis is required for akt - dependent increase in cell size .
fatty acid biosynthesis requires cytoplasmic acetyl - coa , which is generated by the enzyme atp - citrate lyase ( acly ) .
acly function is required for tumor growth in vivo ( bauer et al . , 2005 ; hatzivassiliou et al . ,
inhibition of acly using the inhibitor sb204990 attenuated akt - dependent increase in cell volume in rpe cells ( figures 1f and 1 g ) .
studies in knockout mice have shown selective involvement of srebp1a and 1c in the regulation of lipid biosynthesis ( liang et al . , 2002 ) and promoters of lipogenic genes are preferentially bound by srebp1a in vivo ( bennett et al . , 2004 ) .
mature srebp1 can be detected in nuclear lysates as a diffuse band migrating between 60 and 65 kda .
figures 2a and 2b show that accumulation of nuclear msrebp1 can be detected within 2 hr of akt activation .
induction of msrebp1 precedes the increase in full - length srebp1 ( flsrebp1 ) , which can be detected after 8 hr of akt activation ( figures 2a , 2b , and s3 ) .
an increase in fasn mrna and protein expression can be detected after 4 and 8 hr of akt activation , respectively ( figures 2c and 2d ) .
induction of nuclear msrebp1 as well as fasn expression in response to akt activation is blocked by sterols ( figures 2e and 2f ) .
sterols induce a conformational change in scap that induces insig binding , thereby preventing er / golgi translocation of the srebp / scap complex and srebp processing ( sun et al . , 2005 ) .
the observed induction of flsrebp in response to sterol treatment is most likely due to activation of the liver x receptor ( lxr ) ( repa et al . , 2000 )
akt induces glucose uptake by inducing glucose transporter expression and membrane localization , and enhances glycolytic activity by activating hexokinase ( manning and cantley , 2007 ) .
absence of glucose or treatment with 2-deoxyglucose , an inhibitor of glycolysis , blocked akt - dependent accumulation of msrebp1 ( figure 2 g ) and prevented induction of srebp target genes ( figure 2h ) .
energy depletion after glucose starvation or inhibition of glycolysis is likely to lead to activation of ampk ( hardie and carling , 1997 ) .
activation of ampk by aicar also blocked accumulation of msrebp1 in response to akt activation ( figure 2i ) and prevented induction of srebp target genes ( figure s4 ) .
these results indicate that activation of srebp1 by akt is regulated by cellular energy status .
the activity of mtorc1 is regulated by growth factor signaling as well as nutrient availability and energy homeostasis ( sarbassov et al . , 2005 ) .
nuclear accumulation of msrebp1 in response to akt activation was prevented by rapamycin ( figure 3a ) .
likewise , induction of fasn and acly mrna and protein levels was blocked by rapamycin treatment ( figures 3b and 3c ) .
importantly , treatment with rapamycin completely abolished induction of phosphorylation of the ribosomal protein s6 , but only had a minor effect on phosphorylation of gsk3 or acly , indicating that the activity of the myrakt - er fusion protein is not affected by rapamycin ( figure 3c ) .
furthermore , rapamycin treatment also blocked insulin - dependent induction of fasn protein and mrna ( figures s5a and s5b ) and ectopic expression of mtor was sufficient to induce msrebp1 as well as fasn expression in the absence of myrakt - er activation in u2os cells ( figure s5c ) .
mature srebp proteins are highly unstable and are rapidly degraded through an ubiquitin - dependent pathway ( hirano et al . , 2001 ) .
this phosphorylation enables binding of the fbw7 ubiquitin ligase and induces degradation of the srebp protein .
activation of akt increased levels of transfected myc - tagged wild - type msrebp1 ( amino acids 1490 ) ( figure 3d ) .
interestingly , akt - dependent accumulation of exogenous wild - type msrebp could still be observed in the presence of rapamycin , indicating that mtorc1 function is dispensable for stabilization of msrebp1 in response to akt activation .
furthermore , inhibition of gsk3 using the chemical inhibitor sb216763 did not block akt - dependent accumulation of endogenous msrebp1 , induction of fasn protein , or fasn promoter activity in response to akt activation ( figure s6 ) .
these results indicate that akt - dependent activation of endogenous srebp does not solely involve regulation of the stability of srebp but suggest an additional , mtorc1-dependent pathway that contributes to srebp activation .
it has previously been suggested that rapamycin treatment can also affect the activity of mtorc2 in certain cell types ( sarbassov et al . , 2006 ) .
our own results show that long - term treatment with rapamycin leads to activation of akt phosphorylation by inhibition of a negative feedback loop in the cell line used here ( figure s7 ) .
to address any potential involvement of mtorc2 in the regulation of lipogenic gene expression , we used sirna to selectively abolish expression of raptor or rictor , components of mtorc1 or mtorc2 , respectively . silencing of mtor and raptor significantly reduces s6 phosphorylation ( figure 3e ) , while rictor silencing results in reduction of akt phosphorylation on threonine 450 ( figure s8 ) , which has been shown to be a substrate for mtorc2 ( facchinetti et al . , 2008 ; ikenoue et al . , 2008 )
. silencing of mtor or raptor , but not rictor , prevents akt - dependent induction of fasn and srebp1 expression ( figure 3e ) .
silencing of mtor also blocked akt- dependent fasn and acly induction in u2os cells ( figure s9 ) .
we also addressed the role of srebp in the induction of de novo lipid biosynthesis in response to akt activation .
figure 3f shows that silencing of srebp1 and 2 blocks incorporation of glucose , pyruvate , or acetate into cellular lipids .
furthermore , silencing of mtor or raptor , but not rictor , abolished induction of lipogenesis in response to akt activation ( figure 3f ) .
we next asked whether induction of lipogenic gene expression by srebp is involved in the regulation of cell growth in mammalian cells .
transfection of sirna oligonucleotides targeting both srebp1 and 2 significantly reduced the increase in cell size observed in response to akt activation in rpe cells in a dose - dependent manner ( figures 4a and 4b ) .
likewise , induction of fasn expression in response to akt activation was reduced when srebp1 and 2 were silenced ( figure 4c ) .
similar results were obtained when srebp1 and 2 were silenced using different sirna sequences ( data not shown ) .
these results indicate that activation of srebp and expression of srebp target genes is required for efficient cell growth in mammalian cells in vitro .
hlh106 has been identified as the gene coding for a single srebp isoform in flies , and dsrebp shows significant sequence homology to human srebp1 within its dna binding domain ( seegmiller et al .
we first measured the effect of transient silencing of dsrebp or dfas on cell volume in the drosophila cell line kc167 .
figures 5a and 5b show that silencing of dsrebp or dfas results in a significant reduction in mean cell volume and fsc of g1 cells .
interestingly , insulin treatment of kc167 cells resulted in increased levels of fatty acids and phosphoglycerides similar to the results obtained upon akt activation in mammalian cells ( figure s10 ) . to address the role of dsrebp in the regulation of cell growth in vivo , we generated a series of transgenic flies expressing an inverted repeat rna under the control of the uas promoter .
three different fly lines ( rnai - s1 , rnai - s2 , and rnai - s3 ) were used for the analysis .
induction of dsrebp rnai expression using the daughterless ( da)-gal4 driver resulted in reduced expression of dsrebp and dfas in larvae ( figure 5c ) .
the three rnai lines showed differences in silencing efficiency with rnai - s3 having the strongest effect ( figure 5c ) .
silencing of dsrebp during embryogenesis caused a dose - dependent developmental delay and lethality during larval and pupal stages ( figure s11 ) .
flies reaching adulthood showed a significant reduction in body size , reduced body weight , and wing area ( figures 5d and s12 ) .
the rnai - s3 fly line did not produce any viable offspring when crossed to the da - gal4 driver .
this is consistent with an essential role of dsrebp during larval development ( kunte et al . , 2006 ) .
deletion or overexpression of components of the insulin signaling pathway affects cell size in a cell - autonomous manner ( weinkove et al . , 1999 ) .
we therefore asked whether silencing of dsrebp in specific compartments of the drosophila wing could affect cell and organ size .
figures 5e and 5f show that expression of dsrebp rnai cassette in the dorsal wing compartment ( ms1096-gal4 ) caused a dose - dependent reduction in adult wing area .
similarly , silencing of dsrebp using the engrailed ( en)-gal4 driver caused a specific reduction in the area of the posterior compartment of the wing ( figures 5 g and 5h ) . furthermore
, reduced size of the posterior compartment was accompanied by an increase in density of wing epithelial cells ( figures 5i and 5j ) indicating that silencing of dsrebp causes a reduction in cell size .
we also investigated whether inhibition of dsrebp function by means other than gene silencing would cause similar growth inhibition .
deletion of the n - terminal transactivation domain in mammalian srebp results in dominant inhibition of srebp function ( sato et al . , 1994 ) .
ms1096-gal4-driven expression of n - terminally deleted dsrebp ( dn75 ) or a mutant in which the transmembrane domain has been deleted in addition to the n terminus ( dn75 t ) resulted in a substantial reduction in wing area ( figure s13 ) .
we next asked whether the pi3k / akt pathway regulates the activity of srebp in flies .
transient silencing of the catalytic subunit of pi3k , dp110 , or dakt reduced mrna abundance of dfas and dsrebp in kc167 cells ( figure 6a ) .
conversely , silencing of dpten resulted in increased expression of both transcripts ( figure 6a )
. ubiquitous expression of dp110 using the da - gal4 driver resulted in enhanced dsrebp and dfas expression in second instar larvae compared to controls ( figure 6b ) , indicating that the pi3k / akt pathway activates dsrebp function .
expression of mature dsrebp ( m - dsrebp ) using the ms1096-gal4 driver resulted in significant lethality when flies were reared at 25c ( data not shown ) .
when flies were reared at 18c ( resulting in a reduced activity of the gal4/uas system ) , severe deformation of the wing was observed ( figure 6c ) .
expression of the full - length form of dsrebp ( fl - dsrebp ) in the wing caused less severely misshapen wings even when flies were reared at 25c .
however , coexpression of dp110 and full - length dsrebp resulted in severely deformed wings , a phenotype that is similar to that caused by expression of m - dsrebp ( figure 6c ) .
this result suggests that the activity of full - length dsrebp is enhanced by pi3k signaling .
expression of dp110 causes an overgrowth phenotype in the wing , indicated by a 15%20% increase in surface area of the wing ( leevers et al .
silencing of dsrebp attenuated the increase in wing size induced by expression of dp110 ( figure 6d ) .
similar results were obtained using a nonoverlapping rnai sequence targeting dsrebp or by heterozygous deletion of the dsrebp gene ( figure s14 ) .
expression of a kinase domain mutant of pi3k ( dp110[kd ] ) decreases cell and organ size in drosophila ( leevers et al . , 1996 ) .
expression of dp110[kd ] using the decapentaplegic ( dpp)-gal4 driver resulted in a 20%30% reduction in the size of the dpp compartment .
expression of the dsrebp rnai hairpin resulted in a small but significant decrease in wing area ( figure 6e ) .
however , coexpression of dsrebp rnai with dp110kd did not further decrease the size of this compartment .
, these results suggest that dp110 and dsrebp are components of the same pathway in the regulation of cell growth in drosophila .
cell growth is tightly controlled by mitogenic signaling pathways and requires the activation of biosynthetic pathways for the generation of macromolecules , including proteins and lipids .
activation of the mtor complex 1 ( mtorc1 ) increases translation and ribosome biogenesis ( wullschleger et al . ,
2006 ) , but effects of mtorc1 on other anabolic processes are less well understood .
we show here that ectopic activation of akt induces a significant increase in cell volume in the absence of mitogens and that akt - dependent size increase is blocked by the mtorc1 inhibitor rapamycin .
analysis of medium metabolites by nmr revealed that akt activation results in increased uptake of glucose and amino acids as well as secretion of lactate .
the involvement of mtorc1 in akt - dependent translocation of glucose and amino acid transporters to the plasma membrane has been demonstrated before ( edinger and thompson , 2002 ) .
we observed significant accumulation of fatty acids and phosphoglycerides in response to akt activation and enhanced incorporation of labeled glucose , pyruvate , or acetate into the lipid fraction in cells with activated akt , all of which was blocked by rapamycin .
these results show that akt activates de novo lipogenesis and that mtorc1 activity is required for this effect .
hepatocytes show increased srebp1c mrna levels in response to insulin treatment or akt expression ( azzout - marniche et al . , 2000 ;
we have previously shown that akt activation results in nuclear accumulation of msrebp1 and enhances expression of a number of srebp target genes ( porstmann et al . ,
, we show that nuclear accumulation of msrebp1 is rapid and precedes the induction of transcription of srebp target genes as well as the increase in flsrebp1 .
it should be noted that the srebf1 gene is a target of positive feed - forward regulation , since a functional sre has been identified in its promoter region ( amemiya - kudo et al . , 2000 ) .
thus , induction of flsrebp after akt activation could be due to enhanced sre - dependent transcription of the gene .
activation of srebp1 by akt was completely blocked by glucose starvation , inhibition of glycolysis , or ampk activation by aicar .
ampk regulates lipid metabolism by phosphorylating acc and induces a switch from fatty acid biosynthesis to fatty acid oxidation ( hardie and carling , 1997 ) .
thus , regulation of srebp activity by ampk could reflect an additional adaptation to energy restriction .
activity of mtorc1 is regulated by mitogens but also responds to cellular energy status ( sarbassov et al . , 2005 ) .
ampk activates tsc2 resulting in inhibition of mtorc1 ( inoki et al . , 2003 ) .
it seems likely that inhibition of srebp1 activity following glucose deprivation or ampk activation is mediated by mtorc1 .
our results show that nuclear accumulation of srebp1 and induction of srebp target genes requires mtorc1 activity .
it has been suggested that induction of fasn and acc expression by her2 in breast cancer cells involves increased protein translation ( yoon et al . , 2007 ) .
however , we observed an increase in fasn and acly mrna abundance in response to akt activation .
induction of fasn and acly mrna was blocked in the presence of rapamycin indicating that mtorc1 is involved in the regulation of srebp - dependent transcription .
furthermore , silencing of mtor or raptor , but not rictor , abolished activation of expression of srebp1 , fasn , and acly , thus confirming that regulation of srebp - dependent gene expression is exclusive to mtorc1 .
it has been shown that gsk3 regulates the stability of msrebp1 by inducing the association of the ubiquitin ligase fbw7 resulting in rapid degradation of srebp1 ( sundqvist et al . , 2005 ) .
we observed an increase in the stability of exogenous msrebp in response to akt activation . this was dependent on the presence of two gsk3 phosphorylation sites but was still observed in the presence of rapamycin .
however , our results indicate that akt also regulates srebp1 activity in a gsk3-independent , mtorc1-dependent manner .
it has been shown that dominant negative akt prevents er / golgi translocation of scap ( du et al . , 2006 ) .
tor has been implicated in vesicle transport in yeast ( neufeld , 2007 ) and localization of mtor to the er and the golgi plays an essential role in its regulation and downstream signaling ( drenan et al . , 2004 ) .
thus , it seems possible that mtorc1 , or proteins downstream of mtorc1 signaling such as s6k or 4-ebp , could be involved in the regulation of srebp processing within these compartments .
silencing mtor or raptor , but not rictor , is sufficient to abolish akt - dependent incorporation of glucose , pyruvate , or acetate into cellular lipids .
this effect seems to be most pronounced for the incorporation of acetate , which only requires the activity of acc and fasn .
an involvement of raptor in the regulation of lipogenesis is also consistent with its essential function during mouse development ( guertin et al . , 2006 ) .
interestingly , silencing of srebp1 and 2 was sufficient to prevent akt - dependent lipogenesis and significantly reduced cell growth in response to akt activation .
this result confirms that srebp - dependent gene expression represents an important mechanism in the regulation of lipogenesis during cell growth .
consistent with a role for srebp in cell size regulation in mammalian cells , we observed that silencing of dsrebp or its target gene dfas resulted in a significant decrease in cell size in drosophila kc167 cells .
interestingly , dsrebp or dfas silencing completely blocked the increase in cell volume caused by silencing of tsc2 .
silencing of dsrebp in different compartments of the developing wing imaginal disc caused a reduction of the respective compartment in adult flies and was caused by a reduction in cell size rather than cell number .
it is somewhat surprising that expression of m - dsrebp in the wing causes a severe growth defect .
it has been shown before that overexpression of msrebp1a causes cell - cycle arrest through induction of the cdk inhibitors p21 , p27 , and p16 ( inoue et al . , 2005 ;
it is also possible that overactivation of srebp causes an imbalance between protein and lipid biosynthesis and suggests that srebp activity needs to be tightly controlled to allow normal tissue development .
kunte and coworkers have detected dsrebp activation in the developing fat body , the midgut as well as in oenocytes , a specialized cell type that have hepatocyte - like functions in lipid metabolism ( gutierrez et al . , 2007 ; kunte et al . ,
this expression pattern suggests a role in lipid metabolism similar to the function of srebp1c in mammalian liver .
our results are consistent with a cell - autonomous role for dsrebp and suggest that dsrebp is required for normal growth of peripheral larval tissue .
dsrebp silencing restricted tissue growth induced by overexpression of dp110 in the wing and did not enhance the reduction in size of a wing compartment expressing kinase - dead dp110 .
these results suggest that both factors are components of the same pathway and indicate that activation of dsrebp is required for the induction of cell growth by the pi3k pathway in flies
. it will be interesting to investigate whether regulation of dsrebp downstream of pi3k requires tor .
further investigations are required to elucidate the exact mechanism of regulation of dsrebp activity by signaling pathways involved in growth regulation in flies
. taken together , our data are consistent with a model that places srebp activation downstream of the intracellular signaling pathway that integrates mitogenic signaling with energy status and nutrient availability ( figure 7 ) .
it also suggests that protein biosynthesis and lipogenesis are regulated in a concerted manner during cell growth .
rpe myrakt - er and u2os myrakt - er cells have been described before ( porstmann et al . , 2005 ) .
rpe myrakt - er cells were maintained in dmem / hams f12 ( 1:1 ) medium with 10% fcs , glutamine , and sodium bicarbonate .
u2os myrakt - er cells were grown in dmem with 10% fcs and glutamine . for culture at low sterol concentration , cells were grown in medium with 1% lipoprotein deficient serum ( lpds , intracel ) .
kc167 cells were cultured in schneider 's medium ( gibco ) with 10% fcs , penicillin / streptomycin , and glutamine .
a list of antibodies , chemicals , and plasmids is provided in the supplemental experimental procedures .
rpe myrakt - er cells were detached using trypsin , washed , and resuspended in pbs .
median electronic cell volumes were determined in triplicate using a z2 coulter counter ( multisizer ii , beckman - coulter ) .
kc167 cells were collected by centrifugation and analyzed as above . for g1 specific cell size ,
four hundred and fifty microliters of medium samples were mixed with 50 l d2o , and 25 l of 10 mm sodium tsp were added for chemical shift calibration and quantification .
intracellular metabolites were extracted using a dual - phase - extraction method as previously described ( tyagi et al . , 1996 ) .
lipid extracts were reconstituted in 600 l deuterated chloroform , and 0.008% v / v tetramethylsilane was used as a chemical shift reference ( 0 ppm ) .
water - soluble metabolites were reconstituted in 600 l d2o , and 25 l of 1 mm tsp was added for quantitation and chemical shift calibration .
proton nmr spectra were acquired using a bruker 600 mhz ( bruker avance ) nmr system .
spectral peaks for water - soluble and lipid metabolites were assigned according to sitter et al .
metabolite levels were normalized to cell number , and each experiment was repeated at least six times .
cells were grown in medium with 1% lpds in the presence of 100 nm 4-oht or ethanol for 24 hr .
2.5 ci / ml d-[6-c]glucose ( 45 m final concentration , amersham ) , 2.5 ci / ml [ 2-c]-pyruvate ( 166 m final concentration ) or 10 ci / ml [ 1-c]-acetate ( 85 m final concentration , both perkin elmer ) was added , and cells were incubated for 4 hr at 37c .
lipids were extracted by successive addition of 2 ml methanol , 2 ml chloroform , and 1 ml dh2o .
lipids were dissolved in ultima gold lsc cocktail ( perkin elmer ) and counted on a beckman ls 6500 scintillation counter .
each measurement was performed in duplicate , results were normalized to total protein content , and experiments were repeated at least twice .
for total cell lysis , cells were washed with ice - cold pbs and dissolved in lysis buffer ( 1% triton x-100 , 50 mm tris ph 7.5 , 300 mm nacl , 1 mm egta , 1 mm dtt , 1 mm navo4 , and protease - inhibitor [ roche ] ) .
nuclear lysates were prepared as described ( porstmann et al . , 2005 ) .
for detection of msrebp , cells were treated with 25 g / ml alln for 1 hr prior to lysis .
equal amounts of cell lysates were separated on sds - page and blotted onto pvdf membrane ( immobilon ) .
total rna was isolated from rpe myrakt - er or kc267 cells using the rneasy kit ( qiagen ) and treated with dnase i ( qiagen ) .
for preparation of rna from drosophila larvae , 1020 s instar larvae were homogenized in liquid nitrogen and dissolved in buffer rlt ( qiagen ) followed by rna purification according to manufacturer 's protocol .
two - and - a - half micrograms of total rna was used for first - strand cdna synthesis with superscript ii rt and oligo dt primer ( invitrogen ) .
real - time pcr was performed with sybr green pcr master mix ( applied biosystems ) .
rpe - myrakt - er cells were transfected using fugene ( roche ) according to manufacturer 's instructions .
twenty - four hours post transfection , cells were placed into medium containing 1% lpds and induced with 100 nm 4-oht as indicated . for gene - silencing experiments
, cells were transfected using dharmafect reagent 1 ( dharmacon ) following a reverse transfection protocol .
twenty - four hours post transfection , cells were split and divided into parallel cultures for different treatments .
sirna sequences are provided in the supplemental experimental procedures . for depletion experiments in kc167 cells ,
dsrnas were generated by in vitro transcription using the megascript t7 transcription kit ( ambion ) .
silencing was performed using 25 g of dsrna , and cells were analyzed after 5 days .
a sequence comprising 700 bp of the 5 end of the dsrebp gene was amplified by pcr from genomic dna and inserted as an inverted repeat into pwiz - uast ( lee and carthew , 2003 ) .
uas - dp110wt , uas - dp110[kd ] , and dpp - gal4/uas - dp110[kd ] fly lines were described previously ( weinkove et al . , 1999 ) .
ms1096-gal4/uas - dp110wt flies were a gift from ernst hafen ( eth , zuerich ) .
dsrebp flies were a gift from robert b. rawson ( university of texas southwestern medical center , dallas ) .
wings of male flies were dehydrated in ethanol , mounted in euparal ( agar scientific ) , and photographed .
cell number was determined by counting wing hairs in an area of fixed size in anterior or posterior wing compartments . unless stated ,
p values were calculated using a two - tailed student 's t test assuming equal variance . | summarycell growth ( accumulation of mass ) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules .
the pi3-kinase / akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin ( torc1 ) .
here we show that akt - dependent lipogenesis requires mtorc1 activity .
furthermore , nuclear accumulation of the mature form of the sterol responsive element binding protein ( srebp1 ) and expression of srebp target genes was blocked by the mtorc1 inhibitor rapamycin .
we also show that silencing of srebp blocks akt - dependent lipogenesis and attenuates the increase in cell size in response to akt activation in vitro .
silencing of dsrebp in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dpi3k .
our results suggest that the pi3k / akt / tor pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth . |
vascular complications after percutaneous renal biopsy are uncommon with macroscopic hematuria occurring in up to 3.5% of patients .
most are self - limited with blood transfusion needed in only about 0.9% and radiologic intervention in 0.6% cases .
the pathology seen on angiography in most cases is either renal arterial pseudoaneurysm ( psa ) or arterio - venous fistula ( avf ) .
rarely , aorta , lumbar artery and superior mesenteric artery may be injured during biopsy . but injury to left colic artery is very rare with only one case reported in literature .
we report a case of left colic artery psa developing after renal biopsy , which was successfully embolized using microcoils .
a 60-year - old female presented to the nephrology outpatient clinic with history of pedal edema and periorbital swelling and abdominal distension of 3 months duration .
lab investigation revealed blood urea of 124 mg / dl , serum creatinine of 3.5 g / dl and estimated glomerular filtration rate of 35 ml / min/1.73 mm .
subsequently a renal biopsy was done in the nephrology ward by conventional technique ( without ultrasound guidance ) .
after renal biopsy , she developed abdominal pain and gradually increasing swelling in left flank region .
the patient 's monitor revealed tachycardia ( pulse rate 132/min ) and low blood pressure ( 100/70 mm hg ) .
there was no obvious psa in the left kidney . with a suspicion of renal artery psa , digital subtraction angiography ( dsa ) of left renal artery
when even after 48 h patient 's symptoms did not improve , computed tomography ( ct ) angiography was done , which showed a large hematoma in the left retroperitoneal region and a psa arising from a branch of inferior mesenteric artery [ figure 1 ] .
the psa was not seen in the arterial phase and was better seen on the venous phase due to its narrow neck .
the patient was immediately shifted to angiography suite and dsa was done through transfemoral route .
inferior mesenteric artery dsa showed the psa arising from the ascending branch of left colic artery [ figure 2a and b ] .
a 2.7 fr microcatheter was used to reach distal to the neck of psa and the segment adjacent to the neck of psa was embolized using microcoils .
post embolization , dsa showed no opacification of the psa [ figure 2c ] . the vascular arcade supplying the colon showed normal opacification through collaterals .
( a ) arterial phase computed tomography ( ct ) scan shows a large hematoma ( * ) in the left anterior pararenal space .
( b ) axial and ( c ) coronal ct images in venous phase shows the enhancing psa on the anterior aspect of the hematoma ( arrow ) .
an attenuated artery is seen feeding the psa ( arrow heads ) digital subtraction angiography .
( a ) inferior mesenteric artery angiogram shows pseudoaneurysm ( psa ) arising from the left colic branch ( arrow ; psa not fully included in the frame ) .
( b ) selective angiogram with microcatheter shows the narrow neck ( arrow head ) of the psa ( arrow ) .
( c ) angiogram after placement of microcoils ( black arrow ) shows complete embolization of the psa .
vascular arcade of the colon is preserved , seen through collateral circulation ( white arrows )
percutaneous renal biopsy , when done under ultrasonographic guidance , is rarely associated with complications .
perinephric or retroperitoneal hematoma is seen in up to 17% patients when an ultrasonography is done in post - biopsy period .
higher rates of bleeding are seen with the use of larger bore needles , higher serum creatinine values and presence of acute kidney injury .
the incidence of psa and avf developing after percutaneous renal biopsy is rare , occurring in about 12% cases .
most of the avfs remain asymptomatic or resolve spontaneously on follow - up , and intervention is necessary only if they become symptomatic .
pseudoaneurysms developing after renal biopsy are very rare , although exact incidence is not known in literature . as with other psa ,
renal psa are also unstable and may often rupture into the pelvicalyceal system or perinephric space resulting in hematuria or hematoma .
psa of extra - renal arteries are very rare , with a few case reports of injury to aorta , superior mesenteric artery , lumbar artery and left colic artery .
the left colic artery arises from the inferior mesenteric artery and divides into ascending and descending branches and the former courses anterior to the left kidney toward the descending colon and anastomoses with the branches of middle colic artery and first sigmoid artery to form the vascular arcade . due to its location close to left kidney
, it may rarely be at risk of injury during biopsy , especially when biopsy is done without guidance .
the patients of extra - renal arterial involvement develop retroperitoneal hematoma or hemoperitoneum rather than hematuria and may present with hypotension , pain / swelling and/or drop in hemoglobin .
although ultrasonography with color doppler may be useful in some cases , most of the patients with persistent symptoms beyond 2448 h need ct angiography for evaluation of renal and extra - renal arteries .
adequate precautions are needed while performing ct scan as iodinated contrast may further deteriorate the already compromised renal function .
these include adequate hydration , use of iso - osmolar contrast agent and to perform hemodialysis after the scan .
ct angiography will demonstrate any psa or avf and/or any leak of contrast and also helps in providing a road map for angiography and embolization .
dsa can also be done as a first line of investigation without ct angiography as catheterization and angiography of the renal artery will provide information about the abnormality better than ct scan and also allows embolization in the same sitting .
also , it helps in avoiding additional dose of iodinated contrast agent used in ct scan .
however , if small accessory renal or extra - renal artery is the source of bleed , it may be missed , as angiogram of only renal artery of the involved kidney is done and it would be a tedious process to search for other arteries .
superselective catheterization of the branch / artery supplying the psa must be done for better demonstration of neck of psa and for successful embolization with minimal complications .
embolization agent most widely used is coil / microcoil . as the renal artery is an end artery ,
however , in case of mesenteric arteries , due to collateral supply , occlusion of distal and proximal segment across the neck of psa is needed for successful embolization .
also , coil embolization of shortest segment of the mesenteric artery feeding the psa prevents ischemia to the bowel due to collateral circulation . in our case , we initially did not expect extra - renal artery involvement and hence we performed left renal dsa without ct angiography to avoid administering higher iodine contrast and contrast induced nephropathy .
but since the patient did not improve , ct angiography was needed which showed psa from left colic artery , which was successfully embolized using microcoils . however ,
such complications are very rare and ct angiography as first line of investigation should be done only in selected patients .
extra - renal artery psa after renal biopsy is extremely rare and should be considered in a patient with retroperitoneal hematoma and without hematuria . | vascular complications after percutaneous renal biopsy are uncommon and may require interventional management . in most of these cases ,
the pathology is a renal arterial pseudoaneurysm ( psa ) or an arterio - venous fistula .
injury to other vessels like aorta , lumbar arteries or mesenteric arteries is rare with only one case of left colic artery psa reported in literature .
we report a case of a 60-year - old female , who developed left colic artery psa after renal biopsy , which was successfully embolized through endovascular route using microcoils . |
superior mesenteric artery ( sma ) syndrome , also called wilkie 's syndrome , is a rare clinical phenomenon believed to be caused by compression of the third portion of the duodenum by the overlying superior mesenteric artery . an abnormally acute aorta - sma angle , or high retroperitoneal attachment of the ligament of treitz , or both ,
this phenomenon has been recognized as a named clinical entity after its original description in 1861 .
the incidence of duodenal compression within the aorto - sma angle has been estimated to be as high as 0.3% from upper gastrointestinal barium swallow studies , but the incidence of clinically significant and appropriately confirmed disease has been estimated to be much lower ( range , 0.010.08% ) .
acceptance of this cause as a clinically reversible pathology has been controversial , but recent evolution of the literature seems to be clarifying the issue .
medically refractory cases have in the past been treated by open release of the ligament of treitz , allowing mobilization of the duodenum or by enteric bypass . as we move into the minimally invasive era , operative intervention for the disease can and
, we describe laparoscopic duodenojejunostomy to successfully treat sma syndrome and review the literature on the subject .
a 32-year - old woman presented with a 5-year history of vague epigastric pain that intermittently radiated to the back .
she had at that time been found on 2 occasions to have elevated pancreatic enzymes and was treated conservatively for pancreatitis .
extensive workup at that time including ct scan , mri , and ercp revealed no identifiable anatomic abnormalities . at the time of presentation to our institution , 3 years had passed , and the patient had persistent chronic pain and progressively worsening nausea . over the previous 6 months
her epigastric pain increased with meals , and she subsequently developed profuse vomiting after large meals without antecedent symptoms . in an attempt to alleviate these symptoms , she would eat smaller meals and consume fewer liquids , which led to dehydration , fatigue , and constipation .
a ct scan revealed a 4-cm dilated duodenal bulb with abrupt decompression to normal duodenum beyond the sma ( figure 1 ) .
the distance between the sma and aorta at the level of the duodenal lumen was 7.2 mm .
an upper gastrointestinal swallow study revealed a dilated proximal duodenum with an abrupt vertical cutoff ( figure 2 ) . during fluoroscopy , there was significant to - and - fro peristalsis of the second and third portions of the duodenum against an apparent obstruction , with small jets of contrast squirting through .
after contrast went through , a narrow lumen at the point of obstruction with distal decompressed bowel could be seen ( figure 3 ) .
the diagnosis of sma syndrome was assumed , and the patient was counseled on laparoscopic duodenojejunostomy . computed tomography of the abdomen revealed dilated proximal duodenum ( large white arrow ) , decompressed distal duodenum ( large black arrow ) , and narrow distance between the superior mesenteric artery and aorta ( small double arrow ) .
contrast study before surgery demonstrating dilated proximal duodenum with a sharp vertical cutoff ( arrow ) .
delayed images showing narrowed duodenal lumen at the level of obstruction ( large arrow ) with distal decompression ( small arrow ) . at operation , the patient was placed in the supine position with both arms tucked .
insufflation was attained by using an insufflation needle ( ethicon pneumoneedle , ethicon endo - surgery inc . ,
a 10-mm optiview port ( ethicon non - bladed trocar 10/12 , ethicon endo - surgery , cincinnati , oh ) containing a 10-mm , 0-degree scope with the camera focused to the tip of the port was used to enter the abdomen under direct visualization .
a 12-mm port was then placed in the left mid subcostal region with a 5-mm port between the two .
the camera was then switched for a 10-mm , 30-degree lens , and the handle was attached to the robotic endoscopic positioning system for audio - activated control ( aesop , computer motion co. , sunnyvale , ca ) .
omentum and transverse colon were retracted cephalad , revealing the large dilated duodenum bulging through a thin , attenuated mesocolon , which was incised to expose the surface of the duodenum .
the ligament of treitz was identified , and the proximal jejunum was run approximately 20 cm to the first loop that could be brought easily to the dilated duodenum without tension . utilizing a 2 0 surgidac suture on the laparoscopic stitching device ( ussc endostitch 173016 , us surgical corp . , norwalk , ct ) ,
a running suture was placed to secure the 2 limbs of bowel and serve as the back row in preparation for a 2-layer , side - to - side anastomosis .
the duodenum and jejunum were entered with the harmonic shears ( ultracision harmonic scalpel , ethicon endo - surgery , inc . ,
cincinnati , oh ) approximately 3 mm above the posterior row , and the stapling device ( 2.5 mm/45 mm ) was deployed .
the defect was closed with running absorbable suture using the suture device to complete the inner layer of the anastomosis .
the outer layer was then completed with a running nonabsorbable stitch joining the ends of the originally placed posterior row .
one intraperitoneal drain was left at the anastomosis and brought out through the right subcostal port site .
a swallow study performed on postoperative day 1 revealed no evidence of leakage or stenosis , and the patient was started on a clear liquid diet and advanced to pureed foods over the next 2 days prior to discharge on day 3 . on follow - up
a 32-year - old woman presented with a 5-year history of vague epigastric pain that intermittently radiated to the back .
she had at that time been found on 2 occasions to have elevated pancreatic enzymes and was treated conservatively for pancreatitis .
extensive workup at that time including ct scan , mri , and ercp revealed no identifiable anatomic abnormalities . at the time of presentation to our institution , 3 years had passed , and the patient had persistent chronic pain and progressively worsening nausea . over the previous 6 months
her epigastric pain increased with meals , and she subsequently developed profuse vomiting after large meals without antecedent symptoms . in an attempt to alleviate these symptoms , she would eat smaller meals and consume fewer liquids , which led to dehydration , fatigue , and constipation .
a ct scan revealed a 4-cm dilated duodenal bulb with abrupt decompression to normal duodenum beyond the sma ( figure 1 ) .
the distance between the sma and aorta at the level of the duodenal lumen was 7.2 mm .
an upper gastrointestinal swallow study revealed a dilated proximal duodenum with an abrupt vertical cutoff ( figure 2 ) . during fluoroscopy , there was significant to - and - fro peristalsis of the second and third portions of the duodenum against an apparent obstruction , with small jets of contrast squirting through .
after contrast went through , a narrow lumen at the point of obstruction with distal decompressed bowel could be seen ( figure 3 ) .
the diagnosis of sma syndrome was assumed , and the patient was counseled on laparoscopic duodenojejunostomy . computed tomography of the abdomen revealed dilated proximal duodenum ( large white arrow ) , decompressed distal duodenum ( large black arrow ) , and narrow distance between the superior mesenteric artery and aorta ( small double arrow ) .
contrast study before surgery demonstrating dilated proximal duodenum with a sharp vertical cutoff ( arrow ) .
delayed images showing narrowed duodenal lumen at the level of obstruction ( large arrow ) with distal decompression ( small arrow ) .
at operation , the patient was placed in the supine position with both arms tucked .
insufflation was attained by using an insufflation needle ( ethicon pneumoneedle , ethicon endo - surgery inc . ,
a 10-mm optiview port ( ethicon non - bladed trocar 10/12 , ethicon endo - surgery , cincinnati , oh ) containing a 10-mm , 0-degree scope with the camera focused to the tip of the port was used to enter the abdomen under direct visualization .
a 12-mm port was then placed in the left mid subcostal region with a 5-mm port between the two .
the camera was then switched for a 10-mm , 30-degree lens , and the handle was attached to the robotic endoscopic positioning system for audio - activated control ( aesop , computer motion co. , sunnyvale , ca ) .
omentum and transverse colon were retracted cephalad , revealing the large dilated duodenum bulging through a thin , attenuated mesocolon , which was incised to expose the surface of the duodenum .
the ligament of treitz was identified , and the proximal jejunum was run approximately 20 cm to the first loop that could be brought easily to the dilated duodenum without tension . utilizing a 2 0 surgidac suture on the laparoscopic stitching device ( ussc endostitch 173016 , us surgical corp . , norwalk , ct ) ,
a running suture was placed to secure the 2 limbs of bowel and serve as the back row in preparation for a 2-layer , side - to - side anastomosis .
the duodenum and jejunum were entered with the harmonic shears ( ultracision harmonic scalpel , ethicon endo - surgery , inc . ,
cincinnati , oh ) approximately 3 mm above the posterior row , and the stapling device ( 2.5 mm/45 mm ) was deployed .
the defect was closed with running absorbable suture using the suture device to complete the inner layer of the anastomosis .
the outer layer was then completed with a running nonabsorbable stitch joining the ends of the originally placed posterior row .
one intraperitoneal drain was left at the anastomosis and brought out through the right subcostal port site .
a swallow study performed on postoperative day 1 revealed no evidence of leakage or stenosis , and the patient was started on a clear liquid diet and advanced to pureed foods over the next 2 days prior to discharge on day 3 . on follow - up , the patient denies pain and nausea and enjoys a regular diet without symptoms .
the abnormally narrow duodenal lumen as it passes under the sma may be secondary to an acute angle between the sma and aorta or a high - riding ligament of treitz wedging the intersecting segment of duodenum tight into the angle .
the angle is between 38 degrees and 65 degrees in normal individuals , but may be well less than 10 degrees in patients with sma syndrome .
expressed another way , the distance between the vessels at the level of the duodenum is normally between 10 mm to 28 mm , whereas patients with symptomatic compression have been found to have a mean distance of 6 mm .
excessive wasting of retroperitoneal fat has also been attributed to the development of this rare syndrome .
reports of abnormally low body fat causing sma syndrome need to be viewed with caution given that the gastrointestinal difficulties of these patients leading to weight loss could blur the cause - effect relationship . if dangerously low body weight were a primary cause , it could be plausibly explained by decreased retroperitoneal fat allowing the small bowel and sma to lie more posterior , narrowing the vascular angle .
reported cases of successfully reversing symptoms with aggressive caloric augmentation have been attributed to reversing this variable and simultaneously lend credence to the notion that in fact low body weight may be a primary cause .
the vascular angle may be iatrogenically narrowed after an operation that fixes the sma into a more posterior position , and this syndrome is reported after ileo - anal anastomosis .
expectedly , because a normal , functional intersection between the duodenum and the vascular angle is dependent upon normal anatomy in 3 dimensions , spinal deformities have been associated with the disease . particularly , surgical correction of congenital spinal deformities alters the natural position of the aorta via iatrogenic hyperlordosis , thereby narrowing the angle .
recent epidemiologic evidence analyzing patients who underwent posterior spinal fusion for scoliosis identified those patients whose weight percentile for height is under 5% to be at risk for developing postoperative sma syndrome .
paralysis and full - body casts have been associated with the development of acute sma syndrome in previously asymptomatic patients .
casted / paralyzed patients may experience duodenal compression by the overlying sma when they suddenly are subjected to prolonged supine positioning .
this notion is supported by the development of sma syndrome in other types of patients subjected to prolonged supine periods , such as trauma and burn patients .
further , immobilized patients with newly diagnosed sma syndrome have been successfully treated by simply altering positions .
patients with idiopathic sma syndrome also experience relief from postural changes , although this will not alleviate the disease .
however , as many as 40% of patients have no apparent explainable cause , similar to the case described here . in these patients , it is unclear why onset is delayed until early adulthood without evidence of an anatomy - altering event . the great majority of patients will present before age 50 .
perhaps natural changes in the gi tract and body habitus with time produce the syndrome in patients fated to have it .
familial cases have been reported including a recent report of monozygotic twins who developed idiopathic sma syndrome at ages 28 and 29 .
diagnosis currently rests mostly on upper gastrointestinal series and computed tomographic ( ct ) scans .
features of sma syndrome on upper gastrointestinal series are a dilated proximal duodenum and vertical or oblique compression of the third portion of the duodenum .
flouroscopy during the swallow study was highly suggestive in this case with a to - and - fro pattern of peristalsis proximal to the obstruction and only small jets of contrast squirting beyond the obstruction .
this specific fluoroscopic pattern has been previously described with sma syndrome , and it appears to offer a strong indication for the presence of the disease . historically , angiography was recommended .
however , it currently offers little information that can not be obtained by ct , it poses significantly greater risks , and it was unnecessary in this case . with appropriate contrast
, the ct angiography can be generated in difficult cases with coronal and sagittal reconstruction showing the precise anatomic associations with the vascular angle , the measurement of this angle , and the distance between the vessels . in our case , abdominal ct with intravenous contrast and upper gastrointestinal series were the only studies required for diagnosis .
although endoscopy is of minor positive diagnostic value , we feel it is mandatory in all patients to rule out intraluminal pathology before either making the diagnosis or initiating treatment .
management of sma syndrome can be initially conservative with nasogastric decompression , intravenous rehydration , and aggressive nutritional support .
symptoms in immobilized patients first should be considered related to position and treated accordingly . in idiopathic cases similar to ours of patients who are functioning outside of the hospital but are symptomatic , nonoperative options for definitive treatment are not very realistic , and upon confirming the diagnosis , we feel it is appropriate to discuss surgical options .
current surgical options for the disease involve 2 separate concepts : bypass of the obstruction or lysis of the ligament of treitz .
liberation of the ligament of treitz has been historically used with success in small numbers of patients . while this operation is advantageous because enterotomy is avoided , the largest comparative study between the 2 operations demonstrated that 21% of patients failed to respond to lysis of the ligament , whereas all patients treated with bypass experienced resolution of symptoms .
recurrence after ligament liberation occurs simply as a result of postoperative adhesions tethering the bowel into a similar position as the ligament of treitz had done previously .
recurrence has been described after duodenojejunostomy when the entire anastomosis migrated under the sma recreating the obstruction , obviously an unusual event . since the introduction of minimally invasive surgery , the results of the open era have thus far been replicated with laparoscopy , albeit in smaller numbers .
laparoscopic lysis of the ligament has been successful in 3 of 4 cases ( 75% ) .
there have now been multiple published cases of laparoscopic bypass that have been performed with sucess .
our experience reinforces the idea that laparoscopic duodenojejunostomy should be offered to patients with a diagnosis of sma syndrome early in the course , because conservative management is unlikely to provide long - term satisfaction .
sma syndrome is a real anatomic clinical pathology resulting in chronic , consistent obstructive symptoms .
laparoscopic duodenojejunostomy should be considered the treatment of choice for these patients because it offers a high likelihood of an excellent outcome based on the current literature . | background : superior mesenteric artery ( sma ) syndrome , also called wilkie 's syndrome , is a rare clinical phenomenon believed to be caused by compression of the third portion of the duodenum by the overlying superior mesenteric artery .
we present the case of a 32-year - old female who presented with epigastric pain , weight loss , and vomiting.methods:her workup included a normal upper endoscopy as well as an abdominal ct scan and upper gi contrast study that confirmed the diagnosis of superior mesenteric artery syndrome .
the patient was taken to the operating room and underwent successful treatment with laparoscopic duodenojejunostomy.results:the patient achieved complete relief of her symptoms and is able to eat a regular diet without difficulty .
sma syndrome is a real anatomic clinical pathology resulting in chronic , consistent obstructive symptoms .
an upper gi series and ct scan with contrast can confirm the diagnosis.conclusion:laparoscopic duodenojejunostomy should be considered the treatment of choice for these patients , because it offers a high likelihood of excellent outcome based on the current literature . |
nowadays , the number of individuals with cerebral palsy ( cp ) that survive into adulthood has increased , since life expectancy of children with cp has increased dramatically and their mortality rate has significantly reduced ( 1 ) .
consequences of cerebral palsy in adulthood can affect physical , psychological capabilities and quality of life ( qol ) ( 1 ) .
one of the most common motor impairments in individual with cp is spasticity ( 2 ) .
uncontrolled spasticity affects on joint alignment , activity of daily living and social participation ( 4 ) .
studies have shown that adults with cp suffering from degenerative joints diseases , musculoskeletal pain , fatigue , joint deformities and functional mobility deterioration ( 5 , 6 ) .
moreover , other problems of adults with cp are job finding , marriage and aging ( 8) .
the most commonly applied physiotherapy techniques for cp patients are based on the reduction of impairments and improvement of function however both functional status and quality of life of cp patients are affected ( 9 )
. world health organization quality of life group defines qol as individuals perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals , expectations , standards and concerns ( 10 ) .
qol scores of adult with cp are significantly lower than healthy people ( 11 ) .
studies showed that cp children with better gross motor functioning perceived a higher physical qol ( 12 , 13 ) .
ophiem et al reported that physical component correlated negatively with musculoskeletal pain in adults with cp , and there was hardly any correlation between pain and psychological component ( 14 ) .
vanderslot et al showed a positive relationship between level of motor function and physical aspect of health related quality of life ( hrqol ) ( 15 ) .
furthermore , young et al reported level of motor function was responsible for 45/2% of variance in qol scores in adults with cp ( 16 ) .
in contrast , tarsuslu et al and gaskin and morris found no correlation between hrqol and the functioning level in adult population ( 17 )
. therefore results of studies were conducted in adult with cp in the field of association between qol or hrqol and level of motor function are inconsistent .
also seldom studies exist that have explored relationship between spasticity and qol in adult cp people .
determination of the association between spasticity and the level of motor function with qol could be an evidence for the usefulness of therapeutic interventions .
the purpose of the present article was to investigate the relationship between quality of life with spasticity and level of motor function in community dwelling iranian young adults with spastic cerebral palsy .
seventy - seven adults with cp were enrolled in this cross - sectional and analytical study .
inclusion criteria were diagnosis of spastic cp by a physician , age between 20 and 40 years and lack of any other neurological condition including neuropathies and myopathies .
subjects were excluded if they had a history of using inhibitory plasters , fractures , sprain or strain injury of their legs in the past 6 months and scored less than 18 on the folstein mini - mental status test ( mmse ) ( 18 ) .
all assessments were completed by a physiotherapist during a single session and were performed in raad rehabilitation goodwill complexes .
the ethic committee of the tehran university of medical sciences and health services approved the study .
demographic data including age , gender , height , weight , marital status and type of spastic cp ( unilateral and bilateral ) , according to the classification system by of surveillance of cerebral palsy in europe ( scpe)(19 ) and history of taking anti spasticity drug or injection of botolinum toxin type a or any other surgery within the 12 month period were obtained through patients records . to determine the level of motor function or severity of motor impairment
the gmfcs is a 5-level classification system which describes motor performance on the basis of functional sitting and walking ability and need for assistive technology and wheeled mobility .
individuals in level 1 walk independently while an individual in level 5 has no independent mobility except with significant assistive devices ( 20 ) . in recent years
, it has been reported that gmfcs is a reliable and valid tool for classifying gross motor functioning in adults with cp ( 20 ) .
mts grades the quality of muscle reaction into 5 levels ( table 1 ) ( 21 ) . to measure spasticity ,
when the grade of muscle reaction reached two or more , movement was repeated to measure an angle of catch or clonus with hand held gonoiometer ( 21 ) .
quality of muscle reaction ( gracies et al 2001 ) . to test spasticity of knee flexor muscles ,
participants were lied in supine position on a bed , and their hips flexed at 90 and the knees allowed to flex fully under gravity . while one hand of the therapist stabilized the thigh , and the other held under the ankle , so the knee could be moved into the position of maximal extension with a fast stretching velocity ( as fast as possible ) .
when mts score was detected two or more , movement was repeated to obtain an angle of spasticity . in order to apply goniometer ,
axis was put over the lateral epicondyle of the femur , with stationary arm parallel to long axis femur and movable arm parallel to long axis of tibia . an assistant held lower limb in this angles and the rater measured given angle by goniometer .
the affected knee was tested in adults with unilateral involvement while the more affected knee was measured in those with bilateral involvement .
the quality of life was measured by the world health organization quality of life questionnaire ( whoqol- bref ) ; it consists of 26 items and envelops four qol dimensions : physical health ( 7 items ) , psychological health ( 6 items ) , social relationships ( 3 items ) , and environmental health ( 8 items ) along with two overall qol and general health items . to score each item within every domain
the whoqol - bref has been shown to have a good - to - excellent reliability and an acceptable validity in various groups of participants in iran ( 23 ) .
participant filled out the whoqol - bref questionnaire on their own . in this study ,
spasticity of knee flexor muscles was measured because knee flexor muscles spasticity can greatly interfere with mobility .
each participant was given a familiarization trial before the first test session and all assessments were performed randomly .
in addition , the relationship between quality of life with spasticity , contracture and level of motor function were assessed using pearson 's product moment correlation coefficient ( r ) and spearman 's rank correlation coefficient .
mann - whitney u and independent sample t tests were used to compare mean scores of qol between the two genders .
analyses were performed with spss 19 for windows . the kolmogorov - smirnov test was used to test the normal distribution of variables . in addition , the relationship between quality of life with spasticity , contracture and level of motor function were assessed using pearson 's product moment correlation coefficient ( r ) and spearman 's rank correlation coefficient .
mann - whitney u and independent sample t tests were used to compare mean scores of qol between the two genders .
table 2 presents demographic and clinical characteristics of 77 young adults with spastic cp . in a pilot study on 30 participants ,
the inter - rater reliability of spasticity angle was very high ( intraclass coefficient ( icc ) value was 0.93 ( p=0.000 ) ) .
also , the intra - rater reliability of spasticity angle in 10 participants was investigated in two sessions over one week .
demographic and clinical characteristics of 77 young adults with spastic cp sd : standard deviation ; n ( % ) : number ( percent ) gmfcs : gross motor function classification system among the 77 spastic cp participants , 43 ( 56% ) had spasticity in knee flexor muscles .
no correlation was found between the severity of knee flexor muscles spasticity with the four domains of qol and the two general items in these 43 individuals ( p > 0.05 ) ( table 3 ) .
correlations between the qol and spasticity in 43 individual with spasticity in knee flexor muscles in addition , no correlation existed between qol and the level of motor function ( p > 0.05 ) .
table 4 shows value of r and signification of the relationship between qol and spasticity and level of motor function in 77 participants with cerebral palsy .
correlations between the qol and level of motor function in 77 individual with cerebral palsy
table 5 presents the results of mann - whitney u and independent sample t tests for comparing mean scores of qol 's domains in two genders .
comparison of mean scores ( sd ) of the qol domains between two genders ( n=77 )
our results showed that spasticity and level of gross motor function were not correlated with qol .
adaptation of our study population to their condition and acceptance of their disability may have resulted due to lack of association between spasticity and qol domains .
in contrast , welmer et al reported a low correlation between the physical aspect of hrqol and spasticity in stroke population ( r < 0.4 , p < 0.05 ) .
they applied the sf-36 and mas to assess hrqol and spasticity of upper and lower extremities in the people 18 months after first stroke ( 26 ) .
therefore , this discrepancy could be related to differences in the measurement tools , type of population and culture .
additionally , wiegerink et al reported that young adults with cp had problems in their sexual relationship ( 27 ) .
while , our subjects did not report any difficulty in the social aspect , because our participants were single and most of them did not answer the sex item in the qol questionnaire .
hence , if all participants answered the sex item of the social domain and we did not have to employ the mean of two other items in this aspect of qol as the score of the sex item , the result of the social domain would have been different . in our study
changes in the level of motor function could not alter qol . although the severity of disability is considered as one of the main factors affecting functional status in both children and adults with cp ( 28 ) .
it seems that adults perception of qol is discordant with the severity of their disability .
furthermore , according to albrecht 's balance theory , the experience of high qol is contingent on the person 's achievement of balance between body , mind and spirit ( 29 ) .
we believe that lack of a relationship between qol and the level of motor function could indicate this balance in spastic cp adults .
our results are consistent with previous studies which examined the relationship between the level of motor function and qol in cp populations ( 17 , 30 ) . on the contrary , vandersolt et al ,
reported that bilateral spastic cp adults who had a better gmfcs showed a higher physical hrqol ( 15 ) .
the hrqol is a subdomain of qol that focuses on physical health and psychological health .
in addition , vargus- adams and ko found that lower gmfcs levels were associated with lower physical health scores ( 12 , 13 ) .
this difference could have resulted from different study samples , qol assessment tools and people who completed qol questionnaire . moreover in our study no difference was found between mean scores of qol in females and males . according to the social and cultural condition of iran , disabled people are supported by family and others who are responsible for performing their roles . thus difference between males and females were disappeared .
our results were consistent with the findings of van der slot et al and ko et al ( 13 , 15 ) .
this study supports lack of relationship between spasticity and level of motor function with qol of community dweller adults with cp . in other word
adults who are community dweller , the need for a family and financial support , employment and improve function in order to enhance their qol .
future studies should focus on the impacts of other impairments ( such as muscle weakness and impaired balance ) on the qol , to identify which impairment can be affect on qol of adult cp who are community dweller .
furthermore , larger studies should be conducted on other age groups and other kinds of cp to determine the relationship between these variables and qol .
also , the relationship between spasticity of other muscle groups in upper and lower limbs and qol should be investigated .
the cross sectional design , convenience sampling and relatively small sample size may be factors affecting the interpretation of the results .
also we can not generalize our results to married adults with spastic cp , people who are imprisoned in the house and other types of cerebral palsy .
this study indicated that although spasticity is a common impairment in adult cp patients , it could not change the qol of adults with spastic cp .
in addition , it seems that limitations in gross motor function did not affect the qol in adults with spastic cp . | backgroundconsequences of cerebral palsy in adulthood can affect physical , psychological capabilities and quality of life .
the purpose of this study was to investigate the relationship between quality of life with spasticity and level of motor function in iranian young adults with spastic cerebral palsy who were community dweller.methodsin an analytical cross sectional study , 77 participants with spastic cerebral palsy ( 44 women , 33 men ) with age range of 20 to 40 years ; ( mean age 26.195 yr ) took part in this study .
they were enrolled from three raad rehabilitation goodwill complexes in tehran and karaj cities .
all subjects were recruited through convenient sampling .
severity of spasticity for knee flexors was measured with modified tardieu scale .
in addition , the level of motor function , and quality of life were assessed respectively through gross motor function classification system and world health organization quality of life questionnaire ( whoqol- bref ) . to analyze data ,
pearson and spearman correlation coefficient was used.resultsno correlation found between quality of life with knee flexor muscles spasticity and level of motor function ( p > 0.05).conclusionquality of life as a multi dimensional concept has been impacted by many factors such as physical status , environmental issues and culture .
possibly , severity of spasticity and level of function have a less pronounced effect on quality of life in community dwelling adults with cerebral palsy . |
they work by competitively inhibiting hmg - coa reductase , the enzyme involved in the conversion of hmg - coa to mevalonate , an early rate - limiting step in the synthesis of cholesterol in the liver . besides the lipid lowering effect of statins in the primary and secondary prevention of coronary artery diseases and heart failure ,
they have been shown to have nonlipid lowering effects , such as in the treatment of infections , alzheimer 's disease , stroke , and rheumatoid arthritis .
therefore , statins have become an area of research in the battle for management or prevention of many detrimental diseases .
the most frequently used statins in saudi arabia are simvastatin and atorvastatin . both medications proved to have a ldl - c lowering effect of > 25% and 66% , respectively .
osteoporosis is a leading public health problem and one of the most common diseases among saudi population as it has a prevalence of 23% .
osteoporosis is defined as the reduction in the strength of bone leading to an increased risk of fractures .
the world health organization operationally defines osteoporosis as a bone density that falls 2.5 standard deviations ( sd ) below the mean for young healthy adults of the same gender , also referred to as a t - score of 2.5 .
it is a prohormone rather than a true hormone , because it must be further metabolized to its active form , 1,25-dihydroxyvitamin d , in order to gain biologic activity . in figure 1 , 1,25-dihydroxyvitamin d stimulates the intestinal absorption and decreases urinary excretion of calcium and phosphate , the major mineral constituents of bone .
factors such as uv exposure , race , and dietary intake are all known to affect concentrations of 25ohd in humans .
vitamin d deficiency is defined by most experts as a 25ohd level of less than 25 nmol / l . a level of 25ohd of 25 to 75
nmol / l can be considered an indication of a relative insufficiency of vitamin d , and a level greater than 75 nmol / l can be considered sufficient .
statins have been reported to exempt an unexpected additive effect of stimulating bone formation in animal studies . moreover ,
an improvement in bone mineral density ( bmd ) has been indicated in human participants .
the link between 25ohd status in patients taking statins was illustrated by the significant decrease in vitamin d deficiency after the use of atorvastatin was illustrated in the significant decrease in vitamin d deficiency after the use of atorvastatin .
on the other hand , another study did not support the general beneficial effect of simvastatin on bone .
it is hypothesized that , since cholesterol is a precursor of vitamin d , inhibiting the synthesis of cholesterol will also inhibit the synthesis of vitamin d ( figure 1 ) .
one of the objectives of this study is to assess bone health and statins intake in relation to osteoporosis in hypercholesterolemic patients .
another objective is to evaluate the efficacy of atorvastatin in comparison to simvastatin in relation to bone health .
these objectives will be achieved through evaluating statins ' effect on 25ohd level and bmd at the lumbar spine and femoral neck .
the study protocol was approved by the irb of the center of excellence for osteoporosis research in king fahd medical research center , jeddah , saudi arabia .
participants were recruited randomly from king khalid national guard hospital , king abdulaziz university hospital , and primary healthcare centers .
the primary endpoint of this study is the effects of statins on 25ohd level , bmd at lumbar spine , and bmd at femoral neck .
secondary endpoints include effects on serum 25ohd and bmd at lumbar spine and femoral neck caused by statin dose variation , variation in their duration of use , and intake of calcium and vitamin d supplementation .
participants eligible for the study were aged 18 years or older , on simvastatin or atorvastatin , in a dose range of 1060 mg / day of either medication , and as a monotherapy for hypercholesterolemia for duration of one year or longer .
we excluded participants on simvastatin or atorvastatin for less than one year , hypercholesterolemic subjects who are on lipid lowering medications in addition to atorvastatin or simvastatin or on statins other than the study medications .
pregnant or breastfeeding women and participants with abnormal liver or renal function tests were also excluded from the study .
subjects were recruited from king abdulaziz university hospital , king khalid national guard hospital , and various primary healthcare clinics ( figure 2 ) .
participants in both groups were matched in terms of age , sex , body mass index , and baseline laboratory values .
study participants were divided into a study group , hypercholesterolemic participants taking statins , and a control group , participants not taking statins .
participants in the study and control groups were further subdivided based on calcium and vitamin d supplementation .
four milliliters of venous blood was withdrawn and collected in coagulated tube under standardized conditions .
serum was prepared by centrifugation at 2500 g ( 1957 rpm ) for 10 minutes .
the samples were stored at 85c within 30 minutes of centrifugation until being analyzed for 25ohd levels and other analytes .
bmd ( g / cm ) of the anteroposterior lumbar spine ( l1l4 ) and the means of the proximal right and left femurs ( total and subregions ) were measured using dual - energy x - ray absorptiometry ( dxa ) ( lunar prodigy model , model 8743 bx-1l , lunar corp .
a calibration procedure was performed daily to determine instrument variation using a phantom supplied by the manufacturer .
precision error of the phantom was 0.3% and was less than 1.2% for the spine and less than 2.0% for femoral regions in vivo .
moreover , there was no significant drift over the period of study using the dxa system .
quality control procedures were carried out in accordance with the manufacturer 's recommendations as explained previously .
bmd reference values were based on who criteria : a t - score between 1 and 2.5 indicates osteopenia and a t - score equal to or less than 2.5 indicates osteoporosis .
serum tests were conducted using vitros 250 chemistry system autoanalyzer ( ortho - clinical diagnostics
ldl - c level was obtained by calculation from hdl - c level [ total cholesterol hdl - c ] .
serum 25ohd was measured by direct competitive chemiluminescence immunoassay ( clia ) using a liason autoanalyzer ( diasorin inc . , stillwater , mn , usa ) by inserting 250 l of serum in the enzyme - linked immunosorbent assay ( elisa ) .
other biochemical analytes measured were determined using kits and reagents supplied by ortho - clinical diagnostics , usa , using vitros 250 chemistry autoanalyzer ( ortho - clinical diagnostics
the planned sample size of 172 participants with a ratio of control to study 1 : 1 was calculated on the basis of assumption that the study would have a statistical power of 90% .
sample size calculation based on estimated power was done using gpower version 3.1 ( heinrich - heine - university dsseldorf , dsseldorf , germany ) .
t - test ( independent 2-samples t - test ) was used to compare the results of study and control groups , as well as for within - group analyses .
one way analysis of variance ( anova ) testing was used to compare within - group differences in terms of different doses and durations of statins use .
significant two - sided alpha ( p value ) was set at 0.017 as a correction for multiple testing because three outcome variables ( serum 25ohd , lumbar spine bmd , and femoral bmd ) were studied .
a linear regression model was constructed with each of the three variables as dependent variables and study group , statin type and presence of supplementation ( calcium and vitamin d ) as independent ones .
all statistics were calculated using spss version 17.0 ( spss , inc . , chicago , illinois , usa ) .
out of total 165 participants , 51 were excluded from the study because they did not meet the matching criteria . the remaining 114 participants ( 69% )
were included in the study and were divided into a matching of study and control groups with a ratio of 1 : 1 . as a result ,
there was no significant difference between the two groups in terms of age , body mass index , liver function , serum calcium , magnesium , and inorganic phosphate .
distribution of study group participants among different doses and durations of statins use was similar between simvastatin and atorvastatin groups ( figure 4 ) .
data from lipid profile tests obtained for the study group had confirmed the compliance of most of the participants on statins which was obtained earlier in the study by personal interviews ( figure 5 ) .
results of serum 25ohd of the study group showed a mean of 29.32 18.07 , while the results of the control group were 31.67 17.03 .
no statistical difference was found between the two groups ( p = 0.47 ) ( table 2 ) ( figure 6 ) .
data of lumbar spine bmd revealed a significant difference between the two groups ( p = 0.05 ) with a mean of 1.09 0.15 for the study group and 1.03 0.14 for the control group .
similarly , the bmd at the femoral neck also showed a statistical significance between the two groups with p of 0.02 .
the mean of femoral neck bmd of the study group was 0.93 0.15 , while that of the control group was 0.87 0.11 ( table 2 ) ( figure 6 ) .
regression coefficient was used to demonstrate the association of bmd at lumbar spine and femoral neck to the intake of statins . when dividing the study group into two subgroups , simvastatin and atorvastatin groups
, data showed no significant difference in any of the measured variables , 25ohd , lumbar spine bmd , and femoral neck bmd ( p = 0.35 , 0.92 , 0.57 , resp . ) .
the mean value of serum 25ohd in the simvastatin group was 26.92 13.36 compared to 31.47 21.45 in the atorvastatin group .
the simvastatin group displayed a lumbar spine bmd mean of 1.09 0.17 , while the atorvastatin group had a mean of 1.09 0.14 .
the femoral neck bmd was 0.94 0.18 and 0.92 0.12 for the simvastatin and atorvastatin groups , respectively ( table 3 ) ( figure 6 ) .
using anova testing , variation in doses of statins among study group participants did not exhibit an impact on 25ohd level ( p = 0.23 ) , lumbar spine bmd ( p = 0.09 ) , and femoral neck bmd ( p = 0.21 ) .
furthermore , the differences in the duration of statins use were not associated with significant effects on vitamin d levels ( p = 0.97 ) , lumbar spine bmd ( p = 0.82 ) , and femoral neck bmd ( p = 0.94 ) .
results of this study demonstrated that 25ohd level may be decreased by using statins . within the groups
analyses showed that participants on supplementation ( calcium and vitamin d ) in the control group had higher mean 25ohd level ( 38.14 20.42 ) than those who were not taking supplements in the same group ( 28.17 13.98 ) ( p = 0.06 ) .
in contrast , in the study group , there was no significant difference between the two subgroups with regard to adding calcium and vitamin d supplements ( p = 0.13 ) .
participants who were taking supplements had a serum 25ohd mean of 33.41 21.24 , while those who were not taking supplements showed a mean of 25.89 14.37 ( table 4 ) ( figure 7 ) .
interestingly , statins intake along with supplementation had a positive effect on lumbar spine bmd but not on femoral neck bmd .
the bmd at the lumbar spine of the study group participants who were taking supplementation showed a mean of 1.03 0.13 versus 1.14 0.15 for those who are not on supplementation ( p = 0.004 ) . on the other hand ,
the mean bmd at the femoral neck was 0.9 0.13 for those taking supplements and 0.95 0.17 for those who were not taking supplements ( p = 0.19 ) ( table 4 ) ( figure 7 ) . among control group participants ,
no significant difference was found between subjects on supplementation and those who are not taking supplements in relation to bmd at lumbar spine ( p = 0.41 ) and femoral neck ( p = 0.62 ) .
the mean value of lumbar spine bmd for participants on supplementation was 1.01 0.14 , whereas the femoral neck bmd displayed a mean of 1.05 0.14 .
participants who are not taking supplements had a mean bmd of 0.86 0.11 at the lumbar spine and 0.88 0.11 at the femoral neck ( table 4 ) .
fifty - three participants were excluded , all from the control group , since they could not be matched with participants in the study group based on age and bmi .
data of this study showed that statin intake has no positive effect on 25ohd status .
in fact , cholesterol is the precursor of 7-dehydrocholesterol , which is consequently converted to the active form of vitamin d after the exposure to ultraviolet light ( figure 1 ) .
based on this fact , the inhibition of cholesterol synthesis by statins will lead to further inhibition of vitamin d synthesis .
in contrast , this is in odds with the explanation and conclusion by prez - castrilln et al .
, where he postulated that the increase in 25ohd level by atorvastatin is a result of inhibition of the hmg - coa reductase enzyme , which may increase levels of 7-dehydrocholesterol and increase the synthesis of 25-hydroxycholecalciferol .
although 25ohd level was not improved by statin intake , the bmd values of lumbar spine and femoral neck of participants in the statins group in this study were higher than those of the control group , which can support the theory that statins can improve bone health .
possible mechanisms by which statins increase bmd were illustrated by cruz and gruber , where statins inhibit hmg - coa reductase and consequently will inhibit the cascade that leads to the activation of osteoclasts , bone resorbing cells .
the other possible mechanism is that statins increase the expression of bone morphogenetic protein-2 mrna .
this protein , bone morphogenetic protein-2 , is a growth factor that stimulates osteoblasts proliferation , maturation , and thus , formation of new bone .
in addition , this benefit was also confirmed recently ; rejnmark et al . found that the use of statins is associated with improvement in forearm bmd only , but not in the bmd at lumbar spine and femoral neck , which is in contrast to our study result .
this finding might be due to the specific type of population included in the study by renjmark et al . as they specifically included postmenopausal osteopenic women .
this study revealed that there are no significant differences between the two studied statins , simvastatin and atorvastatin , including their different dosages and durations of use , among study group participants in relation to 25ohd level and bmd at lumbar spine and femoral neck .
given statins ' pleiotropic effects , researchers have hypothesized that those effects might be due to statins stimulating the vitamin d endocrine system either by upregulating 1,25-dihydroxyvitamin d or inhibiting its catabolism . in our study
we only assessed the association between 25ohd level and statin intake ; therefore , such hypothesis could not be rejected . however , a prospective cohort study is warranted to establish a cause - effect relationship
. limitations of this study also included the unmatching between participants regarding their comorbidities and currently used medications other than simvastatin or atorvastatin .
also , liver and kidney functions , diet , and sun exposure were not taken into consideration , which may play important roles in the activation and metabolism of vitamin d. post hoc analysis showed that this study had a power of 76% .
simvastatin and atorvastatin , at any dose for duration of more than one year , have no additive effect on 25ohd level but have a positive effect on the bmd at the lumbar spine and femoral neck . | background . besides lipid - lowering effect of statins
, they have been shown to have nonlipid lowering effects , such as improving bone health .
an improvement in bone mineral density ( bmd ) has been indicated in some studies after the use of statins , in addition to an increase in 25-hydroxyvitamin d ( 25ohd ) level .
the aim of this study is to explore the association between statins and bone health taking into consideration 25ohd level and bmd . methods .
this is a randomized , cross - sectional comparative study .
subjects were divided into two groups , hypercholesterolemic participants taking simvastatin or atorvastatin as the study group and a matched control group not taking statins .
all participants were assessed for serum 25ohd and bmd at lumbar spine and femoral neck .
results .
a total of 114 participants were included in the study , 57 participants in each group .
results of serum 25ohd showed no significant difference between study and control groups ( p = 0.47 ) , while bmd results of lumbar spine and femoral neck showed significant difference ( p = 0.05 and 0.03 , resp . ) .
conclusion .
simvastatin and atorvastatin , at any dose for duration of more than one year , have no additive effect on 25ohd level but have a positive effect on the bmd . |
statistically , urbanization levels are higher in countries that have higher income , and with many studies suggesting a strong correlation between urbanization and income level , it has been a long - standing notion that urbanization promotes economic growth . because of this , accelerating urbanization has been viewed as an important instrument for economic development and reducing regional income disparity in some developing countries including china .
showed that demographic urbanization levels , measured as the share of population living in cities , have no causal effect on national wealth examined across 180 countries . also using demographic indicators , other studies obtained similar conclusion in africa and asia .
however , using demographic data to represent urbanization level might be inappropriate for such analysis because the varying and changing definition of the urban population often makes it incomparable over time and across countries .
urbanization is a multifaceted phenomenon , with profound changes in land use and the built environment , economic structure , social organizations , behavioral and consumption patterns , political and administrative arrangements . even assuming the result obtained by bloom et al . and other studies
do establish that there is no causal linkage between demographic urbanization level and economic growth , this is not sufficient to establish there is no causal linkages between urbanization and economic growth , as there may be causal linkages between other aspects of urbanization and economic growth when measured by different , and perhaps more suitable indicators .
land conversion is one of the key processes that characterizes urbanization , and many studies reveal the process , speed , magnitude , and consequences of land use change accompanying urbanization .
we use the term landscape urbanization to describe and differentiate this process from demographic urbanization which is based on the size or share of population living in cities .
a landscape urbanization indicator , such as the total built - up area in a city , may be a useful substitute for demographic urbanization indicators as it has clear and unified definition , and therefore provides new potential for conducting cross - country time - series analysis , using either statistical data or satellite remote sensing data .
in addition to describing urbanization processes in general , landscape urbanization indicators may reveal linkages between urbanization and economic and ecological processes and environmental consequences that demographic data can not .
many studies argue that economic growth stimulates urban expansion in china or vice versa , but these arguments are generally based on a positive correlation between these two indicators . yet , correlation does not necessarily mean causation .
recent study on land - use drivers in the pearl river delta during 19881996 found a causal linkage from foreign direct investment to urban expansion but not vice versa .
du et al . found a bidirectional causality between urban land expansion and economic growth in an economic and technological development zone in tianjin .
yet , these studies focus on specific city region or urban districts , and there is no analysis illuminating the causal relationships between urban land expansion and economic growth across a range of chinese cities and regions . in this work , we re - examine the causal relationship between urbanization and economic growth in the chinese context , using built - up area as a landscape urbanization indicator .
we assess patterns of urban land expansion and economic growth according to differences in city size , conduct long - term and short - term causal analysis between urban land - use expansion and economic growth , both at city level and provincial level .
to examine whether landscape urbanization boosts economic growth at the provincial and urban level , and whether economic growth causes further landscape urbanization , we examine how built - up area and economic growth changed in different cities , and conduct causality analysis between built - up area and gdp per capita . in this paper
, we use the definition of causality proposed by granger , where variable x is said to be the granger cause of y if at time t , yt+l is better predicted by using past values of x than by not doing so .
our causality test consists of four stages ( see figure 1 for the flowchart ) : ( 1 ) heterogeneous panel unit root tests to test the order of integration of variables ; ( 2 ) pedroni s panel cointegration test to examine the long - run relationship between the variables given that all variables are first - order integrations ; ( 3 ) panel - based vector error correction model ( vecm ) to examine both short- and long - run causality ; and ( 4 ) heterogeneous panel causality test on the first difference of all variables to further verify short - run causality . decisions about statistical significance of test were made on a 5% level .
as previously discussed , we use built - up area as indicator of landscape urbanization . in china ,
built - up area is defined as a largely continuous area covered by urban facilities but may include some isolated facilities such as airports .
it is generated by the ministry of housing and urban - rural development ( mohurd ) based on two sources : ( i ) annual incremental data based on the actual land area that is granted the permission to be developed into urban land in each city each year ; ( ii ) state bureau of surveying and mapping performs actual measurements annually or every other year , integrating aerial photo and/or other remote sensing data and ground surveys .
the department of urban social and economic survey at the national bureau of statistics performs sample investigation in selected cities , to calibrate the initial data produced by the mohurd .
the resulting measure is considered as the best proxy of the real urban area in china.according to he et al . , the relative error between the derived total urban land area and the statistical built - up area data at national scale was less than 2% in 1992 , 1996 , and 1998 , and maximum relative error at province scale did not exceed 10% with most of the provinces less than 3% .
three panels of urban data and one panel of provincial - level data are used for the causality test .
it is common in china that a city includes some counties which are largely rural . in order for the city data to reflect only urban component and
exclude the counties within the city administrative boundaries , our data set exclude county level cities / towns .
the gdp , year - end population , and built - up area excluding counties for the 174 chinese cities ( 19901998 ) in panel u1 come from a single source , cities china 19491998 , and the data for the 135 chinese cities ( 19972006 ) in panel u2 are from the china city statistical yearbook series .
see figure 2 for their spatial distribution map , and supporting information for the list of cities in each panel .
we excluded those cities that do not have the full range of data for the period of interest , and those that have apparent data errors in the original database .
panel u3 consists of 121 cities that appear in both panels u1 and u2 , and thus have time range of 19902006 .
all the gdp data are converted into comparable prices , and gdp per capita is calculated from gdp divided by the year - end population .
we also test whether built - up area expansion contributes to regional economic growth , with a panel ( p ) of built - up area and gdp per capita data ( 19972006 ) for all 34 provincial regions except hong kong , macau , and taiwan , compiled from the china statistical yearbook series .
panel u3 ( 19902006 ) consists of 121 cities that appear in both u1 and u2 panel .
rapid urban expansion is a spatial manifestation of this process , and has become the most significant characteristic of land - use change in china .
urban land conversion is regarded as one of the primary reasons for agricultural land loss . during 1997 to 2006 in china , a total of 12 869 km of land was converted into built - up area . in the same period ,
the average annual growth of built - up area in 135 cities was 5.7 km every year .
for example , during this period , shenzhen city added 66 km of built - up area each year on average .
based on remote sensing image interpretation , growth in urban built - up area of shenzhen city from 2000 to 2007 was dramatic ( figure 3 ) . in order to achieve a better understanding of growth pattern across different cities ,
we have examined the growth of 135 cities in panel u2 during 19972006 according to cities initial size in 1997 .
the average annual growth of gdp per capita , built - up area , and population increase are significantly positively correlated with initial gdp per capita , built - up area , and population size in other words , richer cities , in terms of gdp per capita , and larger cities , in terms of demographic size and landscape size , also gain more income , have larger built - up area expansion , and attract more population than poorer and smaller cities ( figure 4 ) .
it is important to note the cross - effect here , for example , larger cities are gaining more per capita income , and grow faster in terms of total gdp ; while richer cities are expanding more in terms of size .
we have also examined whether this tendency in total amount translates into difference in normalized or marginal growth rate in all measured indicators according to city size .
our results indicate no significant correlation in either case , suggesting that overall , there is no general trend that either smaller cities or larger cities grow faster in terms of growth rate ( see supporting information for the analysis results ) .
rapid urban expansion of shenzhen city from 2000 to 2007 based on remote sensing image interpretation .
red color for urban land , yellow for bare land , green for land with vegetation , blue for water body .
etm+ image on november 1st 2000 , tm image on september 15th 2000 , etm+ slc - off composite data on december 7th and november 30th 2007 are used for this comparison .
average annual growth of gdp per capita , built - up area , and population of 135 chinese cities during 19972006 .
we perform granger causality test to further verify the existence and direction of causality between built - up area and gdp per capita in china .
the results of heterogeneous panel unit root tests indicate that built - up area and gdp per capita in provincial panel and three urban panels are all integrated at order one .
the results from pedroni s method indicate the existence of cointegration between gdp per capita and built - up area in all panels , meaning there is a long - run equilibrium relationship between gdp per capita and built - up area .
once we established that the two variables are cointegrated , we use a panel - based vecm to conduct granger causality tests .
the results indicate that there are long - run bidirectional causalities between gdp per capita and built - up area in all panels ( table 1 ) . in the short - run
, provincial panel has bidirectional causality between built - up area and gdp per capita , namely the growth in built - up area cause growth in gdp per capita , and vice versa .
the results suggest the existence of long - run bidirectional causalities between built - up area and gdp per capita in all panels ; no short - run causality in panel u1 ; bidirectional short - run causalities in panel u2 and panel p ; short - run causal effect from built - up area to gdp per capita in panel u3 .
panel u3 consists of 121 cities that appear in both u1 and u2 panel , annual data during 19902005 .
bu and pgdp stand for built - up area and gdp per capita , respectively .
cases with probability levels ( shown in parentheses ) lower than 0.05 reject the null hypothesis . to further verify the short - run causal linkages , we perform hurlin s heterogeneous panel granger causality test .
the results suggest bidirectional causality between built - up area and gdp per capita for the provincial panel .
panel u1 is too short to apply hurlin s method , the causal effects in u2 panel are not significant , and causal effect from gdp per capita to built - up area in panel u3 only under lag2 model ( table 2 ) .
the results indicate a bidirectional short - run causality between built - up area and gdp per capita in panel p , and a causal effect from gdp per capita to built - up area in panel u3 only under lag2 model .
panel u3 consists of 121 cities that appear in both u1 and u2 panel , annual data during 19902005 .
bu and pgdp stand for built - up area and gdp per capita , respectively .
lag1 , lag2 , lag3 , and lag4 mean hurlin s test model of lag order 1 , 2 , 3 , and 4 , respectively .
cases with probability levels ( shown in parentheses ) lower than 0.05 reject the null hypothesis .
first , there is a long - run bidirectional causality between gdp per capita and built - up area in all panels .
the three urban panel results suggest that there is a positive feedback , where urban land expansion brings about growth in gdp per capita which in turn increases urban land expansion .
this positive feedback may explain the mechanism behind the trends in figure 3 , indicating an accelerating growth in gdp per capita with the growth in the city size .
the provincial panel consists of total urban land expansion within the province and overall gdp per capita of the province including city and rural , so the result at the provincial panel means urban land expansion not only contributes to the city gdp , but also to the overall gdp growth of the province .
this means the effect of urban expansion extends beyond the city boundary , and has spillover influences to the region surrounding the city .
second , our results indicate that the provincial panel has short - run bidirectional causality but do not sufficiently confirm a short - run relationship in any of the three urban panels .
a careful study of the original data suggests that city level built - up area data sometimes increase abruptly due to the effect of large built - up area projects , while at the provincial level these steps are smoothed out by multiple cities . since the short - run relationship measures year - to - year variation , this difference in data trends can cause the lack of a short - run causal effect .
in addition , our urban panels consist of cities that have a full range of data and thus exclude cities that have become prefectural level cities after 1990 ( in u1 and u3 ) ( for example , there were 31 new prefectural level cities between 1990 and 1997 ) or 1997 ( in u2 ) and small cities and towns below prefectural level , while the provincial panel naturally includes all cities within the province regardless of their times of emergence .
it is likely that these new prefecture level cities and small towns and cities below prefectural level have contributed toward a stronger short - run causal effect at the provincial level .
china s legal system on land ownership and land - use rights might contribute to the significant role of landscape urbanization in economic growth and the positive feedback between them in recent decades .
land is publicly owned in china , and land supply becomes a powerful macro - economy intervention tool for chinese governments especially since 2003 .
china has a system of long - term lease of land - use rights , which means government could issue a one - off land lease for a duration of 5070 years , and raise considerable revenue from the leasing . during the six year period of 1999 to 2006 , the total income arising from land - use leasing of state - owned land , which often means converting other land - use types into urban built - up land , grew 15 times , reaching 808 billion yuan , raising its proportion in china s government financial income 3.6 times , reaching nearly 21% , its proportion in china s gdp 5 times to 3.8% .
the income from land leases can account for 3070% of a city s financial revenue .
this income of government has a designated use , with 50% tied to improvement of the leased land such as providing basic infrastructure including water , electricity , internet , wastewater treatment , gas , roads and heating .
these investments directly create jobs in the construction sector , which attracts more people into cities and thus stimulates further urbanization . a better urban infrastructure , in turn , attracts further investment in industries and thus contributes to the economic growth of the city and the region in the long run .
therefore , land acquisition has been used heavily by chinese local governments to fuel urban development and finance infrastructure provision . under this circumstance
our results have significant policy implications for china , revealing a tension between urbanization , environment and economic growth .
landscape urbanization brings economic growth , but at the same time could cause negative environmental impacts leading to serious environmental problems in some chinese cities . on the one hand ,
our results confirm that the national policy of promoting urbanization to enhance economic growth does hit the mark .
since by definition built - up area expansion requires infrastructure investment , our results might highlight the importance of infrastructure investment for urbanization to deliver the anticipated economic growth . on the other hand ,
the positive feedback suggests that china s policy to arrest the loss of agricultural land might be challenged by its own policy to promote economic growth through urbanization .
urbanization takes up large amounts of arable land which presents a major threat for sustainable agricultural production and food security in china . in 2006 , china had 0.09 ha of arable land per capita , which was less than 40% of global average ; meanwhile it lost 0.31 million hectares of arable land , of which 84% was changed into construction land . at this rate of urban expansion , the government designated food security bottom line of 120 million hectares of arable land , is fast approaching .
the close linkages between urban expansion and economic growth indicate that the current economic growth model in urban china is highly dependent on natural resource input , in this case , land .
it is likely that eventually this resource - intensive growth mode will change toward a more service - oriented one and thus require less land input , as experienced in other developed countries .
however , it might be very difficult for china to control urban expansion without sacrificing economic growth , at least under its current economic growth model . while the result only reflects the tendency on an aggregated level and
therefore does not dictate the behavior of each and every individual city should follow the same pattern , empirical evidence does seem to reinforce our findings .
for example , nearly half of the first designated greenbelt in beijing was already changed into built - up area in 2005 , suggesting a strong tendency of urban land - use expansion and the difficulty of preventing it .
empirical evidence in the macro - economic history of china also supports the close linkages between urban expansion and economic growth . in 1999 , the chinese government strived to keep the economic growth rate at 8% after the economic recession caused by the southeast asia financial crisis , and found that urbanization was a good instrument for promoting economic growth . in 2004 ,
the state council subsequently decided to tighten its control over permission for new land development , which has brought about an economic slowdown in some cities and regions .
this challenge is likely to continue into the future . as part of its economic growth strategy
, the chinese government aims to increase demographic urbanization from 43% in 2005 to 47% by 2010 , with an economic growth rate of 7.5% .
this will inevitably increase demand for built - up areas for infrastructure , industry , and residential use .
given the agricultural land scarcity in the country , there is an urgent need for china to identify an alternative pathway that eases the heavy dependent on land resource input . while it is not clear to what extent the causal effect between urban expansion and economic growth is applicable to other contexts than china , putting urban expansion under control is a universally challenging task .
for example , the greenbelt in seoul aimed at containing expansion within its ring resulted in leapfrog development .
bad urban planning or poor implementation of such is often blamed for uncontrolled urban growth .
assuming the existence of a feedback between urban expansion and economic growth is a widespread phenomenon , then the driving force is coming from a different dimension and urban planning alone might not be sufficient to achieve the goal .
our findings imply that compared to a demographic urbanization indicator , a landscape indicator has the potential to better reveal the complex linkages between urbanization and accompanying economic and environmental processes .
built - up area expansion is a spatial manifestation of population , economic , and environmental dynamics of urbanization . as discussed by bloom et al .
, there are different types of urbanization in terms of drivers and economic outcomes , among which demographic indicators alone can not distinguish .
built - up area might differentiate them to some extent . while urban population growth with built - up area expansion might indicate a direct association with economic activities , urban population growth without built - up area expansion
in addition to economic aspects , built - up area also reveals strong environmental linkages through concentration of materials , and varying energy use and ghg emissions according to different urban density and transportation system .
our analysis on urban growth pattern shows larger cities gain more per capita gdp growth , and more and faster in terms of total gdp , which endorses betterncourt et al.s result .
it also shows that richer and larger cities ( both in terms of population size and built - up area ) are expanding more .
these suggest the relationship between city size and wealth creation is not only nonlinear but also bidirectional and complex , which in turn suggests the existence of feedback mechanisms in the growth of cities .
the positive feedback between landscape urbanization process and economic growth identified in this study may reveal partly the complex mechanism of such nonlinear , accelerated growth in city size and wealth .
urban land expansion is not only the consequences of economic growth in cities , but also drivers of such growth .
while it seems urban land expansion can trigger such feedback within the chinese contexts , there might be other types of such positive feedback in work in other context , involving other factors .
uncovering these dynamics and mechanisms of the interaction among different components within an urban system is an important step toward understanding the complexity of cities and urbanization processes .
this will in turn inform a more coherent urbanization , economic and environmental policy system that reflects the true dynamics of cities . | accelerating urbanization has been viewed as an important instrument for economic development and reducing regional income disparity in some developing countries , including china .
recent studies ( bloom et al . 2008 ) indicate that demographic urbanization level has no causal effect on economic growth . however , due to the varying and changing definition of urban population , the use of demographic indicators as a sole representing indicator for urbanization might be misleading . here , we re - examine the causal relationship between urbanization and economic growth in chinese cities and provinces in recent decades , using built - up areas as a landscape urbanization indicator .
our analysis shows that ( 1 ) larger cities , both in terms of population size and built - up area , and richer cities tend to gain more income , have larger built - up area expansion , and attract more population , than poorer cities or smaller cities ; and ( 2 ) that there is a long - term bidirectional causality between urban built - up area expansion and gdp per capita at both city and provincial level , and a short - term bidirectional causality at provincial level , revealing a positive feedback between landscape urbanization and urban and regional economic growth in china .
our results suggest that urbanization , if measured by a landscape indicator , does have causal effect on economic growth in china , both within the city and with spillover effect to the region , and that urban land expansion is not only the consequences of economic growth in cities , but also drivers of such growth .
the results also suggest that under its current economic growth model , it might be difficult for china to control urban expansion without sacrificing economic growth , and china s policy to stop the loss of agricultural land , for food security , might be challenged by its policy to promote economic growth through urbanization . |
they consist of fibrous giant cells that are specifically designed for efficient nerve - controlled shortening .
to switch the contractile machinery on and off , these cells raise and lower their cytoplasmic ca concentration ( for review see melzer et al . , 1995 ) . to allow the concentration increase to occur with sufficient speed ,
skeletal muscle fibers are equipped with a unique mechanism to activate ca release from their ca storage compartment , the sr .
the mechanism involves tight coupling of the probability that ca release channels ( ryanodine receptors [ ryrs ] ) in the sr membrane are open to cell membrane voltage . the muscle fiber membrane is rapidly depolarized by action potentials that propagate from the postsynaptic region of the nerve
muscle endplate throughout the cell surface and into the cell by means of transversal tubular infoldings ( t system ) .
the signal transmission from the t system to the sr is performed by an assembly of proteins in the contact region between the two membrane systems .
a modified l - type ca channel protein ( dihydropyridine receptor [ dhpr ] ) acts as the voltage sensor and transmits a conformational command signal to the ryr ( dirksen , 2002 ) .
besides activating the contractile filaments , the released ca exerts a positive feedback on the opening of the release channels in a process termed ca - induced ca release ( cicr ) .
time - resolved confocal microscopy using fluorescent probes revealed localized ca release events termed ca sparks ( klein et al . , 1996 ) that result from clustered openings of ryrs , probably under the participation of cicr .
in frog muscle , their frequency was found to increase with fiber membrane depolarization , supporting the hypothesis of a voltage - dependent recruitment of ca release quanta ( klein et al . , 1997 ) .
however , to the research community 's disappointment , such quantal events were not detectable in skeletal muscle fibers of mammals , shedding doubts on a general physiological role of spark - based quantal ca release in skeletal muscle .
only muscle fibers whose outer membranes were permeabilized by saponin ( which leaves the sr membrane functional ) showed sparklike events ( kirsch et al . , 2001 ) .
sparks are also more readily seen in developing muscle cells but not in regions of these cells that respond with ca release to membrane depolarization ( shirokova et al . , 1999 ) .
it has been proposed that the dhpr ryr alignment prevents sparks from occurring ( zhou et al . , 2006 ) .
the search for conditions to elicit ca sparks in intact mammalian muscle finally led to success when wang et al .
( 2005 ) demonstrated localized ca fluctuations in isolated mouse muscle fibers challenged with external solutions of different osmolarities .
an increase of osmolarity to 150% , which was synchronous with an increase of the external ca concentration ( by raising cacl2 from 2.5 to 50 mm ) , elicited shrinkage and localized ca transients in the periphery of the fibers .
such signals could also be observed on return to a normosmotic bath solution after a temporary decrease in osmolarity to 60% , which caused cell swelling . in normal mouse muscle fibers ,
in contrast , fibers from the mdx mouse , an animal model of human duchenne muscular dystrophy resulting from lack of the cytoskeletal protein dystrophin , showed persistent , apparently irreversible spark activity .
a defective ca response has long been suggested to be responsible for the death of muscle fibers in this devastating muscle disease ( tutdibi et al . , 1999 ) .
in the present issue of this journal , a new study from the ma laboratory ( weisleder et al . , 2006 ) reports experiments on muscle fibers from mice close to the end of their natural life span ( 2 yr ) .
these fibers exhibit just the opposite effect to that seen in dystrophic muscle ( i.e. , a drastic shortening of the period of spark activity after brief hyposmotic stimulation ) .
weakening of skeletal muscle is one of the burdens accompanying the aging process , and part of the loss of muscle force in old age has been attributed to alterations in the ca release machinery .
for instance , it has been shown that the dhpr / ryr ratio decreases with age ( renganathan et al . , 1997 ) .
the now discovered drastic changes in osmotic stress - provoked local ca response is a new facet of altered ca regulation in aged muscle .
interestingly , weisleder et al . ( 2006 ) observed a very similar phenomenon in young mice deficient in the protein mg29 .
this protein normally sits in the membrane area of the sr that faces the t system ( i.e. , in a region critical for ca handling ) .
it resembles the synaptophysins , which are found in the membrane of synaptic vesicles , and is one of several small proteins that are suspected to modulate the ca release mechanism .
mg29 has been reported to affect store - operated ca entry and the susceptibility of muscle to fatigue ( pan et al . , 2002 ) . because the level of mg29 was found to be reduced by 50% in skeletal muscle of old mice , weisleder et al .
( 2006 ) propose that this protein plays a role in the weaker ca response to osmotic stimulation of aged muscle fibers .
they suggest that the substantially altered spark activity might result from a reduced sensitivity of the ryrs to cicr .
another contributor to the rapid decline of spark frequency could be the reduced load of ca in the sr that was found for muscle fibers of both aged and mg29-deficient mice .
evidence from reconstituted ryrs and from single cell experiments indicates that the concentration of ca on the luminal side of the sr affects the opening probability of ca release channels ( herrmann - frank and lehmann - horn , 1996 ; launikonis et al . , 2006 ) .
calsequestrin , the major luminal ca - binding protein of the sr , has been suggested to serve as a ca sensor to modulate ryr under the participation of triadin and junctin , two further sr proteins of the t system sr junction ( beard et al . , 2005 ) .
( 2006 ) include muscle atrophy and characteristic changes in ultrastructure , indicating a disruption of the alignment of the sr and t system and fragmentation of sr regions . the structural changes and the altered force response to depolarization and caffeine led to the suggestion that the t system loses control of sections of the sr , possibly as a result of the loss of mg29 as an essential component in maintaining sr
t system alignment . in view of the similarity of mg29 with synaptophysin , a membrane protein of still unknown function concentrated in the clear and dense core synaptic vesicles ,
the study of muscle might ultimately be useful for the understanding of regulated exocytosis in neurons and neurosecretory cells .
there is no doubt that the age - related dysfunction of muscle is a complex process , and it remains to be established whether mg29 is indeed a key element in this process , as the new results seem to indicate .
mg29 's possible involvement in the generation of sparks is intriguing and may open a new avenue to understanding the mechanism that links osmotic changes to ca signaling in muscle .
are the sparks observed under these conditions indeed comparable with the stereotyped signals found in other muscle preparations ?
they may simply be artifacts caused by a mechanical perturbation of the membranes leading to local leaks of ca into the cytoplasm .
alternatively , are they part of a signal transduction chain involving specific mechanosensitive elements , perhaps even designed to modulate the ca signal during the mechanical stress of muscle contraction ? | sparks are transient local elevations of ca ion concentration observed in different types of muscle cells .
such local ca2 + signals can be provoked in skeletal muscle cells by altering the osmotic pressure of the extracellular solution . in this issue , weisleder et al .
( see p. 639 ) demonstrate that the ca2 + response to osmotic stress is substantially altered in aged muscle .
the study presents evidence for a link between this finding and a reduced expression of mitsugumin 29 ( mg29 ) , a small membrane protein of the sarcoplasmic reticulum ( sr ) . |
esophageal achalasia is a primary esophageal motility disorder characterized by lack of peristalsis and failure of the lower esophageal sphincter ( les ) to relax in response to swallowing .
however , degeneration and loss of ganglion cells in the auerbach s nerve plexus have been reported and result in esophageal motor dysfunction .
the diagnosis of achalasia is based on a careful clinical history and the results of barium esophagography , endoscopy , and esophageal manometry .
dysphagia , regurgitation , chest pain , and weight loss are among the most recognized clinical features of the disease .
achalasia is a rare disease , with an estimated annual incidence of one case per 100,000 people . by esophagogastroscopy , we identified esophageal dilation , atony , chaotic spasticity of the esophageal body , food or fluid retention , or resistance to the passage of the endoscope into the stomach .
the main reason to perform endoscopy was to exclude the presence of other pathologies , such as cancer .
patients with achalasia have an increased risk of developing stasis - related squamous cell cancer .
botulinum toxin injection was developed in 1995 , and has been used for many patients in europe and the united states .
botulinum toxin injection is the most common initial treatment for achalasia in the united states and is more efficacious than other treatments such as pneumatic dilation ( pd ) or laparoscopic heller myotomy ( lhm ) . according to one report of 896 patients with achalasia in the united states ,
botulinum toxin injection was the most frequently used as the first - choice endoscopic therapy in 41% .
in japan , esophageal achalasia has been treated with oral drugs , endoscopic balloon dilation , per - oral endoscopic myotomy ( poem ) , and surgery .
however , local injection of botulinum toxin for esophageal achalasia has not been approved as a treatment for medical insurance in japan . as a result ,
we have reported two cases of esophageal achalasia in japan , in which symptoms were improved by localized botulinum toxin injections . in this study , we conducted a pilot trial to evaluate the effectiveness of local injection of botulinum toxin for esophageal achalasia , and to assess its safety and adverse effects among japanese patients .
between january 2008 and december 2014 , 10 patients were diagnosed with esophageal achalasia in saga medical school hospital , japan .
patients were clinically assessed before treatment , including barium esophagography , esophagogastroscopy , and esophageal manometry .
the definition of achalasia by barium esophagography is dilatation of the esophagus and/or liquid or food retention in the esophagus with the lower esophagus having the appearance of a bird s
beak. the maximum diameter of the thoracic esophagus was measured using barium esophagography , and achalasia was classified into grade i , diameter < 3.5 cm ; grade ii , diameter 3.56.0 cm ; and grade iii , diameter > 6.0 cm , in accordance with the descriptive rules .
esophageal manometry analyzed each swallow for the morphology of the esophagogastric junction , the extent of esophagogastric junction relaxation , the contractile front velocity of peristalsis , vigor of the peristaltic contraction , and abnormality of intrabolus pressure .
esophageal manometry was performed in all patients with four - channel manometry ( gmms-600 ; star medical , tokyo , japan ) .
the manometric assembly was passed via an anesthetized nostril and positioned so that the most distal of the 4 cm interval side holes was 1 cm above the les .
the les pressure was measured by the rapid pull - through method , with the subject in the standing position .
baseline recordings of the lower esophageal sphincter pressure ( lesp ) were made for 5 min , after which primary peristalsis was assessed .
the manometric protocol included 5 min to assess basal sphincter pressure and 5-ml water swallows .
vigorous achalasia was defined by amplitude 37 mmhg , and classic achalasia as amplitude < 37 mmhg .
eckardt score was the sum of the symptom scores for dysphagia , regurgitation , and chest pain ( with 0 indicating absence of symptoms , 1 occasional symptoms , 2 daily symptoms , and 3 symptoms at each meal ) , and weight loss ( with 0 indicating no weight loss , 1 loss of < 5 kg , 2 loss of 510 kg , and 3 indicating loss of > 10 kg ) .
thus , the maximum score on the eckardt scale , indicating the most pronounced symptoms , was 12 .
patients were considered to have reached clinical remission if the total symptom score was 3 .
a total of 100 u botulinum toxin a ( botox ; allergan , irvine , ca ) was divided into eight aliquots ( 25 u per quadrant ) , with four injections every 90 degree at the esophagogastric junction and four injections every 90 degree 1 cm above the esophagogastric junction .
after 1 week , we re - measured the shape of the esophagus using barium esophagography and lesp by esophageal manometry .
after 1 week of follow - up of the local injection , the subjective symptoms for esophageal achalasia were assessed using a visual analog scale ( vas ; no symptom 0 , maximum symptoms 10 ) .
patients were periodically followed up at our outpatient clinic . when symptoms of esophageal achalasia relapsed , local injection of botulinum toxin was repeated according to the patients request . the eckardt score , lesp and subjective symptom score data were analyzed using student s t test .
the study was conducted after the protocol had been approved by the ethics review committee of saga medical school .
there were five men and five women , with a mean age of 55 ( 3372 ) years .
two cases had grade i achalasia ( diameter 4 cm ) and eight were grade ii ( diameter 46 cm ) .
four cases had comorbidity of hypertension , diabetes mellitus , and chronic hepatitis c. cases that were treated effectively with botulinum toxin are shown in fig . 1 .
the lower esophagus had a bird s beak appearance and barium did not pass through the esophagogastric junction for more than a few minutes ( fig .
barium passage was improved in barium esophagography and passage of contrast agent was also improved in all cases . before botulinum toxin injection ,
esophageal manometry showed each swallow for the contractile abnormality of peristalsis and markedly elevated residual lesp .
after botulinum toxin injection , esophageal manometry showed improvement of residual lesp , and abnormal peristaltic waves had disappeared .
mean lesp was reduced from 46.9 ( 2871 ) mmhg before treatment to 29.1 ( 1263 ) mmhg after treatment ( 95% ci , 8.51527.095 , p = 0.002 ) ( fig . 2 ) .
mean eckardt score was reduced from 5.5 ( 29 ) before treatment to 1.6 ( 02 ) after treatment [ 95% confidence interval ( ci ) , 2.3025.498 , p<0.001 ] ( fig .
mean score of subjective symptoms was reduced from 10 before treatment to 3.9 1 week after treatment ( 95% ci , 4.8307.370 , p<0.001 ) ( fig . 4 ) .
during follow - up periods , five cases relapsed at 324 months after treatment ( table 2 ) .
the mean score of subjective symptoms was 5.4 ( 47 ) in five recurrent cases and 2.4 ( 14 ) in nonrecurrent cases .
the scores for recurrent cases were significantly higher than those of the nonrecurrent cases ( 95% ci , 1.3374.663 , p<0.001 ) .
three cases relapsed at 3 , 6 and 24 months after treatment ( cases 3 , 5 and 9 ) and received additional botulinum toxin therapy at their request .
treatment was effective until the end of observation . however , one case received pd therapy 6 months after initial treatment ( case 2 ) , and another received poem 3 months after initial treatment ( case 7 ) because of recurrence .
the therapeutic effect in eight cases was conservatively sustained until december 2014 , with mean follow - up of 36 months .
side effects of botulinum toxin injection were not experienced up to december 2014 in all cases .
first , local injection of botulinum toxin was effective for japanese as well as western achalasia patients .
second , botulinum toxin injection was safe compared with several other therapies for esophageal achalasia .
third , a high score for subjective symptoms after treatment became an important indicator of recurrence .
commercially available botulinum toxin is composed of botulinum toxin component ( botulinum neurotoxin and complexing proteins ) and excipients .
botulinum neurotoxin consists of a heavy amino acid chain connected to a lighter amino acid chain by means of a single disulfide bridge .
after injection into the target tissue , the botulinum toxin binds to glycoprotein structures on the cholinergic nerve terminals , and the light chain , a protease , is internalized and cleaves the soluble n - ethylmaleimide - sensitive fusion attachment protein receptor proteins , thereby disabling vesicles from anchoring to the membrane and releasing acetylcholine . this blockage of the cholinergic synapse forces the neurons to form new synapses ; a process known as sprouting
the most commonly used and studied serotype is botulinum toxin a , and achalasia studies have used it exclusively . however , botulinum toxin b is also commercially available . in the united states ,
botulinum toxin injection was most frequently used as the first - choice endoscopic therapy in 41% of cases , followed by balloon dilation in 21% , savary dilation in 20% , maloney dilation in 10% , rigiflex in 4% , and other modalities in 4% .
a previous study has reported that 1 month after botulinum toxin injection , 31 of 37 patients ( 83.7% ) showed significant improvement .
botulinum toxin injection for esophageal achalasia is also useful in japanese patients , as reported in western countries .
however , some studies have reported that most patients who are treated with botulinum toxin injection experience relapse , usually 612 months after the first treatment , and often require repeated treatment .
a meta - analysis of nine studies indicated that the percentage of patients with symptomatic improvement after one session of botulinum toxin injection was 78.7% at 1 month , 70% at 3 months , 53.3% at 6 months , and 40.6% at > 12 months follow - up .
however , at least a second botulinum toxin injection was required in 46.6% of patients .
therefore , botulinum toxin injection is considered to be effective in the short term , but has a risk of relapse .
our study was a small pilot study and the therapeutic effect was conservatively sustained in eight cases and good progress was achieved .
the mean follow - up was 2 years , but we need to extend the surveillance periods and increase the number of patients .
botulinum toxin injection into the les is an established therapy for achalasia and has few side effects .
reported side effects of botulinum toxin injection include mild chest pain and mild heartburn , which is easily controlled by antacid therapy .
rare systemic side effects such as diffuse neuromuscular jitters and autonomic side effects such as dry mouth and eye accommodation difficulties have been reported , especially with high doses of botulinum toxin b. in the present study , there were no serious side effects .
one study has shown that there was no significant difference in remission rates between pd and botulinum toxin at 4 weeks of intervention .
however , pd appeared to be more effective after long - term follow - up of > 6 months . according to five studies that compared botulinum toxin injection with pd ,
the remission rate was 65.8% for pd compared with 36% for botulinum toxin injection , and the time to relapse after the first intervention was shorter for patients who received botulinum toxin compared with pd .
some perforations can be managed nonoperatively , but free perforation requires urgent laparotomy or thoracotomy .
poem is a new endoscopic procedure to treat achalasia , and is a modification of pasricha s submucosal endoscopic esophageal myotomy . in poem ,
visible complete transmural openings into the mediastinum and peritoneal cavity are created in the majority of patients .
therefore , poem potentially carries the risk of mediastinitis , peritonitis and damage to surrounding organs .
the following complications were reported for poem : perforation ( 4% ) , mucosal injury ( 4% ) , bleeding requiring intervention ( 1% ) , and mediastinal hematoma ( 1% ) .
esophageal perforation is the most common major complication for lhm , with an incidence of 04.6% . in another study ,
lhm had complications such as gastrointestinal perforation ( 1.5% ) , pneumothorax ( 1.9% ) , and pulmonary complications ( 1.3% ) .
the difference in safety between botulinum toxin injection and other invasive treatment options such as pd or lhm is arguably the reason why botulinum toxin is the most commonly used initial therapy for achalasia in the united states .
the outcome of botulinum toxin injection was comparable to the outcome of several other therapies for esophageal achalasia , while injection therapy was safer than other therapies .
in our study , the subjective symptoms score in five recurrent cases was significantly higher than in the nonrecurrent cases . however , there was no difference in eckardt score and lesp between the recurrent and nonrecurrent cases .
patients with high subjective symptom scores are likely to experience recurrence within a short time .
. the limitations of our study were its small number of participants and single center analysis .
in addition , there is no universal gold standard technique for injecting botulinum toxin into the les .
a previous study has reported that relapse of symptoms was evident in 19% of patients who received two injections of 100 u compared with 47% and 43% in the single 50 u and 200 u groups , respectively .
further studies are required to validate botulinum toxin injection for achalasia and establish an adequate method of injection . in conclusion ,
botulinum toxin injection for achalasia was safe and effective , with few complications in japanese patients .
therefore , botulinum toxin could be one of the less invasive treatment modalities for esophageal achalasia in japan .
guarantor of the article : daisuke yamaguchi , md . specific author contributions : principal investigator , subject recruitment , subject evaluation , data collection and manuscript preparation : daisuke yamaguchi ; manuscript preparation and statistical analysis : nanae tsuruoka , yasuhisa sakata , ryo shimoda , kazuma fujimoto , ryuichi iwakiri .
| botulinum toxin injection is an accepted treatment modality for esophageal achalasia in western countries .
this pilot study aimed to clarify the effectiveness of botulinum toxin injection for esophageal achalasia in japanese patients .
we enrolled 10 patients diagnosed with esophageal achalasia between 2008 and 2014 .
a total of 100 u botulinum toxin a was divided into eight aliquots and injected around the esophagogastric junction .
we compared the lower esophageal sphincter pressure before and 1 week after treatment .
scores of subjective symptoms for esophageal achalasia were assessed using a visual analog scale ( vas ) before and after 1 week of follow - up of treatment .
barium passage was improved in barium esophagography and passage of contrast agent was also improved .
mean eckardt score was reduced from 5.5 to 1.6 after treatment ( p<0.001 ) . by esophageal manometric study ,
mean lower esophageal sphincter pressure was reduced from 46.9 to 29.1 mmhg after treatment ( p = 0.002 ) .
one week after treatment , mean vas score was reduced from 10 to 3.9 ( p<0.001 ) .
there were no side effects in any cases .
botulinum toxin injection for esophageal achalasia was safe and effective with few complications .
therefore , botulinum toxin could be used as minimally invasive therapy for esophageal achalasia in japan . |
atherosclerosis ( ath ) is an inflammatory disease characterized by intense immunological activity that involves the formation of lesions in the arteries and is characterized by lipid accumulation , inflammation , cell death , and fibrosis .
recruitment of monocytes and lymphocytes from the peripheral blood to the intima of the vessel wall represents the primary event in atherogenesis . among the factors
involved in ath , the local presence of high amounts of low - density lipoproteins ( ldl ) has a key role .
healthy endothelium is an important barrier to the free passage of ldl and immune cells to the underlying interstitium , and dysfunction of endothelial cells ( ec ) is considered an early step in atherogenesis .
ec dysfunction results in increased activation of these cells , sustained by upregulation of adhesion molecules ( ams ) such as vascular cell adhesion molecule-1 ( vcam-1 ) and intercellular adhesion molecule-1 ( icam-1 ) .
vcam-1 expression precedes lesion formation and correlates positively with the extent of exposure to plasma cholesterol .
cells can then change their shape and migrate at the surface of the endothelium to reach the junctions .
recently , we have confirmed the occurrence of extensive alteration of endothelial layers in surgical specimens of patients submitted to carotid endarterectomy [ 5 , 6 ] . in view of
the role played by ec in the development , progression , and clinical complications of ath , reduction of their activation represents a key point in the prevention of atherosclerotic plaque progression .
lower - extremity peripheral artery disease ( pad ) is a chronic arterial disease of atherosclerotic origin characterized by the occurrence of vascular obstruction in various districts .
although public awareness of pad is extremely low , this condition is related to a very high short - term incidence of myocardial ischemia , stroke , and death . besides an aggressive treatment of cardiovascular risk factors , common femoral artery thrombendarterectomy ( fea ) is among the treatments for pad and represents a safe procedure in pad with acceptable perioperative morbidity , as plaque debulking
moreover , surgery treatment with a statin , aimed at reaching low ldl - cholesterol levels , is highly recommended .
no information exists so far about the modification of inflammatory parameters in pad patients after surgery and/or pharmacological treatment .
the first aim of the present study was therefore to investigate adhesion molecules expression in endothelial cells ( ecs ) of patients with pad submitted to peripheral artery revascularization associated with pharmacological treatment . to this end , we longitudinally measured icam-1 , vcam-1 , and 1-integrin mrna expression in ec isolated from venous blood ( circulating ec , cec ) and purified from femoral plaques ( pec ) .
in addition , as a secondary aim , we investigated in the same patients the expression of pro- and anti - inflammatory cytokines in peripheral blood mononuclear cells ( pbmcs ) , in view of the pivotal role played by immune cell - derived cytokines in endothelial activation .
we enrolled 9 patients ( 2 f , 7 m ) with severe pad scheduled for fea and sent to the lipid clinic ( research center on dyslipidemia , university of insubria , varese , italy ) by the department of surgical and morphological sciences of our university hospital .
only patients , who did not receive statins or steroids and had no ongoing clinical infection and/or the presence of infections in the previous three months , were enrolled .
after inclusion in the study , atorvastatin was prescribed to patients , according to established guidelines to reach recommended ldl - cholesterol levels ( 10 mg / day to 4 patients with ldl < 130 mg / dl , and 80 mg / day to 5 patients with ldl > 130 mg / dl ) .
patients were longitudinally studied at the following : the time of the decision for surgery before the institution of statin therapy ( visit 1);the day of surgery ( visit 2 ) after at least 1 month of statin therapy , before pharmacological preparation for the surgical procedure;2 months ( visit 3 ) 5 months ( visit 4 ) after surgery .
the time of the decision for surgery before the institution of statin therapy ( visit 1 ) ; the day of surgery ( visit 2 ) after at least 1 month of statin therapy , before pharmacological preparation for the surgical procedure ; 5 months ( visit 4 ) after surgery . at each visit ,
a sample of peripheral venous blood was obtained for clinical evaluation and for pbmc and cec isolation .
am mrna expression on cec was evaluated at each visit , whereas cytokine mrna expression in pbmc was analyzed at visits 1 and 4 .
in addition , at visit 2 , after surgery the femoral plaque specimens were collected and subsequently processed to isolate pec ( see below ) .
the study conforms to the principles outlined in the declaration of helsinki for use of human tissues or subjects and the study protocol was approved by the local ethics committee .
venous blood was used to isolate cec by means of immunomagnetic cell sorting . to this end
, venous blood samples were divided in aliquots of 5 ml and each aliquot was washed with 5 ml of pbs / bsa 0.1% .
antibodies targeted to cd146 ( dynal a.s . , oslo , norway ) were added to beads using a target - to - bead ratio 1 : 4 .
after 30 min incubation , beads were washed and incubated for 20 min with 5 ml of blood aliquots .
cells were isolated from the mixture by placing the test tube in a magnetic particle concentrator .
medium was removed and cells were washed three times . for the isolation of ecs from plaque ( pec ) ,
surgical specimens were placed in a solution of edta 0.05 m for 5 min and then washed in pbs solution for 5 min .
subsequently , tissue was placed in hepes containing collagenase type ii 0.2% , elastase 0.01% , and trypsinase inhibitor 0.1% and incubated under gentle stirring at 37c for 90 min .
digested tissue was washed twice with pbs , filtered through a 70-micron cell strainer , and finally pecs were removed from the tissue mixture by immunomagnetic sorting ( as above described for cec ) .
ecs isolated from venous blood or from tissue were stained with ab anti - cd45 and ab anti - cd31 ( specific marker for pecam-1 , which is highly expressed on ec ) and visualized by fluorescence microscopy in order to test the absence of cd45 + positive cells .
typical preparations of cd146-sorted cells were negative for cd45 labelling and positive for cd31 labelling , confirming that immune cells were not present in our sample after cell sorting . a counting chamber was used to enumerate cells , which were finally frozen at 80c until analysis .
pbmcs were separated by ficoll - paque plus density gradient centrifugation from whole blood as previously described .
cells were washed two times in nacl 0.15 m and finally resuspended in appropriate medium for subsequent experiments .
typical pbmc preparations contained about 80% lymphocytes and 16% monocytes , as assessed by flow cytometry .
cell viability , assessed by the trypan blue exclusion test , was always > 99% .
total mrna was extracted from 1 10 cells by perfect rna eukaryotic mini kit ( eppendorf , hamburg , germany ) and the amount of rna extracted was estimated by spectrophotometry at 260 nm .
total rna was reverse transcribed using the high - capacity cdna archive kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer 's instructions .
real - time pcr was performed by means of an abi prism 7000 apparatus ( applied biosystems , foster city , ca , usa ) using the assay - on - demand kits .
cycles included one 2 min hold ( 50c ) , one 10 min ( 95c ) , and 40 cycles according to the following steps : 15 s at 95c ( denaturation ) and 1 min at 60c ( annealing and extension ) .
threshold cycle values ( ct1 ) for the genes of interest were calculated , normalized to 18s rna ( ct2 ) ( housekeeping ) content , and finally expressed as 2 , where ct = ct2 ct1 .
primers were obtained from applied biosystems ( foster city , ca , usa ) , and are shown in table 2 .
comparisons between independent measures were performed using the mann - whitney u test and comparisons between dependent measures were performed using the wilcoxon test .
a commercial software was used for calculations ( graphpad prism version 5.00 for windows , graphpad software , san diego , ca , usa , http://www.graphpad.com/ ) .
as described in table 1 , in the group of patients enrolled , 5 were smokers , 8 had hypertension , 7 had hypercholesterolemia , and 2 were subject with alcohol habitus .
no change occurred during the study in all the clinical parameters evaluated ( e.g. , blood pressure , bmi , and routine exams ) or in habitus that could influence parameters measured ( smoking , diet , etc . ) .
all the patients had clinical symptoms referred to pad and had monolateral ( 5 patients ) or bilateral ( 4 patients ) claudication and showed severe stenosis at angiographic evaluation .
severity of symptoms was evaluated according to rutherford classification system as detailed in table 1 .
pharmacological treatments ( table 1 ) remained unchanged during the study with the only exception of the institution of atorvastatin therapy at the time of enrollment . as expected , atorvastatin treatment and fea significantly affected the lipid profile and hs - crp of all the patients ( table 3 ) and no patient , either on 10 or 80 mg / die statin underwent any adverse event during the follow - up period .
as shown in figure 1(a ) , mrna levels of am in cec progressively decreased from visit 1 to visit 4 .
in particular , both icam-1 and 1-integrin were significantly reduced after 1 month , while reduction in expression for vcam-1 was significant from visit 3 .
the % reduction for all molecule tested at visit 4 with respect to visit 1 varied from 41% to 50% ( figure 1(b ) ) .
no difference was observed in am mrna levels in ec between patients on atorvastatin 10 or 80 mg / day and no relationship was found between am mrna expression and lipid profile or hs - crp ( data not shown ) .
am mrna levels in pec at visit 2 ( day of surgery ) were always detectable and were significantly higher in comparison to those found in cec isolated at the same visit ( table 4 ) ; however , no direct correlation between cec and pec mrna levels was found for vcam-1 ( r = 0.44 ; p = 0.051 ) , icam-1 ( r = 0.005 ; p = 0.564 ) , or 1-integrin ( r = 0.08 ; p = 0.830 ) .
no difference was observed in mrna levels of am in ec between patients on atorvastatin 10 or 80 mg / day ( data not shown ) . as shown in figure 2 , cytokine mrna expression in isolated pbmc
in particular , a significant increase in mrna expression from visit 1 to visit 4 was observed for ifn- and il-4 while mrna expression of il-10 and tgf- remained unchanged .
no difference was , however , observed in the ratio ifn-/il-4 ( 0,229 0,083 at visit 1 versus 0,420 0,151 at visit 4 , p = 0,237 ) .
no significant relationship was found between cytokine mrna levels in pbmc and am mrna levels in either cec or pec as well as between the mrna levels of the different cytokines in pbmcs and all clinical parameters measured through the study ( data not shown ) .
the main finding of this study is the observation that in pad patients , surgery and pharmacological treatment induce in cec a profound reduction of the mrna levels of icam-1 , vcam-1 , and 1-integrin .
in addition , in circulating pbmcs of the same patients mrna levels for ifn- and il-4 increase and mrna levels for tgf- and il-10 remain unchanged . in patients with pad , increased levels of soluble am are associated with the presence , the severity , and the extent of ath in the arteries of the lower limbs and are suggestive of a worse outcome .
previous studies documented that pad is associated with elevated levels of most inflammatory markers , such as crp , cd40 ligand , fibrinogen , and others . despite the potential benefits associated to the lowering of these molecule by therapeutic approaches , this is the first study showing that am expression in cec may be modified even in an advanced atherosclerotic stage .
our patients showed high levels of hs - crp that were reduced 6 months after the beginning of the study .
high hs - crp level is commonly related to increased expression or circulating levels of different adhesion molecules that can be considered as indicators of severity of peripheral ath .
indeed , elevated levels of icam-1 in ec were found to be predictive of future development of pad .
it has been shown that among drugs commonly used for managing cardiovascular disease , statins significantly improve endothelial function .
in particular a 4-week treatment with simvastatin was able to restore the vasodilator ability of endothelium , as assessed by increased vasodilatatory response to ach , or contrasted l - nmma - induced vasoconstriction , indicating that both stimulated and basal vasodilator function of the endothelium were enhanced .
the benefit of statin treatment in patients undergoing vascular surgery was documented by the reduced incidence of adverse cardiac outcome during the perioperative period and reduced adverse cardiac events after surgical procedures . in our patients , statin treatment and surgical revascularization induced in cec a decrease of mrna expression of icam-1 , vcam-1 , and 1-integrins ( evident after 1 month of therapy and much more marked 5 months after surgery ) .
we failed to observe any correlation between am mrna levels in cec and pec ( with the marginal exception of a weakly significant positive correlation for icam-1 ) .
possible explanations may include less sensitivity of pec to the effects of pharmacological treatment and need much more time to observe an effect ( as indirectly suggested by the additional reduction of am mrna levels in cec after 2 and 5 months ) .
moreover , we can not exclude that the small number of patients could have masked any possible correlation .
statins may affect cellular function in advanced atherosclerotic stage , such as in patients scheduled for femoral artery revascularization and therefore affected by marked atherosclerotic lesions .
for example , recently we reported that fea associated with statin treatment in pad patients was able to reduce in circulating polymorphonuclear leukocytes angiotensin ii ( ang ii ) type 1 receptor expression ( a receptor pathway extensively involved in the proinflammatory and proatherogenic effects of ang ii ) .
it is well recognized that inflammation as well as its related immunological events positively correlate with the severity of the pad disease and regulate all the stages of the inflammatory cascade , from endothelial activation to immune cell adhesion and subsequent remodeling of the vascular environment .
cytokines are key glycoproteins which modulate all the aspects of vascular inflammation ; moreover , cytokine balance is involved in plaque morphology and stabilization and in determining the structure of the fibrous cap . in cardiovascular diseases ,
cytokines are usually classified into two categories : proatherogenic and protective , but recently it has been shown that this classification is not exhaustive , because of the complexity and overlapping of the biological roles .
for example il-4 , a cytokine classically produced by th2 cells , has been proposed as protective in ath , but il-4 also increases the expression of p - selectin , vcam-1 , and matrix metalloprotease 1 and 12 , which are implicated in aortic aneurysm formation and in acute coronary syndrome .
ifn- , a typical th1 cytokine , is directly related to ath by contributing to all the events that induce disease progression .
ifn- is associated with increased levels of other proinflammatory cytokines , but it is also able to inhibit smooth muscle cells proliferation and collagen synthesis and to induce foam cells apoptosis within then lipid core , which could contribute to plaque stabilization resulting in beneficial effects in advanced stage of the disease . in our patients ' population
since plaque destabilization has been related to reduced il-4 values and increased level of ifn- , the increase of both ifn- and il-4 might represent a protective effect .
we can not exclude that the absence of effects on il-10 and tgf- may be interpreted as the results of polytherapy differentially affecting specific lymphocyte subsets .
in addition , it must considered that these two cytokines are preferentially produced by a specific t - lymphocyte subset ( the regulatory subset that represents a low percentage of pbmcs ) while in this study we have investigated mononuclear cells as a whole ; another possible explanation of lack of effect on these cytokines is that possibly 6 months are not enough to observe any effect on these parameters .
we acknowledge that a critical point of this study was the low number of patients and the absence of untreated patients ; this latter point in particular is , however , difficult to address , as it is clearly unethical to withdraw drug treatments or delaying therapy in such a kind of patients at extremely high risk for vascular acute events , even for limited periods . as for the limited number of subjects , it was underlined that the strict selection criteria ( absence of statin treatment before the study ) used for the study did not allow the inclusion of a greater number of patients . in our opinion
, this study should be considered a pilot study that aimed to point the attention to cellular aspects in this particular group of patients in a late stage of the atherosclerotic process .
indeed , very few studies so far addressed the possibility to modulate ec and/or immune cell function in the later phases of plaque progression . in conclusion , this study supports the hypothesis that also in patients with an advanced ath status , reduction of endothelial dysfunction and modulation of the immune response may be achieved by a combination of surgical procedures and appropriate pharmacological therapy .
future studies should address the possible relationship of such effects with the long - term clinical outcome of patients . | atherosclerosis is an inflammatory disease characterized by immunological activity , in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage .
we investigated gene expression of the adhesion molecules ( ams ) icam-1 , vcam-1 , and 1-integrin in endothelial cells ( ecs ) isolated from venous blood ( circulating ec , cec ) and purified from femoral plaques ( pec ) obtained from 9 patients with peripheral artery disease ( pad ) submitted to femoral artery thrombendarterectomy ( fea ) .
in addition , in peripheral blood mononuclear cells ( pbmcs ) of the same subjects , we investigated gene expression of ifn- , il-4 , tgf- , and il-10
. patients were longitudinally evaluated 1 month before surgery , when statin treatment was established , at the time of surgery , and after 2 and 5 months .
all am mrna levels , measured by means of real - time pcr , in cec diminished during the study , up to 4150% of initial levels at followup .
am mrna expression was significantly higher in pec than in cec . during the study , in pbmcs , tgf- and il-10 mrna levels remained unchanged while ifn- and il-4 levels increased ; however , the ratio ifn-/il-4 showed no significant modification . in pad patients ,
fea and statin treatment induce a profound reduction of am expression in cec and affect cytokine mrna expression in pbmcs . |
hypertension is more severe and associated with more severe sequelae in blacks when compared with subjects of caucasian extraction [ 25 ] .
studies have also shown that black subjects have lower plasma renin levels [ 68 ] .
it is thought that black subjects have inappropriately elevated levels of aldosterone relative to plasma renin activity .
the activity of the renin - angiotensin - aldosterone system ( raas ) is thought to be the major contributor to the pathogenesis of hypertension and its sequelae .
studies have shown that circulating aldosterone levels within the physiologic range are strongly related to increased risk of cardiovascular mortality , fatal stroke , and sudden cardiac death .
it has also been shown that chronic administration of aldosterone in animal models leads to cardiac fibrosis and left ventricular hypertrophy ( lvh ) .
subjects with primary aldosteronism have early and more severe development of lvh when compared with subjects with essential hypertension .
though previous studies , on predominantly white subjects , had not been conclusive on the relationship between left ventricular mass and aldosterone , very few of these studies have been done on black subjects .
this study therefore attempts to determine the relationship between aldosterone levels and left ventricular mass in black subjects with essential hypertension .
subjects were newly diagnosed but untreated hypertensive subjects recruited from the clinics of the university college hospital , ibadan ; through community screening exercises ; and from among the staff of the university college hospital , ibadan .
subjects with secondary hypertension , diabetes mellitus , symptomatic heart failure , and chronic kidney disease were excluded .
informed consent was obtained from each subject , and ethical approval was obtained from the joint institutional review board ( irb ) of the college of medicine , university of ibadan / university college hospital , ibadan , nigeria .
no caffeine - containing beverages , alcohol , or smoking was allowed for at least 2 h. subjects were not on any medications prior to sample collection .
blood was collected into test tubes on ice after 30 minutes of rest in the supine position .
a 24 h collection of urine was made the preceding day in plastic containers containing hcl ( 80 mmol / l final concentration ) .
high - performance ( cation exchange ) liquid chromatography ( hplc ) was used to determine the plasma renin , plasma aldosterone .
plasma and urinary sodium and potassium concentrations were measured using a flame photometric method .
echocardiographic examination was done using a phillip aloka ssd 4000 ( aloka co. ltd . , tokyo , japan ) machine with a 2.55.0 mhz linear array transducer .
two experienced physicians performed the echocardiography . in our laboratory , the intraobserver concordance correlation coefficient ranged from 0.76 to 0.98 while that of the interobserver concordance ranged from 0.82 to 0.96 .
measurements of lv internal diameter ( lvidd ) , intraventricular septal thickness ( ivstd ) , and posterior wall thickness ( pwtd ) were obtained at end diastole .
lv mass ( lvm ) was calculated using the formula published by devereux et al .
: 0.8 ( 1.04 ( ( lvidd + pwtd + ivstd)3 lvidd3 ) ) + 0.6 , and lvm was indexed to the allometric power of height . relative wall thickness ( rwt ) was measured at end diastole as the ratio 2(pwt)/lvid .
data from continuous variables are presented as mean ( standard deviation ) or as medians with interquartile ranges while categoric data are expressed as percentages .
all statistical tests were two - sided and carried out to a significance level ( p ) of 0.05 .
student 's t - test for independent groups was used to compare normal continuous data while mann - whitney 's test was used to compare nonnormal data .
tertiles of plasma aldosterone were generated and the aldosterone groups were compared with analysis of variance for continuous data , and test was used for categoric data .
pearson correlation analysis was used to determine the correlation between plasma aldosterone and arr and the echocardiographic variables of lv structure . a stepwise linear regression model ( with backward elimination )
table 2 shows the biochemical , hormonal , and echocardiographic parameters of the subjects . plasma sodium and renin were lower in the hypertensive subjects .
the left atrial , lv wall dimensions , and lv mass were higher in the hypertensives .
the hypertensive subjects frequently had more hypertrophic lv geometric patterns when compared with the normal subjects .
there were no differences in the plasma aldosterone levels and the urinary excretion of sodium and potassium .
table 3 shows the biochemical , hormonal , and echocardiographic parameters of the subjects according to plasma aldosterone tertiles .
there was no univariate association between the parameters of lv structure and the plasma aldosterone and aldosterone - to - renin ratio levels ( table 4 ) . in a multivariate model involving lv mass index and age , sex , body mass index ( bmi ) , aldosterone , plasma renin , pulse pressure , and systolic blood pressure ( sbp ) , only age ( p = 0.008 ) , bmi ( p = 0.046 ) , and systolic blood pressure ( p = 0.001 )
were independently associated with the lv mass index ( r = 0.15 , p < 0.0001 ) .
in this group of black african subjects , there was no association of plasma aldosterone with lv mass .
also , no gender - specific differences in the relationship of lv mass index with plasma aldosterone were seen .
only the age of the subjects , their body mass index , and the level of their blood pressure were associated with lv mass .
previous studies on the association between lv mass and circulating aldosterone levels in subjects with essential hypertension had yielded conflicting results .
. found no association between aldosterone levels and lv structure in hypertensive caucasians and nonobese black americans , and vasan et al . found no relationship between lv mass and serum aldosterone in the framingham cohort .
other studies [ 1624 ] had noted associations between lv mass and blood aldosterone levels .
these conflicting results could be due to differences in the study populations , that is , presence of other comorbidities such as diabetes mellitus , long - standing hypertension , differences in dietary salt intake , and the role of drugs that could alter the renin - angiotensin - aldosterone system .
our study subjects were newly diagnosed and treatment - naive hypertensives ; subjects with elevated blood sugar and diabetes mellitus were excluded and were african subjects living in tropical environment .
it is quite probable that significant racial differences exist in the relationship between aldosterone levels and lv mass .
had documented a significant association between plasma aldosterone levels and lv posterior wall thickness and relative wall thickness but no relationship between lv mass index and aldosterone in women , while edelmann et al .
reported a robust and consistent association between echocardiographic parameters of lv structure and serum aldosterone concentration exclusively in women .
these gender differences had been thought to be due to differences in adaptation to pressure overload due to oestrogen signalling in the myocardium .
in contrast to these studies , we did not find any gender related differences in the association of lv mass and plasma aldosterone levels .
it is also probable that envirnomental and dietary factors play a significant role in the modulation of the effects of the renin - angiotensin - aldosterone system in native black africans .
the influence of body mass index on lv mass had been noted in previous studies [ 26 , 27 ] .
mul et al . in their study among subjects with metabolic syndrome suggested that obesity might play a role in the association of lv mass with aldosterone levels .
the influence of the level of the blood pressure especially systolic blood pressure [ 2830 ] on lv mass index had been previously described in several studies .
this is supported by the findings from this study that shows the independent association of systolic blood pressure with lv mass index .
the influence of systolic blood pressure on the development of left ventricular hypertrophy is thought to be a result of an increased end - systolic stress which is mostly related to the systolic blood pressure .
this supports observations from previous studies that target organ damage in hypertension increases progressively with age [ 32 , 33 ] .
though our patients were newly diagnosed with hypertension , many of the subjects could have had longstanding but undiagnosed hypertension .
working in south - west nigeria found that about 42% of their subjects already had left ventricular hypertrophy at diagnosis of their hypertension .
our study is limited in that only a single measurement of aldosterone was made and might be insufficient in estimating the daily exposure to aldosterone .
the determination of 24 hr urinary aldosterone is better suited for the assessment of daily aldosterone load . in conclusion , in our group of black newly diagnosed hypertensive subjects , we did not find an independent association of lv mass index with plasma aldosterone concentration .
further studies relating the 24 hr aldosterone load might be necessary to fully evaluate the association of aldosterone and measures of lv structure . | background . hypertension is the most common cardiovascular disease worldwide and is a major cause of morbidity and mortality .
studies have suggested that the activity of the renin - angiotensin - aldosterone system play a major role in the target organ damage such as left ventricular hypertrophy occuring in hypertension .
we sought to determine the relationship between plasma aldosterone and left ventricular mass in untreated african hypertensives .
methods .
we recruited 82 newly diagnosed and untreated hypertensives and 51 normal controls .
measurements obtained included echocardiographic lv mass index , plasma aldosterone and renin . results .
the hypertensive subjects had lower renin levels ( 21.03[6.974 ] versus 26.66[7.592 ] ng.ml1 , p = 0.0013 ) , higher lv mass index ( 52.56[14.483 ] versus 42.02[8.315 ] g.m2.7p <
0.0001 ) when compared with the controls .
there were no univariate associations between lv mass index and plasma aldosterone ( r = 0.0179 , p = 0.57 ) and between lv mass index and plasma renin ( r = 0.0887 , p = 0.61 ) . in a multivariate model involving lv mass index and age , sex , body mass index ( bmi ) , plasma aldosterone ,
plasma renin and systolic blood pressure ( sbp ) , only age ( p = 0.008 ) , bmi ( p = 0.046 ) , and sbp ( p = 0.001 ) were independently associated with the lv mass index .
conclusions . in this group of hypertensive africans
, there is no independent association of plasma aldosterone with lv mass .
the height of the blood pressure , the body mass index and the age of the subjects determined the lv mass . |
, the medical faculty and deans offices can not operate in isolation but must establish links with existing services run by the university , the city or the country , churches and so on .
for example , kids ( children at university ) , an initiative by the university of heidelberg , the german national association for student affairs ( studentenwerk ) and the equality office collated all key institutions , advisory services , initiatives , childcare and financial support offers on the university s website in cooperation with local authorities and the state in a helpful and comprehensive way ( http://www.uni-heidelberg.de/studium/imstudium/kids/ ) .
the deaneries are responsible for giving advice to students , their individual term plans and planning the academic progress of students .
thus it gives food for thought if 56% of students state a need for advice regarding curriculum planning that 93% of respondents at the same time said that they are not in regular contact with their academic advisor about their study organisation , with 62% of respondents even claiming they do not know that the academic dean s office offers academic advisory services .
everyday experience can not explain the survey results for the dean s office ; particularly in the light of conscious pregnancy planning and raising children while studying advice is vital , sometimes even before pregnancy . from the outset ,
the academic advisors present themselves as a first point of contact for study - related problems and pregnant students and students with children are always made to feel welcome when dropping in and sensitive advice is given when giving advice regarding the extra difficulties of their situation .
perhaps the dean s office is seen by some students primarily as a body dealing with course regulations and examinations rather than as a neutral advisory office offering to take up problems without negative consequences for the students studies . the offers and advisory services the academic dean offers
the dean s office will develop strategies for engaging the target groups more effectively and encouraging them to regularly visit the academic advisor in the dean s office .
increasing the flexibility of degree courses is a legitimate request from student parents ; considering their group placement and exchange requests or the possibility of taking a break from the course and a flexible return to it should go without saying .
similar requests have also been made by many other students who are in comparably difficult situations , for example , ill or disabled students , students caring for relatives , students who must self - finance their studies and foreign students , some of whom require support because of language problems , different learning cultures or problems with immigration issues .
students who began their studies at a later stage of life , often following a first qualification , who resume studying after a break or as extreme long - time students who have exceeded the regular length of study require advice .
many students approach the academic advisory services with special requirements regarding study planning that are not limitations through personal or social hardships but through their additional commitments or special engagements while studying . for example
students who study two degrees in parallel , students on scholarships or students on other forms of sponsorship , students with unusual commitments in competitive sports , art , music , politics or social or scientific projects , as well as students who are active on the student council or hired as tutors by the faculty or aspiring to study abroad . from this angle , students with children are one of many student groups with legitimate needs and prioritisation of special groups is not possible .
in addition , study regulations and principles of equal treatment and achievement of learning targets must apply to all students , even groups of students with special needs or problems . in general
, timetables can not be adjusted according to the wishes and needs of students as many fixed aspects must be taken into account in the organisation of teaching .
the number of teaching hours , examinations and group sizes are often specified by law , there are accommodation and staffing factors and , not least of all , didactic aspects of the curriculum .
grouping together certain types of students into their own groups seems difficult due to the heterogeneity of needs and is also questionable from a didactic point of view .
structured part - time medical studies are also not really an option as extending the period of study , which under normal circumstances is already six years and three months , could not be justified . on the other hand , an individual study extension for students
can be easily arranged as the state examinations must not be taken at fixed dates and credits gained do not expire .
individual solutions for part - time study in the practical year are available upon request for student parents .
the feedback from the prenatal clinic that medical students are under extreme pressure to perform , discouraging pregnant students and sometimes leading to abortions is disconcerting .
a survey carried out in heidelberg on motivation in medical studies ( heimi 2010 ) confirmed that performance pressure and fear of failure concerns many students : 11% of surveyed students are frequently or constantly afraid of not being able the meet the demands of the course , only 31% of students have never been worried about possibly having to quit the course .
the idea of studying at an elite university can lead students to believe that only unburdened students who are able to fully perform permanently are welcome . not only is the quality of teaching important for the motivation of the students but also supportive environments and the certainty that students are not perceived as a nuisance .
the university must recognise it as a special achievement when students manage to combine studying with raising children and that their studies are completed with and for the children .
better advertising of the advisory services offered by dean s office , e.g. by promoting them on the homepage.improved information through networking the dean s office with the advisory services offered by the university for student parentsresponsiveness and flexibility in the organisation of studies for pregnant women and student parentsreducing presence requirements in some blocks and modules of some clinical courses or reducing attendance control .
this is done in those teaching forms and course sections which permit independent study of the contents by the students at home , where possible supported by electronic media.forum on the learning platform for networking and exchange of experiences among the students with childrenensuring prenatal advisory centres inform students that they should also approach academic advisors at the dean s office in case of problems.requesting management to provide changing and nursing facilities in the dean s office better advertising of the advisory services offered by dean s office , e.g. by promoting them on the homepage .
improved information through networking the dean s office with the advisory services offered by the university for student parents responsiveness and flexibility in the organisation of studies for pregnant women and student parents reducing presence requirements in some blocks and modules of some clinical courses or reducing attendance control .
this is done in those teaching forms and course sections which permit independent study of the contents by the students at home , where possible supported by electronic media .
forum on the learning platform for networking and exchange of experiences among the students with children ensuring prenatal advisory centres inform students that they should also approach academic advisors at the dean s office in case of problems . requesting management to provide changing and nursing facilities in the dean s office
| increasing the flexibility of medical degree courses as well as individual advice and curriculum planning are essential needs of pregnant students and student parents .
the results of the ulm study gave the academic advisers in the dean of studies office in heidelberg a large number of suggestions on how to offer sensitive , competent and needs - based advice to this target group .
comprehensive reflection on many years of experience in giving advice and recent survey results leads to a series of concrete measures which will contribute to more family - friendliness in heidelberg s medical school . |
management of intracranial pressure ( icp ) is the main goal for patients with traumatic brain injury .
moreover , direct injury , secondary insult due to post - traumatic increase of icp or decrease in cerebral perfusion pressure are well recognized as potential causes of increasing mortality and morbidity.467 ) decompressive craniectomy ( dc ) is an important method to decreased icp,9 ) and dc is widely performed throughout the world.10 ) this surgical procedure is performed by any neurosurgeons or even residents under training , so it should achieve enough decompression without interoperator differences , but textbook only commented that " the bone flap should not be just as large as possible".3 ) the authors considered that the skin incision could interfere the size of enough decompression area , and considered new skin incision technique what we called the " n - shaped skin incision " .
the purpose of this study is to suggest a new skin incision technique for decompressive surgery to promote greater decompression , and compared the efficacy and safety of this new technique compared to the conventional surgical technique .
the surgical indication of space - occupying severe traumatic brain injury was as follows : a midline shift of the cerebral structures and parenchymal hypodensity in greater than 50% of the middle cerebral artery territory , which are both identified on computed tomography ( ct ) scanning.25 ) a total of 38 patients , who underwent decompressive surgery in single university hospital between may 2012 and december 2014 , were recruited for this study .
these patients were classified into 2 groups : group a ( n=15 ) which the patients were underwent n - shaped skin incision , and group b ( n=23 ) which the patients who underwent the conventional surgical method ( question mark skin incision ) .
all patients were managed promptly after the surgical intervention , such as the medical treatment and acute care from admission to post - operation .
a , an " n - shaped " skin incision was made and included the slightly curved line along the frontal hair line and the parabola line pass through the parietal and temporal regions on the operation side as presented in figure 1a . in group b , a large skin incision was made and included the frontal , parietal , and temporal regions on the side which similar shape to question mark , and the incision was made as wide as possible ( figure 1b ) .
the soft tissue flap was elevated and retracted by the standard techniques in both groups , and the dura was fixed at the edge of the craniotomy to prevent epidural bleeding . the dura was opened with a large asterisk - shaped incision that involved the frontal , parietal , and temporal lobes .
lyophilized cadaver dura was placed underneath the incised dura , and it was secured with several sutures .
the area of the decompressed regions ( cm ) ( figure 2a ) , protruded brain volume ( cm ) ( figure 2b ) out of the skull surface , and the time interval between skin incision and bone flap removal were compared .
the area of the decompression region was approximately calculated by the following equation : a ( area , cm)=d * d * ; " d " was the anteroposterior diameter of the bone flap ; " d " was the perpendicular diameter to d from the superior craniectomy margin to the inferior margin in centimeters
. the protruded brain volume protruding out of the skull surface is simply calculated by the following equation : v ( volume , cm)= 1/3 * a * h ; " a " is the area of the bone flap ; and " h " is the height of the outward of the brain on ct from the level of the center of the bone flap in centimeters .
the time interval between skin incision and decompression time was calculated using the anesthetic medical record ( minutes ) .
patients ' outcomes were clinically evaluated according to the modified rankin scale ( mrs ) for 3 months after surgery .
surgical mortality was defined as death within 30 days after the procedure . a favorable outcome and poor outcome
were defined as mrs 0 to 3 ( moderate disability or better ) , and mrs 4 to 6 ( severe disability or death ) .
chicago , il , usa ) and are expressed as the meanstandard deviation ( sd ) .
the measurements and outcomes between 2 groups were analyzed by mann - whitney u test and chi - square test .
table 1 summarized the demographic data for both groups . no significant differences between the 2 groups regarding age , sex , diagnosis , and
the area of craniectomy was larger in group a ( 389.145.7 cm ) than that in group b ( 318.756.5 cm , p=0.041 ) .
the protruded brain volume out of the skull surface was 151.8117.7 cm in group a and 116.281.3 cm in group b , indicating that more decompression was performed in group a ( p=0.045 ) .
in addition , the time interval between skin incision and bone flap removal was much shorter in group a ( 23.313.7 minutes vs. 29.515.1 minutes ,
p=0.013 ) .
there were no surgery - related complications except for 1 patient in group a and 3 patients who had a postoperative epidural hematoma ( edh ) and subsequent edh evacuation ( p=0.531 ) .
the clinical outcomes throughout the 3 month after surgery were as follows : in group a , 6 ( 40.0% ) out of 15 patients had favorable outcomes ( mrs 0 - 3 ) and 1 ( 6.7% ) patient had expired ; in group b , 5 ( 21.7% ) out of 23 patients had favorable outcomes and 4 ( 17.4% ) patients had expired .
it was considered that group a showed more favorable outcome proportion and lesser mortality cases proportion , but these differences were not significantly observed ( p=0.225 and 0.339 ) due to the limitation of the number of cases .
the role of surgery in the management of traumatic brain injury was often based on subjective criteria or previous experience of each individual surgeon.11 ) surgical evacuation
should be considered when the neurological status which revealed the development of new " brainstem " signs and a sustained increase in icp were suspected .
image studies should be performed urgently to evaluate the progression of intracerebral hemorrhage ( ich ) and brain swelling .
an increase in the size of ich on image studies is also an indication for surgical removal .
decompression could provide an enlargement of the intracranial space , preventing further rise of icp and cerebral herniation.8 ) a craniectomy of greater than 8 cm may be large enough , but to obtain a real decompression , most authors recommend a minimum diameter of 12 cm .
however , skin incision may also play a critical role in the whole hemicranium.1 ) the most well - known common technical skin incision technique is the classical " question mark " flap ( figure 1b ) .
the incision starts at the temporal level , which then goes toward the superior level to preserve blood supply of the flap from the superficial temporal artery .
the advantage of this incision is that most neurosurgeons are accustomed to the anatomical structures ; however , in the posterior segment of the flap , the blood supply is low , which may lead to several complications , especially dehiscent wound or skin necrosis .
indeed , the desired decompression size is achieved by various extensions of the scalp to the outer edge of the incision .
however , our newly supposed " n - shape " incision method shows that the desired decompression has easily been achieved with less time until bony decompression ( figure 2 ) .
this n - shape skin incision included the slightly curved line along the frontal hair line and the parabola line pass through the parietal and temporal regions on the operation side as presented in figure 1a . in this study
, we indicated that the " n - shape " skin incision method has several advantages , such as larger mean area of craniectomy , more brain volume protruding out of the skull surface , and faster time interval between skin incision and bone flap removal .
this incision technique could also provide minimized ischemic complication at the operatory wound site due to the preservation of the occipital artery , and this may be a better blood supply , as presented in figure 3.1 ) there was also greater improvement in the clinical outcomes using this new skin incision method .
the clinical outcomes at 3 months ' post - surgery was as follows : in group a , 6 ( 40.0% ) out of 15 patients had favorable outcomes ( mrs 0 - 3 ) and 1 ( 6.7% ) patient had expired , whereas in group b , 5 ( 21.7% ) out of 23 patients had favorable outcomes and 4 ( 17.4% ) patients had expired .
we consider this to be an excellent clinical result with respect to two decompressive factors , such as a wider decompression size and greater possibility of decompression such as temporal bone .
as well known to neurosurgeons , the vital brain structure is located at the area of the temporal bone ; hence , decompression of this site is crucial to prevent central herniation . the conventional skin incision could provide a visualization of this area ; however , the thickness of the temporalis muscles makes it very difficult .
our new incision method , nonetheless , included this critical point in the incision line , so the vital temporal bone decompression was easier and faster to the conventional method ( figure 4 ) .
indeed , surgery - related complications with the new incision technique showed similar results compared with the conventional technique : 1 ( 6.6% ) patient in group a vs 3 patients ( 13.0% ) in group b with postoperative edh and subsequent edh evacuation .
the crossed line point could be a problem in scalp wound healing , but in this retrospective study , there were only minimal skin problems which were improved after simple dressing .
although this study showed excellent results from using the new skin incision method for dc , there are still limitations worth noting for this study .
first , this study included only a small number of cases and was retrograde reviewed .
secondly , the calculated decompressive size was measured using ct images ; hence , a delicate volume to achieve desired decompression was not exactly presented . despite this limitation , this study provided notable surgical results and clinical outcomes of the " n - shape " skin incision method for dc .
future studies are necessary to further evaluate this new technique ; we , however , believe that this surgical technique would show excellent results , similar to our study , for well - designed studies in the future .
dc using our newly suggested " n - shape " skin incision method is feasible and safe .
additionally , it may even be more beneficial - offering greater ease and reduced time for training neurosurgeons - compared with conventional craniectomy . | objectivedecompressive craniectomy ( dc ) is a useful surgical method to achieve adequate decompression in hypertensive intracranial patients .
this study suggested a new skin incision for dc , and analyzed its efficacy and safety.methodsin the retrograde reviews , 15 patients underwent a newly suggested surgical approach using n - shape skin incision technique ( group a ) and 23 patients were treated with conventional question mark skin incision technique ( group b ) .
two groups were compared in the terms of the decompressed area of the craniectomy , protruded brain volume out of the skull layer , the operation time from skin incision to bone flap removal , and modified rankin scale ( mrs ) which was evaluated for 3 months after surgery.resultsthe decompressed area of craniectomy ( 389.1 cm2 vs. 318.7 cm2 , p=0.041 ) and the protruded brain volume ( 151.8 cm3 vs. 116.2 cm3 , p=0.045 ) were significantly larger in group a compared to the area and the volume in group b. the time interval between skin incision and bone flap removal was much shorter in group a ( 23.3 minutes vs. 29.5 minutes , p=0.013 ) .
but , the clinical results were similar between 2 groups .
group a showed more favorable outcome proportion ( mrs 0 - 3 , 6/15 patients vs. 5/23 patients , p=0.225 ) and lesser mortality cases proportion 1/15 patients vs. 4/23 patients , but these differences were not significantly observed ( p=0.225 and 0.339).conclusiondc using n - shaped skin incision was a feasible and safe surgical technique .
it may be an easier and faster method for the purpose of training neurosurgeons . |
raman scattering from the silicon ( si ) nanostructures ( nss ) has been extensively studied in recent years [ 1 - 4 ] .
observed raman line - shapes from the si nss are asymmetrically broadened and red - shifted from its counterpart for the bulk si .
most authors have fitted the first - order experimental raman band to an asymmetrical line - shape first proposed by richter et al .
and then modified by campbell et al . . in this model , the asymmetry and red - shift in the raman peak
many others have explained the asymmetry and downshift in the raman line - shape in terms of a combined effect of quantum confinement and laser heating .
magidson and beserman have observed the fano interference between photo - excited electrons and discrete phonons in bulk si when raman spectra were recorded using laser power density of 10w / cm .
however , the presence of photo - excited fano interaction in si nss was proposed very recently where detailed photo - excitation - dependent raman studies were carried out on the si nss .
an increase in the asymmetry ratio of raman line - shape was noticed as a result of increasing excitation laser power density in the range 0.221.76 kw / cm . effect of the quantum confinement on fano resonance is not studied yet and needs further studies to elucidate the behavior of fano interaction as a function of si nss size and laser power density .
the purpose of this paper is to study the fano interaction in the si nss as a function of the nss size and laser power density .
the si nss of two different sizes are fabricated by laser - induced etching ( lie ) method by etching using two different etching times for same laser power .
surface morphology is studied by atomic force microscopy ( afm ) to see the formation of quantum confined si nss .
raman spectra are recorded using two different laser power densities of 0.2 and 0.88 kw / cm for both the samples .
raman spectra recorded using 0.2 kw / cm are fitted using phenomenological phonon confinement model to calculate the most probable si nss size . using these nss sizes ,
raman spectra recorded using 0.88 kw / cm are fitted using fano - raman line - shape to find the fano asymmetry parameter to see the effect of si nss size on the fano interaction .
higher fano interaction is seen for smaller si nss as compared to large si nss .
two samples ( samples a and b ) containing si nss are fabricated by the lie method .
the lie is done by immersing a si wafer ( resistivity of 35 cm ) into 48% hf acid and then focusing a 500 mw argon - ion laser beam ( eex = 2.41 ev ) to a circular spot of 120-m on the wafer .
the etching time is 45 min for the sample a and 60 min for the sample b keeping other parameters ( like laser power density , wavelength , and concentration of hf ) the same .
raman scattering was excited using photon energy 2.54 ev of the argon - ion laser at two different laser power densities of 0.2 and 0.88 kw / cm .
the reason for choosing low laser power density is to avoid heating of the sample during raman recording .
raman spectra were recorded by employing an spex-1403 doublemonochromator with hamamatsu ( r943 - 2 ) photomultiplier tube arrangement and an argon ion laser ( coherent , innova 90 ) .
figure 1a , b shows afm images of si nss formed in the samples a and b , respectively . the images shown in fig
. 1 are high - resolution images taken from the pore walls of the laser - etched samples .
figure 1a shows the formation of si nss having sizes in the range of a few nanometers .
higher quantum confinement is expected in sample b as compared to sample a. the possibility of quantum confinement effect in these si nss is investigated by raman experiments .
figure 2a shows raman spectrum from the sample a recorded using an excitation laser power density of 0.2 kw / cm .
raman active optical phonon mode , which is observed at 520.5 cm for the bulk si , shifts toward lower wavenumber ( 518.5 m ) in fig .
the raman line - shape has asymmetry ratio of 2.8 with fwhm of 12.5 cm in fig .
2a . asymmetry and broadening in raman line - shape is attributed to the quantum confinement of phonons in si nss [ 12 - 15 ] .
the asymmetry ratio is defined here as l/h , where , l and hare half widths on the low- and high - energy side , respectively , of the maximum .
figure 2b displays raman spectrum from the sample a when recorded using the excitation laser power density of 0.88 kw / cm .
this raman spectrum has peak at 518 cm with asymmetry ratio of 3.1 and fwhm of 14 cm in fig .
figure 2b shows that asymmetry , red shift , and fwhm in raman line - shape increase by the increasing excitation laser power density .
changes in the raman line - shape are reversible in nature on decreasing the laser power density .
heating effect is ruled out because the laser power density is not high enough to do appreciable heating .
increase in the asymmetry on increasing excitation laser power density is due to fano interaction between electronic raman scattering involving photo - excited electrons within electronic states and usual optical phonon raman scattering . at higher laser power density of 0.88
kw / cm , the electronic raman contribution increases because of more number of photo - excited electronic transitions .
there is no effect of increased laser power density on phonon raman scattering because of absence of the heating effect .
these two effects combine to show high asymmetry ratio in fig . 2b as compared to fig .
afm images showing silicon nss in ( a ) the sample a and ( b ) the sample b raman spectra from samples a and b. the calculated raman spectra are indicated by solid line curves and the experimental data are plotted as discrete points .
phonon confinement model has been used to fit the experimental data in ( a ) and ( c ) whereas eq.1is used to fit the data in the ( b ) and ( d ) laser power density - dependent raman spectra from smaller si nss in sample b are shown in fig .
2c , d. figure 2c , d is the raman spectra from sample b when recorded using excitation laser power densities of 0.2 and 0.88 kw / cm , respectively .
2c has peak at 518 cmwith asymmetry ratio of 2.9 and fwhm of 13.5 cm . higher asymmetry ratio and phonon softening in fig . 2c as compared to fig .
2a is due to higher quantum confinement effect from si nss in sample b than in sample a. the raman spectrum peaked at 517 cmwith asymmetry ratio of 3.7 and fwhm of 16.5 cmis observed in fig .
2d due to photo - excited fano interaction in sample b. in order to quantitatively analyze the above - mentioned effects , the experimental data in the fig .
2 are theoretically fitted with fano line - shape for nanoparticles given by:(1 ) the (k ) is the phonon dispersion relation of the optic phonons of bulk si given by with a = 171,400 cm and b = 100,000 cm .
the term in curly bracket takes care of the fano interaction and the exponential term takes into account the confinement effect on fano interaction in si nss of size l. the
n(l) is a gaussian function of the form , n(l ) [ exp ((l l0)/ ) , included to account for the size distribution of the nanocrystallites .
the l0l1 , and l2 are the mean crystallite size , the standard deviation of the size distribution , the minimum , and the maximum confinement dimensions , respectively . since fano effect is negligible ( |1/q| 0 ) at low laser power density of 0.2 kw / cm due to insufficient number of photo - excited electrons .
2a , c shown as discrete squares are fitted by considering only phonon confinement effect ( eq . 1 of reference ) .
the theoretically obtained value of mean crystallite size ( l0 ) is 4.5 nm for sample a and is 3.0 nm for sample b. this implies that the quantum confinement effects are more pronounced in sample b than in sample a. all the fitting parameters used to fit the experimental raman data in fig .
it shows that distribution in si nss size is very narrow ( = 1 nm ) for both the samples .
qualitatively one can see that sizes from raman results are in consonance with the afm results in fig
. 1 .
different fitting parameters used to fit the raman line - shapes from samples a and sample b in the fig .
2b , d shown as discrete triangles are fitted with fano - raman line - shape of eq .
1 with the appropriate l0l1 , and l2 values obtained earlier for samples a and b as given in table 1 . in order to fit the experimental raman data in fig . 2b , d ,
2b , d show a good fitting for the fano asymmetry parameter |q| equal to 16 and 10 for the samples a and b , respectively .
it reveals higher photo - excited fano interaction in the smaller size nss ( sample b ) as compared to larger size nss ( sample a ) . while fitting raman data , the value of
q was kept constant for a given l0 , where the distribution of size is very narrow ( = 1 nm ) .
the smaller sizes present in the sample b are much smaller in size as compared to the bohr s exciton radius of 5 nm for si .
thus , the confinement effect will be more in sample b in comparison with sample a. quantum confinement of electrons lead to discrete energy levels .
electronic raman scattering may interfere with usual optical phonon scattering when optical phonon energy lies in the region e = e1 e2(where , e1 and e2 are energy of discrete electronic levels ) .
probability of interference increases in the smaller size nss where discrete electronic levels are separated by optical phonon energy .
such type of fano interaction can not be seen in bulk si or larger sized nss .
fano interaction can be seen in the bulk si when doping is above 10 cm .
therefore , size - dependent fano interaction in si nss is due to quantum confinement of electrons and phonons in laser - etched si .
in summary , the raman line - shapes from the si nss are investigated as a function of si nss size and excitation laser power density .
the raman line - shape becomes more asymmetric , wider , and shifts to lower wavenumber when the raman spectra are recorded with higher laser power density .
this behavior is attributed to the fano interference between discrete phonons and photo - excited electronic transitions .
fano interaction is more pronounced for smaller size nss at same laser power density . in other words ,
smaller size nss will start showing photo - excited fano interaction at lower excitation laser power density than for larger size nss .
higher quantum confinement of photo - excited electrons and phonons in smaller si nss is responsible for observation of size - dependent photo - excited fano interaction in laser - etched si nss .
authors acknowledge the financial support from the department of science and technology , goverment of india under the project optical studies of self - assembled quantum dots of semiconductors .
one of the authors ( r. kumar ) acknowledges the financial support from council of scientific and industrial research ( csir ) , india . technical support from mr . | photo - excitation and size - dependent raman scattering studies on the silicon ( si ) nanostructures ( nss ) prepared by laser - induced etching are presented here .
asymmetric and red - shifted raman line - shapes are observed due to photo - excited fano interaction in the quantum confined nanoparticles .
the fano interaction is observed between photo - excited electronic transitions and discrete phonons in si nss .
photo - excited fano studies on different si nss show that the fano interaction is high for smaller size of si nss .
higher fano interaction for smaller si nss is attributed to the enhanced interference between photo - excited electronic raman scattering and phonon raman scattering . |
the three data components of the analysis are age- and sex - specific mortality rates , age- and sex - specific population estimates by weight status , and age- and sex - specific diabetes incidence rates .
mortality rates were calculated based on number of deaths by sex and age from the national vital statistics system ( 9 ) .
population estimates , including information on age , sex , height , weight , and diabetes , were drawn from the national health interview survey ( nhis ) , an ongoing nationally representative cross - sectional survey of the civilian noninstitutionalized population .
the nhis uses multistage probability sampling to select respondents and collects self - reported health information , with marginal response rates of 70% for the adult sample interviews ( 10 ) .
population estimates and number of diabetes cases were classified by age ( 10-year intervals ) , sex , and bmi levels ( normal / underweight , < 25 kg / m ; overweight , 25 to < 30 kg / m ; and obese , 30 kg / m ) .
incident cases of diabetes were identified based on reported age at diagnosis of diabetes that is , being told by a doctor or health care professional that they had diabetes and age at time of interview .
diabetes incidence rates were derived by dividing the estimated number of people in a given age - sex group reporting that they were diagnosed during the year before the survey by the total population in each age - sex group .
individuals diagnosed with diabetes > 1 year before were no longer at risk for developing diabetes and so were not included in the numerator or denominator for rates .
analyses included 143,765 adults in 19801989 and 150,718 adults in 20002004 , of whom 507 and 1,366 , respectively , reported being diagnosed in the previous year .
we estimated the number of years an individual can expect to live without diabetes given their age , sex , and bmi status while also accounting for mortality risks using multiple - decrement life table techniques ( 12,13 ) .
diabetes - free life expectancy must be lower than total life expectancy , as an individual faces the competing risk of diabetes and death , both of which end diabetes - free life .
to determine the contribution of each age and bmi group to changes in total diabetes - free life expectancy between 19801989 and 20002004 , we used a discrete - time decomposition method by arriaga ( 14 ) .
the method calculates the total effect of a change in life expectancy above age x between two points in time as the sum of the direct effect of a change in rates above age x and the sum of the change in life expectancies contributed by the additional person - years lived above age x as a result of the improvements in rates between the two periods .
an extension of the method allows us to decompose changes in life expectancy by type of decrement , with the decrements in this analysis being diabetes incidence and death .
the contribution of each of these two types of decrements to the total decrement at each age is calculated from the proportion of the differences in total decrements it represents between two points in time .
thus , the difference in diabetes - free life expectancy at a given age between two periods is the result of differences in diabetes incidence and mortality rates above that age ( 14,15 ) .
in addition to examining the competing risks of diabetes and all - cause mortality in the entire population , we also performed these calculations for each bmi group separately .
this allowed for a better understanding of the effects of secular changes in weight on diabetes incidence overall and among people of different weights , showing the number of years that a person in each bmi category can expect to live without diabetes . using similar life table techniques ( 12,13 )
, we also estimated the proportion of the population that will eventually be diagnosed with diabetes given their weight status and the current diabetes and mortality rates in the u.s . because data on age at diagnosis were not available for people younger than age 18 and older than age 79 in some nhis waves , analyses were restricted to ages 18 to 79 years .
this approach implies that incidence rates at younger ages are negligible or that the calculations are conditional on not having been diagnosed before age 18 .
we tested this assumption by reallocating in our estimates observed mortality according to risk ratios by bmi group reported in the literature ( 16,17,18 ) .
we tested the possibility that changes in diagnosis detection rather than incidence are driving the results .
the results are robust to each of these sensitivity tests ( supplementary table 1 ) .
the bmi - specific calculations do not model the transition of individuals between bmi categories . to relax this assumption , we estimated diabetes - free life expectancy for each bmi group at ages 18 , 40 , and 60 years , which shows how the changing weight composition of the population at each age may affect diabetes - free life expectancy in bmi groups .
on the basis of nhis data and vital statistics , we calculated that life expectancy at birth in the u.s
. in 19801989 was 70.6 years for men and 77.4 years for women , and it had increased to 74.3 years for men and 79.0 years for women by 20002004 .
remaining life expectancy at age 18 also increased by 3.1 years for men and 1.1 years for women ( supplementary table 1 ) .
in contrast , diabetes - free life expectancy at age 18 decreased by 1.4 years among men ( from 50.2 to 48.8 years ) and 1.7 years among women ( from 54.2 to 52.5 years ) ( table 1 and supplementary fig .
1 ) . these reductions in diabetes - free life expectancy in the context of otherwise increasing life expectancy in the u.s . indicate declines in the proportion of the lifetime spent without diabetes .
the proportion of the lifetime spent without diabetes fell from 0.93 to 0.85 for men and from 0.90 to 0.85 for women between 19801989 and 20002004 .
years of diabetes - free life expectancy by age and bmi levels , 19801989 vs. 20002004 , u.s .
data are from national vital statistics system of the national center for health statistics , centers for disease control and prevention , and single - year data are from the nhis . the role of diabetes incidence in these reductions in diabetes - free life expectancy
can be better appreciated by standardizing mortality levels : if mortality rates had remained the same between 19801989 and 20002004 instead of improving by 2.4 years for men and 0.8 years for women , diabetes - free life expectancy would have fallen even more by 3.8 years for men and 2.5 years for women ( not shown ) .
in fact , mortality rates decreased , so reductions in diabetes - free life expectancy result solely from increases in diabetes incidence or diagnoses rather than from increases in mortality . while overall diabetes - free life expectancy decreased between the 1980s and the 2000s , there are substantial differences between bmi groups . diabetes - free life expectancy at age 18 among normal / underweight individuals increased from 51.6 to 53.5 for men and from 57.3 to 59.0 for women , as shown in table 1 .
the diabetes - free life expectancy of overweight individuals was slightly lower than those of normal / underweight individuals at both time periods .
however , the diabetes - free life expectancy of overweight individuals also increased , from 50.4 to 51.4 for men and from 52.2 to 54.9 for women .
obese individuals had by far the lowest expected diabetes - free lifetimes in both time periods .
furthermore , this was the only group to experience decreases in diabetes - free life expectancy between the 1980s and the 2000s , and these decreases were large : 5.6 years for men and 2.5 years for women .
thus , all of the observed reductions in diabetes - free life expectancy at the population level were actually due to increases in diabetes only among obese individuals , a situation that also holds for diabetes - free life expectancy at ages 40 and 60 .
in fact , when we decomposed the contributions of each bmi group to changes in the u.s .
population overall , we found that improvements in the other weight groups countered these reductions in diabetes - free life expectancy without completely offsetting them .
, obese 18-year - old men and women could expect to live 13.7 and 19.1 fewer years without diabetes than normal and underweight 18-year - old men and women , respectively .
these numbers represent increases in the difference in diabetes - free life expectancy between obese and normal / underweight individuals of 7.5 years for men and 4.2 years for women compared with the 1980s .
our decomposition of the contribution of each age group to changes in diabetes - free life expectancy shows that losses in years of life without diabetes occurred generally at younger ages for men than for women ( fig .
normal and underweight women saw improvements at all ages , with the greatest improvements at ages 18 to 39 ; overweight women also experienced improvements at most ages . on the other hand
, obese women saw reductions in diabetes - free life expectancy at all ages > 30 .
for men , the gains in diabetes - free life expectancy among normal / underweight and overweight men were less even , with some losses in the 3rd decade of life .
obese men in all age groups experienced decreases in diabetes - free life expectancy , with the greatest losses among men younger than age 50 .
contribution of age groups to changes in diabetes - free life expectancy between 19801989 and 20002004 in the u.s .
the proportion of 18-year - olds who would develop diabetes increased greatly between 19801989 and 20002004 ( fig .
23.3% of 18-year - old women and 17.8% of 18-year - old men would eventually have diabetes given the current mortality and diabetes incidence rates . in the 2000s
, this proportion had increased to just over one - third of the population , specifically 33.8% of women and 34.7% of men , suggesting closing gender gaps in diabetes incidence .
proportion of 18-year - olds in the u.s . who will develop diabetes , by sex , bmi , and period .
normal / underweight , overweight , and obese individuals experienced different changes in lifetime risk of diabetes .
for women , in the 1980s , 12 , 31 , and 53% of normal / underweight , overweight , and obese 18-year - olds , respectively , could expect to be diagnosed with diabetes . by 20002004 ,
these risks did not change for normal / underweight and overweight women but did increase for obese women to 66% . during the 1980s , normal / underweight men had similar risks of developing diabetes in their lifetimes to those faced by women ( 12% ) . however , heavier men were at considerably lower risk than women : 17% for overweight and 37% for obese . by the 2000s , the proportion of men who would develop diabetes in their lifetime increased for all weight groups to 16 , 27 , and 63% for normal / underweight , overweight , and obese men , respectively .
the calculations above assume that there were no differences in mortality by diabetes or weight status .
allowing for differential mortality between bmi groups and by diabetes status does not change the finding that diabetes - free life expectancy decreased and that this decrease resulted from increasing diabetes incidence among obese individuals ( supplementary table 1 and supplementary fig .
1 ) . it could be that fewer obese individuals with diabetes were undiagnosed in the 2000s than in the 1980s , meaning that diabetes incidence would appear to be higher even if there were no change in incidence .
previous research has shown that the proportion of diagnosed cases has increased significantly only for those with bmis > 35 ( 19 ) .
the resulting diabetes - free life expectancy at age 18 is 3 years lower than implied by diagnosed incidence rates , falling in the 2000s to 46.0 years for men and 49.4 years for women .
accounting for changes in undiagnosed diabetes does not fully explain the decreases in incidence among normal / underweight and overweight men and women ( with the possible exception of overweight men ) , nor does it explain the increases in diabetes incidence among the obese population .
thus , even if diabetes detection has improved disproportionately in obese patients , obese individuals still bear the burden of decreases in diabetes - free life expectancy that occurred during the past 20 years .
on the basis of nhis data and vital statistics , we calculated that life expectancy at birth in the u.s
. in 19801989 was 70.6 years for men and 77.4 years for women , and it had increased to 74.3 years for men and 79.0 years for women by 20002004 .
remaining life expectancy at age 18 also increased by 3.1 years for men and 1.1 years for women ( supplementary table 1 ) .
in contrast , diabetes - free life expectancy at age 18 decreased by 1.4 years among men ( from 50.2 to 48.8 years ) and 1.7 years among women ( from 54.2 to 52.5 years ) ( table 1 and supplementary fig .
1 ) . these reductions in diabetes - free life expectancy in the context of otherwise increasing life expectancy in the u.s . indicate declines in the proportion of the lifetime spent without diabetes .
the proportion of the lifetime spent without diabetes fell from 0.93 to 0.85 for men and from 0.90 to 0.85 for women between 19801989 and 20002004 .
years of diabetes - free life expectancy by age and bmi levels , 19801989 vs. 20002004 , u.s .
data are from national vital statistics system of the national center for health statistics , centers for disease control and prevention , and single - year data are from the nhis . the role of diabetes incidence in these reductions in diabetes - free life expectancy
can be better appreciated by standardizing mortality levels : if mortality rates had remained the same between 19801989 and 20002004 instead of improving by 2.4 years for men and 0.8 years for women , diabetes - free life expectancy would have fallen even more by 3.8 years for men and 2.5 years for women ( not shown ) .
in fact , mortality rates decreased , so reductions in diabetes - free life expectancy result solely from increases in diabetes incidence or diagnoses rather than from increases in mortality . while overall diabetes - free life expectancy decreased between the 1980s and the 2000s , there are substantial differences between bmi groups . diabetes - free life expectancy at age 18 among normal / underweight individuals increased from 51.6 to 53.5 for men and from 57.3 to 59.0 for women , as shown in table 1 .
the diabetes - free life expectancy of overweight individuals was slightly lower than those of normal / underweight individuals at both time periods .
however , the diabetes - free life expectancy of overweight individuals also increased , from 50.4 to 51.4 for men and from 52.2 to 54.9 for women .
obese individuals had by far the lowest expected diabetes - free lifetimes in both time periods .
furthermore , this was the only group to experience decreases in diabetes - free life expectancy between the 1980s and the 2000s , and these decreases were large : 5.6 years for men and 2.5 years for women .
thus , all of the observed reductions in diabetes - free life expectancy at the population level were actually due to increases in diabetes only among obese individuals , a situation that also holds for diabetes - free life expectancy at ages 40 and 60 .
in fact , when we decomposed the contributions of each bmi group to changes in the u.s .
population overall , we found that improvements in the other weight groups countered these reductions in diabetes - free life expectancy without completely offsetting them .
, obese 18-year - old men and women could expect to live 13.7 and 19.1 fewer years without diabetes than normal and underweight 18-year - old men and women , respectively .
these numbers represent increases in the difference in diabetes - free life expectancy between obese and normal / underweight individuals of 7.5 years for men and 4.2 years for women compared with the 1980s .
our decomposition of the contribution of each age group to changes in diabetes - free life expectancy shows that losses in years of life without diabetes occurred generally at younger ages for men than for women ( fig .
normal and underweight women saw improvements at all ages , with the greatest improvements at ages 18 to 39 ; overweight women also experienced improvements at most ages . on the other hand
, obese women saw reductions in diabetes - free life expectancy at all ages > 30 .
for men , the gains in diabetes - free life expectancy among normal / underweight and overweight men were less even , with some losses in the 3rd decade of life .
obese men in all age groups experienced decreases in diabetes - free life expectancy , with the greatest losses among men younger than age 50 .
contribution of age groups to changes in diabetes - free life expectancy between 19801989 and 20002004 in the u.s .
the proportion of 18-year - olds who would develop diabetes increased greatly between 19801989 and 20002004 ( fig .
23.3% of 18-year - old women and 17.8% of 18-year - old men would eventually have diabetes given the current mortality and diabetes incidence rates . in the 2000s , this proportion had increased to just over one - third of the population , specifically 33.8% of women and 34.7% of men , suggesting closing gender gaps in diabetes incidence .
proportion of 18-year - olds in the u.s . who will develop diabetes , by sex , bmi , and period .
normal / underweight , overweight , and obese individuals experienced different changes in lifetime risk of diabetes .
for women , in the 1980s , 12 , 31 , and 53% of normal / underweight , overweight , and obese 18-year - olds , respectively , could expect to be diagnosed with diabetes . by 20002004 ,
these risks did not change for normal / underweight and overweight women but did increase for obese women to 66% . during the 1980s , normal / underweight men had similar risks of developing diabetes in their lifetimes to those faced by women ( 12% ) .
however , heavier men were at considerably lower risk than women : 17% for overweight and 37% for obese . by the 2000s , the proportion of men who would develop diabetes in their lifetime increased for all weight groups to 16 , 27 , and 63% for normal / underweight , overweight , and obese men , respectively .
the calculations above assume that there were no differences in mortality by diabetes or weight status . in robustness check ,
we incorporated risk ratios from the literature into our estimates . allowing for differential mortality between bmi groups and by diabetes status does not change the finding that diabetes - free life expectancy decreased and that this decrease resulted from increasing diabetes incidence among obese individuals ( supplementary table 1 and supplementary fig .
1 ) . it could be that fewer obese individuals with diabetes were undiagnosed in the 2000s than in the 1980s , meaning that diabetes incidence would appear to be higher even if there were no change in incidence .
previous research has shown that the proportion of diagnosed cases has increased significantly only for those with bmis > 35 ( 19 ) .
the resulting diabetes - free life expectancy at age 18 is 3 years lower than implied by diagnosed incidence rates , falling in the 2000s to 46.0 years for men and 49.4 years for women .
accounting for changes in undiagnosed diabetes does not fully explain the decreases in incidence among normal / underweight and overweight men and women ( with the possible exception of overweight men ) , nor does it explain the increases in diabetes incidence among the obese population .
thus , even if diabetes detection has improved disproportionately in obese patients , obese individuals still bear the burden of decreases in diabetes - free life expectancy that occurred during the past 20 years .
in this study , we used demographic techniques and large national datasets to examine changes in diabetes - free life expectancy between 19801989 and 20002004 for adult men and women in the u.s . and to examine the changing role of body mass for diabetes risks .
these methods offer the advantage of allowing us to examine the changing incidence of diabetes while holding the improvements in mortality during the period constant .
they also allow us to estimate the contributions of changing diabetes incidence rates for different levels of body weight to overall diabetes - free life expectancy , which makes it possible to better understand the observed changes in overall diabetes risks and the differing risks in the population .
we found that the proportion of 18-year - olds who would develop diabetes in their lifetimes increased by almost 50% among women and almost doubled among men between the 1980s and the 2000s . while life expectancy for men and women in the u.s .
increased , diabetes - free life expectancy at age 18 decreased by 1.7 years for women and 1.4 years for men , indicating declines in the proportion of the lifetime spent without diabetes .
this pattern points to the emergence of a prolonged period of morbidity rather than longer healthy life spans , but only among obese individuals .
, obese 18-year - old men and women could expect to live 13.7 and 19.1 fewer years without diabetes , respectively , compared with normal / underweight 18-year - old men and women .
increases in diabetes incidence among obese people led to reductions in diabetes - free life expectancy so large that the overall diabetes - free life expectancy of the adult u.s
. population decreased in spite of improvements in diabetes - free life expectancy among nonobese individuals , who represent the majority of the population .
in the main models , we standardized mortality across bmi groups to isolate the effect of changes in diabetes incidence .
the approach allowed us to determine that changes in diabetes incidence rather than mortality were responsible for the decrease in diabetes - free life expectancy .
it also allowed us to identify the ages at which the greatest changes in diabetes incidence occurred .
sensitivity analyses show that our results are robust to adjusting mortality rates by diabetes and weight status to the rate ratios reported in the literature .
since our estimates did not directly model transitions between bmi groups , we have not shown the effect of gaining or losing weight on the expected life span without diabetes , as was also the case for previous studies ( 20 ) .
the measures yield cumulative estimates of diabetes - free life expectancy for people starting at a given bmi level at age 18 .
because the bmi composition of cohorts tends to change over time , with people gaining weight as they age , we also relaxed this assumption to account for weight increases by estimating diabetes - free life expectancy for each bmi group at age 40 and 60 .
shifts in the bmi composition of the population likely have reinforced changes in diabetes risks within bmi categories ; that is , as the proportion of the population who is obese has increased , higher incidence rates of diabetes seen among heavier individuals affect a larger proportion of the population , leading to increases in diabetes cases .
there are several possible explanations for our finding that the decreases in diabetes - free life expectancy have been borne almost exclusively by the obese population .
one explanation is that rates of diabetes detection have changed , specifically among obese individuals .
robustness checks indicate that changes in undiagnosed diabetes in obese patients relative to others explain part but not all of the reductions in diabetes - free life experienced by this group .
another reason may be changes in weight distributions within bmi groups , because our analyses indicate that weight increases were greatest among obese men and women : the average obese american was > 3.4% heavier in the 2000s than in the 1980s . since diabetes risk increases with bmi ( 2,3 ) , that the average obese person was heavier in the 2000s explains some of the increases in diabetes risk .
another consideration could be changes in the racial composition within each bmi category , since previous studies showed that the prevalence of diabetes has increased more among non - hispanic blacks and hispanics relative to non - hispanic whites in the normal and overweight categories but less in the obese category ( 21 ) .
nhis data show that the change in racial composition of bmi groups was lowest among obese individuals , leading to a relatively higher proportion of whites in the obese category .
thus , the racial distribution in the bmi categories is not likely to explain the observed trends .
another possible explanation is that diabetes risks are higher with younger age of obesity onset ; indeed , the prevalence of obesity has tripled among children since the 1970s ( 22 ) .
our estimates are not conditional on bmi level before the age to which they pertain , so earlier onset of obesity could explain some of the increases in diabetes risk for obese individuals .
a limitation of this study is our reliance on self - reported data on diabetes diagnosis , weight , and height , which have been shown to be biased ( 23 ) and may be systematically different by sex , ethnicity , and age ( 24 ) .
in addition , nhis data are collected via rigorous in - person interviews , and the differences in reported and measured bmi are not large and do not affect health risk estimates , including those associated with diabetes ( 24 ) .
furthermore , in spite of this limitation , the nhis is the only dataset with sufficient sample size to conduct this analysis . since 34% of the u.s .
adult population is obese ( 25 ) , these findings entail that more than one - third of americans can expect to develop diabetes , even if they reached adulthood without diabetes ; this proportion stands at approximately two - thirds of obese men and women .
these estimates suggest a large future increase in the prevalence of diabetes and its complications , especially among obese individuals .
our results suggest that in the face of budgetary or logistic constraints , new efforts to prevent diabetes can have the greatest impact among obese individuals , because those who are not obese generally have experienced decreases in risks during the past 2 decades with current prevention efforts .
this study highlights the growing prevalence of diabetes , implying greater future health care demand at younger ages and for longer life spans .
this will necessitate medical and public health professionals with training in diabetes management and facilities well equipped for the management of diabetes and treatment of comorbidities among obese individuals .
the many dimensions of changing population and individual health must be well understood and tracked with methods such as those demonstrated in this study to improve health care planning and health .
| objectivewith increasing life expectancy in the u.s .
, it is important to know whether a longer life expectancy means a longer healthy life span or a prolonged period of later - life morbidity .
this study examines changes in lifetime without diabetes , a leading cause of morbidity in later life.research design and methodsusing demographic methods and nationally representative data , we estimated changes in diabetes - free life expectancy between 19801989 and 20002004 for adult men and women in the u.s . , estimated the contribution of changes in age - specific diabetes rates , and examined the changing effects of weight status on diabetes risks.resultswhile life expectancy at age 18 for men and women increased between the 1980s and the 2000s , diabetes - free life expectancy at age 18 decreased by 1.7 years for men and 1.5 years for women .
the proportion of 18-year - olds who would develop diabetes in their lifetimes increased by almost 50% among women and almost doubled among men .
obese individuals experienced the greatest losses in diabetes - free life expectancy during this period , estimated at 5.6 years for men and 2.5 years for women.conclusionsdiabetes-free life expectancy decreased for both men and women between 19801989 and 20002004 , and these decreases are almost entirely attributable to large increases in diabetes incidence among obese individuals . |
sexually transmitted / transmissible infections ( stis ) also referred to as sexually transmitted diseases ( stds ) or venereal diseases ( vds ) are infections that can be transmitted from one person to another through sex ( vaginal intercourse , anal sex , or oral sex ) .
sexually transmissible infections ( stis ) are common . a number of these infections in addition to being acquired as a result of sexual contact can also be acquired by other means , such as contact with blood or blood products .
stis have major demographic , economic , social , and political impact on many populations , particularly sub - saharan africa and asia . in 1993 ,
the world bank estimated that stis ( excluding hiv ) were the second most common cause of healthy life lost among women aged 1544 years after maternal morbidity and mortality .
it is estimated that up to 1 million people are newly infected with stis daily .
the pattern and distribution of stis vary considerably between countries , but also within countries there are significant geographical variations . despite sharing a common route of transmission , stis constitute a group of diverse infections with multiple causative agents including viruses ( e.g. , human immunodeficiency virus , hiv ; herpes simplex virus , hsv ; human papilloma virus , hpv ; and hepatitis b virus , hbv ) , bacteria ( e.g. , gonococcus , chlamydia , and syphilis ) , and protozoa ( e.g. , trichomoniasis ) . preventive strategies for these infections should be based on the involved pathogens .
the majority of the preventive measures for hiv and other stis that have been used for many years were dependent on male controlled methods despite the fact that more than 90% of the infections worldwide result from heterosexual sex .
most of these traditional methods have been shown to poorly prevent hiv and other stis [ 5 , 6 ] .
this minireview aims at discussing new technological advances and new strategies for the prevention of hiv and other stis .
human papilloma viruses ( hpv ) are dna viruses that potentially infect basal epithelial cells of the skin or mucous membranes .
there are about 100 different identified genotypes of hpv ; out of these 40 are implicated in genital infection .
genital hpv genotypes are classified into high risk types ( 16/18/31/33/35/39/45/51/52/56/58/66/68 ) when associated with cervical cancer and low risk types ( 6/11/40/42/43/44/54/61/72 ) when only associated with condyloma acuminata .
the prevalence of hpv has been shown to be inversely related to age in many countries , but in some countries , which are very poor , the prevalence has been shown to be high across all age groups .
genital hpv is transmitted primarily by skin - to - skin contact , usually during sexual intercourse .
virtually all cervical cancer cases arise from genital hpv infection which necessitates the prevention of genital hpv .
hpv vaccines are prepared from protein shells called virus - like particles through recombinant gene technology .
most of the currently available vaccines are bivalent and designed to target hpv genotypes 16 and 18 which are the genotypes associated with most cases of cervical cancer .
there are also quadrivalent vaccines which have a protective effect against the low risk viral genotypes , 6 and 11 .
hpv vaccines provide prophylaxis by preventing infections and consequent disease in at - risk individuals .
available data show that there is no protective effect among women who have already been exposed to hpv 16 and hpv 18 before vaccination .
the vaccines however have shown to be highly effective in the prevention of hpv infection in clinical trials . in clinical trials , nearly 100% of vaccinated women develop detectable levels of antibodies against each hpv genotype present in the vaccine [ 13 , 14 ] .
there is also some degree of cross - protection against hpv 31 and hpv 45 which are closely related to hpv 16 and hpv 18 , respectively .
studies show that the vaccine continues to be immunogenic and well tolerated after 9 years of follow - up . in vivo studies
have shown that the antibody produced by the vaccine prevents virion binding to the cervicovaginal basement membrane .
the advisory committee on immunization practices ( acip ) of the centers for disease control and prevention ( cdc ) recommends that routine hpv vaccination should be initiated at the age of 11 or 12 years .
it also recommends that the vaccine should be given as a 3-dose series , the second dose being administered at least 1 to 2 months after the first dose and the third dose at least 6 months after the first dose .
the recommendation is that the series can be started at the age of 9 years . for females or males who have not been vaccinated previously or who have not completed the 3-dose series ,
it is recommended to administer the vaccine at the age of 13 through 26 years and 13 through 21 years , respectively .
the main one is their negligible prophylactic effect against other oncogenic genotypes that are not targeted by the vaccine .
an effective vaccine therefore should be multivalent and take into account all the high risk hpv genotypes .
overall the uptake of the vaccine has been low due to relatively high cost , parental concerns , lack of provider recommendations , and limited enthusiasm from some health care providers .
invasive cervical cancer is rated as the third most common tumor in women throughout the world .
the incidence of and mortality from cervical cancer vary across the globe ; estimates show that more than 85% of invasive cervical cancers occur in low to middle income countries .
persistent infection with high risk genotypes of hpv , though not a sufficient cause , is considered a necessary cause .
screening for cervical cancer using a cytology based test has been fundamental for decreasing the incidence and associated mortality in countries with wide screening coverage . in asymptomatic women ,
the use of a single cytological screen however has low sensitivity that can be as low as 5060% . despite the high specificity of the test in ruling out cervical cancer among healthy women ,
the test requires repeated rounds of screening to detect cervical intraepithelial neoplasm ( cin ) 3 lesions among women with cervical cancer [ 21 , 22 ] .
recent studies have demonstrated that hpv dna tests have higher sensitivity than cytological screening for the detection of cin3 [ 225 ] .
the negative predictive value ( i.e. , the reassurance of not developing cin3 and invasive cervical cancer among those that have a negative screening test ) is higher with the hpv dna test than in cytology screening .
there is sufficient evidence from studies to recommend the hpv dna test for triaging women with atypical cytology and close monitoring after treatment of cervical intraepithelial neoplasm lesions .
because of its high sensitivity , careful choices of which hpv types should be targeted and the threshold for a positive result are required to optimize the clinical specificity of hpv testing while maintaining its sensitivity for cin3 detection .
in sub - saharan africa , where a majority of world hiv cases are , women account for about 59% of all infected adults .
the number of hiv - positive women aged between 15 and 24 years is threefold higher than that of their male peers representing 76% of hiv cases in that age group .
these figures demonstrate the vulnerability of women in acquiring hiv as compared to their male counterparts .
several factors contribute to the higher risk of acquiring hiv in women than in men .
apart from the known biological factors [ 30 , 31 ] , some sexual behavior patterns such as low marriage rates , partnering with older men , inconsistent condom use , multiple partners , and limited skills in negotiating safer sex put women at an increased risk of acquiring hiv . as a result , measures to prevent mucosal transmission of hiv to women are needed .
the use of topical microbicides has an advantage because women can initiate and control its use unlike most of the other preventive measures .
research on developing potent microbicides is ongoing and several candidates are undergoing effectiveness trials to assess their impact in hiv prevention .
most of the tested microbicides however were found to have disappointing results in protection against hiv [ 36 , 37 ] ; some were found to be potentially harmful .
the microbicide trials network voice trial , which tested daily dosing of tenofovir ( tfv ) gel , oral tdf , and oral combination of tenofovir and emtricitabine ( truvada ) in women of south africa and east african countries of uganda and tanzania , found no significant hiv protective effect in any of the intervention groups .
however , drug level testing in these women showed very low adherence rates that could have influenced the findings .
recently , research in developing microbicides for prevention of mucosal hiv transmission has focused on using antiretroviral agents in various formulations and dosing strategies .
tenofovir , a nucleotide analog antiretroviral , has been shown to be safe with an overall hiv protective effect of 39% ( 54% in women with high adherence ) and 51% against herpes simplex virus type 2 ( hsv-2 ) . comparing the results of voice trial and results of the centre for the aids programme of research in south africa ( caprisa ) 004 trial ,
it is evident that adherence plays a critical role in the success or failure of arv - based microbicides .
initially it was formulated for oral use , but , because of its efficacy , long half - life , and favorable safety profile , it was considered as an ideal drug for making microbicides .
results of the ( caprisa ) 004 trial therefore demonstrate that tfv gel could potentially fill the hiv prevention gap especially for women who can not negotiate safe sex .
a rectal microbicide is a topical substance , which may be prepared in the form of a lube , anal douche , or a wash .
rectal microbicides are developed and tested to reduce a person 's risk of acquiring hiv and other stis through anal intercourse . despite the fact that anal intercourse increases the risk of hiv infection by as much as 10 to 20 percent compared to vaginal intercourse
scientists have tried the use of vaginally formulated 1% tfv gel product for rectal protection ; however , it was found to be neither safe nor acceptable for rectal use .
studies in nonhuman primates have demonstrated that antiretroviral gels provide rectal protection [ 48 , 49 ] .
the results of a phase ii trial ( mtn 017 ) designed to evaluate the rectal safety , drug absorption , and acceptability of a reduced glycerin formulation of tfv gel which is a reformulated version of vaginal tfv gel as well as oral truvada have been reported .
most side effects from study products were minor , and there were no significant differences in adverse events with the gel regimens ( daily and on demand ) compared to oral truvada .
the results of mtn-017 support further studies on the use of reduced glycerine 1% tfv gel as a rectal microbicide for hiv prevention among men who have sex with men ( msm ) and transgender women ( tgw ) .
a double blind randomized phase 2a study of dapivirine 0.05% gel applied rectally in hiv-1 seronegative adults ( mtn-035 ) is currently in development stage .
the fight against hiv / aids can not be achieved with care and treatment alone . as the efforts in finding an effective vaccine remain unsuccessful , several alternative methods for preventing hiv have been proposed .
as previously discussed , there is a need for female - controlled hiv prevention strategies which includes the development of vaginal microbicides : compounds designed to achieve a topical preexposure prophylaxis . however , as discussed , the microbicides which were studied did not appear to be very effective . there were also issues of adherence .
the rings are coated with a nonnucleoside reverse transcriptase inhibitor dapivirine and replaced every month . in a recently published study which was conducted in multiple sites in sub - saharan africa (
aspire study ) , women who used the ring were 27 percent less likely to become infected with the hiv virus .
another sister trial ( the ring study ) found that the ring was 31% effective ; women who used the ring were 31 percent less likely to become infected with the hiv virus .
the efficacy increased with increasing level of adherence . in the aspire study , when the scientists excluded data from 2 sites where many women were not returning for study visits or using the ring consistently , the ring reduced the risk of hiv infection by 37% .
the open label extension studies for the two trials are underway . in order to achieve success in prevention of hiv among the women worldwide
, there should be a diversity of microbicide delivery systems that takes into account the varied populations of women all over the world .
a number of microbicide products are in various stages of their development ; some of the microbicides being developed include films , nanofibers , and tablets which have better drug delivery systems compared to vaginal gels .
vaginal tablets are easily formulated and manufactured ; however , they may leave a grainy residue in the vaginal cavity after dissolution .
vaginal films are also relevant for pericoital use . they have demonstrated capability of delivering physicochemically diverse agents and exhibit enhanced product stability compared to vaginal gels that are semisolid .
polymeric films provide rapid drug release and bioadhesive properties that may increase retention time at the target tissue .
the use of nanoparticle encapsulation is also being investigated as a possible drug delivery system for a microbicide .
nanoparticles formulated from the biodegradable copolymer poly ( lactic - co - glycolic acid ) ( plga ) have been shown to be effective when used as a drug delivery system .
animal studies on rapidly disintegrating vaginal tablets containing tfv either alone or in combination with emtricitabine demonstrated favorable vaginal tissue and fluid concentrations of both drugs [ 56 , 57 ] .
currently , a phase i placebo controlled safety of vaginal tablets in hiv negative women is ongoing .
another trial compared the safety , drug absorption , and drug distribution of dapivirine containing vaginal films and vaginal gels .
the preliminary results of the trial ( fame 02 ) show that plasma levels of dapivirine were comparable across the film and gel arms , suggesting that both products can deliver drugs with similar efficacy .
there are also ongoing trials ( fame 04 ) on a cellulose - based film containing tenofovir ( currently in a phase i ) and on biodegradable electrospun nanofibers containing agents including tenofovir , griffithsin , or carrageenan with activity against hiv , hsv , and hpv .
one of the highest priorities in the hiv pandemic response is to develop an efficacious and protective hiv vaccine .
an efficacious and protective vaccine would be the best long term tool for the control of hiv infection .
the search for an efficacious hiv vaccine has been an ongoing exercise for the past two decades ; however results have been disappointing [ 59 , 60 ] .
the main obstacles include the high mutation rate of the virus resulting in high viral amino acid sequence variability and the concern of causing an autoimmune phenomenon following cross - reactivity between viral and host proteomes .
studies have shown that there is very high peptide identity pattern between hiv and humans which increases the risk of cross - reactivity in the course of hiv-1 immune responses [ 63 , 64 ] . to develop a safe , effective , and universally acceptable vaccine ,
unique viral peptide signatures should be used to minimize potential for harmful collateral cross - reactions .
however , the immune correlation and the quality and magnitude of the immune responses needed for a protective effective against hiv remain unclear .
typically , vaccine development is based on ability of the vaccine to induce immune responses involving both neutralizing antibodies and activation of cytotoxic t lymphocytes as it would occur in nature following an infection .
an initial approach in vaccine development tried to use vaxgen 's aidsvax which is a recombinant form of glycoprotein 120 ( gp 120 ) that forms part of the hiv envelop .
however this vaccine did not show any protective effect against hiv because it failed to induce formation of broadly neutralizing antibodies .
the vaccine that has demonstrated some encouraging findings is the one that is being tested in thailand . in the thai clinical trial (
rv 144 ) , a combination vaccine composed of priming doses of vcp1521 , a recombinant canarypox viral vector , followed by a boosting dose consisting of both the vector and vaxgen 's aidsvax has been tested .
the vaccine has been shown to be able to induce antibody responses against the second variable ( v2 ) loop of gp 120 of multiple hiv subtypes [ 66 , 67 ] .
the conclusion from the findings of this trial was that the prime - boost vaccine may reduce the risk of hiv acquisition in a community - based population with heterosexual transmission as a main route of hiv transmission .
despite the modest level of efficacy observed by rv 144 trial in thailand , it provides a platform for future directions in vaccine search by providing evidence that a safe and effective hiv protective vaccine is possible .
treatment as prevention includes the use of art in hiv - infected individuals to prevent transmission of hiv to unaffected partners .
a study in uganda demonstrated that there is a significant dose - response relation of increased hiv transmission with increasing viral load .
similar findings were obtained in another meta - analysis ; hiv transmission was reduced in patients using art .
it is logical therefore that the lower the viral load , the lower the chances of hiv transmission . the highest evidence for this is provided by hptn 052 , a large multicontinental trial involving hiv serodiscordant couples .
the trial evaluated the impact of early versus late art initiation in these couples . in this study , it was found out that there was 96% reduction in transmission associated with early art initiation .
the results show that early art initiation could have the best results in preventing hiv transmission than any other biomedical measures studied to date .
the same protective effect could be found in other hiv risk groups such as men who have sex with men ( msm ) .
adherence to the arvs has been shown to play a key role in reaching the goal . in view of these findings ,
several countries in europe and the regional guidelines recommend the use of early initiation of art as a strategy to minimize hiv transmission . a cluster - randomized trial ( hptn 071 ( popart ) ) aiming to test whether widespread provision of art is feasible and can substantially reduce population - level hiv incidence is currently underway in zambia and south africa .
this is another strategy in which arvs are used for the prevention of hiv transmission . in this strategy ,
the concept of using arvs as part of preexposure prophylaxis was derived from the same concept used for the prevention of mother to child transmission ( pmtct ) .
drugs with proven efficacy and safety for use as prep include a tenofovir ( tdf ) and emtricitabine ( ftc ) coformulation ( truvada ) .
evidence for this comes from the iprex trial involving a number of men who have sex with men ( msm ) in 6 different countries taking daily truvada . in this trial , men randomized to the truvada arm were 44% less likely to become infected with hiv than in the placebo arm .
those who reported > 90% adherence had 68% efficacy while those who reported < 50% adherence had only 16% efficacy .
studies in heterosexual partners also using truvada and tenofovir as prep have shown significant reduction in hiv transmission . a study done in uganda and kenya involving
serodiscordant heterosexual couples showed that couples randomized to tenofovir and truvada arms were 67% and 75% less likely to become hiv - infected , respectively , compared to those in placebo ( partners prep ) .
the risk reduction was 62.2% in another study done in botswana using truvada involving heterosexual couples ( tdf-2 ) .
another trial assessed whether taking a combination of tenofovir and emtricitabine before and after sexual activity ( on - demand prep ) is effective in preventing hiv transmission among men at high risk for hiv-1 infection . despite an increase in the gastrointestinal and renal side effects , there was a relative risk reduction of 86% in the tdf - ftc group .
there is enough evidence to warrant development of guidelines for the use of truvada for hiv prevention in msm and serodiscordant couples .
however , issues such as development of drug resistance , cost , potential behavioral changes , and risk compensation should not be overlooked .
a number of ecological and observational studies have suggested hiv protective effect of male circumcision [ 7981 ] . since then , several clinical trials have been performed to increase the strength of evidence related to this hypothesis .
three separate trials have demonstrated that male circumcision could reduce male hiv acquisition by up to 60% [ 8284 ] .
in addition to hiv , male circumcision also showed protective effects against other stis such as hpv , hsv-2 [ 82 , 83 ] , and mycoplasma genitalium infections .
male circumcision however has not been shown to reduce hiv to female partners ; the same applies for other stis such as hpv , hsv-2 , and mycoplasma genitalium [ 86 , 87 ] .
several countries in southern and eastern africa with a high prevalence of hiv and low levels of male circumcision are scaling up male circumcision as part of hiv prevention programs .
female - controlled method of protection remains the most promising way to address heterosexual hiv transmission .
the female condom remains the only female initiated barrier contraceptive which can protect against hiv , unwanted pregnancy , and other sexually transmitted diseases . despite worldwide increase in the distribution of female condoms , it remains as the most underutilized reproductive health technology .
evidence suggests that the underuse of female condoms could have been contributed by social stigma and lack of correct information about the product .
the new second - generation female condoms have been developed to improve the acceptability and reduce the cost as well .
the second - generation female condoms ( fc2 ) are made from synthetic nitrile that diminishes distracting crinkling sounds produced by polyurethane .
they have been shown to be effective in the prevention of hiv and other stis and are available at a lower cost compared with the first - generation female condoms .
fc2 have been shown to be a cost - effective method for hiv and other sti prevention even at low adherence .
the protective effect of female condoms for the prevention of stis and pregnancy has been shown to be similar to that of male condoms .
female condoms could have a more protective effect against syphilis , genital herpes , and human papilloma virus infections because it covers more of the female genitalia than male condoms . with proper use of the female condom
the improved female condoms may provide enhanced sensation for men as compared to male condoms ; they are hypoallergic and hence minimize the risk for those allergic to rubber latex .
it may also be inserted hours before sexual intercourse because they do not depend on an erect penis for insertion . the condom is well lubricated and warms to body temperature .
studies have shown that fc2 performed well in short - term acceptability and crossover studies with fc1 [ 91 , 92 ] .
some challenges may continue to limit its roll - out , including the higher cost of female condoms compared with male condoms , the need to learn how to properly use the female condom , the distracting noises associated with the original version , and the visibility of the outer ring outside the vagina .
its use also requires some skill that needs to be learned . also the reported crinkling noises during sexual intercourse in the original version may have put off the potential users .
the outer ring of the condom is also visible outside the vagina , another factor that may affect its uptake .
however reports show increased update of the female condom with good social marketing and health education [ 93 , 94 ] .
postexposure prophylaxis is the use of short - term art to reduce the risk of hiv acquisition following exposure .
existing guidelines recommend initiation of art within 72 hours after exposure to be used for a total of 28 days .
pep has been used for some time for occupational exposures ( since 1990s ) and is becoming available for nonoccupational exposures including sex .
evidence for pep comes from animal studies and retrospective case control analyses of pep for occupational exposure as well as from the use of art for preventing mother to child transmission [ 9698 ] .
existing evidence suggests that pep following nonoccupational exposure may be cost - effective , especially in certain population subgroups [ 99 , 100 ] despite lack of enough evidence on its clinical effectiveness .
there is enough evidence demonstrating that the presence of stis increases the risk of hiv transmission .
biologically , the assumption is that reducing genital tract inflammation will reduce hiv infectiousness as well as susceptibility in hiv - uninfected individuals .
based on this , treatment of stis will presumably result in reduction of the risk of hiv transmission .
a study done in mwanza , tanzania , in mid-1990s demonstrated a 38% reduction in hiv incidence by using a syndromic approach for the treatment of stis .
however , two studies from uganda [ 103 , 104 ] and another one from kenya among female sexual workers did not have similar findings .
one of the possible reasons could be the low levels of treatable stis in ugandan cohorts and presence of a mature hiv epidemic in uganda .
the successes observed in the mwanza trial and the benefits from sti programs necessitate the inclusion and integration of sti treatment as part of an hiv / aids response despite lack of enough evidence on its benefits .
biological interventions provide new hopes for much more effective hiv prevention . some of these biomedical interventions have proven efficacy in hiv prevention , while some are still being researched .
these new biomedical interventions should therefore be combined with behavioral approaches to maximize the hiv preventive effect .
literature review for this article however was not a systematic review , but rather a minireview that meant to give a synopsis of key papers and studies in the field .
a summary of the trials for key papers that summarize these interventions is provided in table 1 . | sexually transmitted infections remain to be of public health concern in many developing countries .
their control is important , considering the high incidence of acute infections , complications and sequelae , and their socioeconomic impact .
this article discusses the new biomedical technologies and strategies for the prevention of hiv and other sexually transmitted infections . |
presynaptic monoamine transporters such as the dopamine transporter ( dat ) regulate signal transduction by controlling neurotransmitter concentrations available for receptor binding at the postsynaptic site .
disturbances in dat function have been implicated in a variety of disorders including schizophrenia , major depressive disorder , bipolar depression , tourette s syndrome , attention deficit / hyperactivity disorder , and anxiety disorders ( for reviews , see nikolaus and colleagues 13 ) .
moreover , dat function is severely impaired in movement disorders including idiopathic and early - onset parkinson s disease , multiple system atrophy , progressive supranuclear palsy , corticobasal degeneration , lewy body dementia , and parkinson s disease dementia ( for reviews , see nikolaus and colleagues 4,5 ) . as dat function
parkinson plus syndromes , but not affected in essential tremor 6 , dat imaging with either pet or single - photon emission computed tomography ( spect ) has become an important tool for differentiating between movement disorders .
symptoms of parkinson s disease can be ameliorated by treatment with l-3,4-dihydroxyphenylalanine ( l - dopa ) .
thereby , l - dopa bypasses the blood brain barrier through a saturable transporter and is converted to dopamine ( da ) by aromatic l - amino acid decarboxylase ( aadc ) mainly within the presynaptic terminals of striatal da neurons ( for reviews , see okereke 7 ) .
there is conflicting evidence as to the displaceability of dat radioligands by endogenous da : in parkinsonian patients , no significant effect on either [ i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ( [ i]fp - cit ) 8 or [ i]methyl-3-(4-iodophenyl)tropane-2-carboxylate ( [ i]-cit ) 9,10 binding was observed after long - term treatment with l - dopa in doses of either 150800 mg / day ( plus carbidopa , dose not specified ) for 1272 months 8 , 300 mg / day ( plus 75 mg / day carbidopa ) for up to 46 months 9 , or 150 , 300 , and 600 mg / day ( plus 37.5 , 75 , and 150 mg / day carbidopa ) for 10 months 10 .
11 after long - term treatment with l - dopa ( 200400 mg / day plus carbidopa or benserazide , dose not specified ) for 3 months and by contin et al .
12 after acute l - dopa challenge ( 100 mg / day plus 25 mg benserazide ) with 2-carbomethoxy-3-(4-[f]-fluorophenyl)tropane ( [ f]cft ) and [ i]fp - cit as radioligands .
in contrast , guttman et al .
13 observed a significant 1622% reduction in [ c]3-(4-iodophenyl)tropane-2-carboxylic acid isopropyl ester ( [ c]rti-55 ) binding after chronic treatment with 300 mg / day
similarly , ex - vivo dissection studies on rats using [ tc]trodat as the radioligand revealed reductions in dat binding by 19 and 59% after application of 125 and 150 mg / kg l - dopa ( plus 25 mg / kg benserazide ) , respectively , and a 44% reduction in dat binding after application of amphetamine ( 1.35 mg / kg , 14 ) .
15 assessed dat binding in nonhuman primates after intravenous infusion of l - dopa ( 250 mol / kg plus 5 mg / kg benserazide ) or amphetamine ( 10.8 mol / kg ) . in this study , l - dopa was not found to decrease [ i]-cit binding , whereas amphetamine reduced [ i]-cit binding by 50% .
16 treated mptp - lesioned monkeys for 3 months with intravenous infusion of l - dopa ( 20 mg / kg twice daily ) observing no effect on [ tc]trodat binding compared with controls . in contrast , in a recent small animal pet study on 6-hydroxydopamine ( 6-ohda)-lesioned rats , chronic l - dopa ( 10 mg / kg plus 15 mg /
kg benserazide for 4 weeks ) reduced [ c]methylphenidate binding by 7% relative to the pretreatment state in the unlesioned contralateral striatum 17 .
interestingly , on the lesioned side , [ c]methylphenidate binding was increased by 6% relative to the state before the initiation of treatment .
the finding of reduced dat binding after pretreatment with l - dopa or amphetamine may be indicative of a competition between the dat ligand and synaptic da .
if endogenous da can displace exogenous radioligands and/or compete with them for presynaptic binding sites , this may have implications for dat imaging studies on neurologic as well as psychiatric patients receiving daergic medication .
thus , the rationale of the present study was the assessment of [ i]fp - cit binding to the striatal dat after challenge with therapeutic doses of l - dopa ( 5 or 10 mg / kg ) . in clinical practice , l - dopa is administered together with aadc inhibitors such as carbidopa or benserazide to prevent its peripheral conversion to da ( for reviews , see di stefano et al .
as microdialysis studies have shown that the sole application of benserazide ( 50 mg / kg ) may elevate striatal da concentrations in normal rats 19 , we further assessed [ i]fp - cit binding to the striatal dat after pretreatment with benserazide .
l - dopa is known to affect motor behavior 20 ; therefore , we additionally set out to investigate the association between striatal dat binding and parameters of motor and exploratory behavior after challenge with 5 or 10 mg / kg of l - dopa .
a total of 41 adult male wistar rats ( tva , heinrich - heine university , dsseldorf , germany ) weighing 39852 g ( meansd ) underwent dat imaging studies at baseline , after treatment with the aadc inhibitor benserazide , and/or after treatment with either 5 or 10 mg / kg l - dopa plus benserazide . out of these rats ,
19 were subjected to behavioral measurements after administration of 5 or 10 mg / kg l - dopa with subsequent assessment of dat binding .
on three other rats , images of bone metabolism , soft - tissue perfusion , and brain perfusion were acquired with tc - labeled tracers to delineate the striatal and cerebellar target regions as previously published 21,22 .
the study was approved by the regional authority ; it was carried out in accordance with the principles of laboratory animal care ( nih publication no .
86 - 23 , revised 1985 ) and the german law on the protection of animals
. the small animal tomograph ( tierspect ) that was used in the study has been described in detail elsewhere 23 .
tomographic resolutions ( full - width at half - maximum ) amount to 3.4 and 2.8 mm for i and tc , respectively .
sensitivities are 16 cps / mbq ( i ) and 22 cps / mbq ( tc ) . in the present study , a low - energy ultrahigh - resolution parallel - hole collimator ( 3710.2 mm )
data were acquired in a 128128 matrix with a pixel width and a slice thickness of 0.664 mm .
reconstruction was performed using an iterative ordered - subset expectation maximization algorithm ( three iterations , four subsets / iteration ) .
an attenuation correction of 0.11 /cm was implemented for both i and tc , assuming a uniformly attenuating medium .
dat binding was assessed after challenge with 5 mg / kg l - dopa methylester ( sigma - aldrich , taufkirchen , germany ; n=15 ) plus benserazide ( sigma - aldrich ; dose , 10 mg / kg ) or 10 mg / kg l - dopa methylester plus benserazide ( n=16 ) .
of the 15 rats pretreated with 5 mg / kg l - dopa / benserazide , all animals underwent dat imaging at baseline ; six animals were scanned further after challenge with benserazide .
of the 16 rats pretreated with 10 mg / kg l - dopa / benserazide , 14 animals were scanned at baseline , whereas 13 animals additionally underwent dat imaging after benserazide .
baseline measurements and measurements after pharmacological challenges were performed in randomized order , and were 117 days apart .
challenges with benserazide ( concentration , 10 mg / ml ; vehicle , 0.9% saline ) and l - dopa ( concentrations , 5 , 10 mg / ml ; vehicle , 0.9% saline ) plus benserazide ( concentration , 10 mg / ml ; vehicle , 0.9% saline ) were applied intraperitoneally 30 min before radioligand application , as previous investigations in rats had shown that maximum striatal da concentrations are reached at 30 min after intraperitoneal application of l - dopa and remain stable for 2 h 24 . in the l - dopa / benserazide condition , l - dopa and benserazide
were administered simultaneously as previous investigations had shown that pretreatment with benserazide up to 3 h before l - dopa administration did not alter motor responses to l - dopa compared with concomitant application with the latter compound 25 .
thirty minutes after l - dopa / benserazide or benserazide application , animals received intraperitoneal injections of 0.9 ml / kg ketamine hydrochloride ( ketavet ; pharmacia gmbh , erlangen , germany ; concentration , 100 mg / ml ) and 0.4 ml / kg xylazine hydrochloride ( rompun ; bayer , leverkusen , germany ; concentration , 2% ) . into the lateral tail vein , 233 mbq [ i]fp - cit ( datscan ; ge healthcare , munich , germany ; concentration range , 0.070.13 g / ml , specific activity range , 2.54.510 bq / mmol at reference time ) was injected using a winged infusion needle set .
the tube was rinsed with 1 ml 0.9% saline amounting to a total injection volume of 1.3 ml . as previous investigations on rats
had shown that equilibrium of [ i]fp - cit binding is reached at 2 h post injection with the ratio of specific to nonspecific striatal uptake remaining stable through the following 4 h 26 , spect measurements were started 2 h after radioligand application .
imaging data were recorded in a step - and - shoot mode over a circular orbit in angular steps of 6 ( 60 projections , 60 s / projection ) using a 65 mm radius of rotation .
the 15% energy window was centered on the 159 kev gamma photopeak of i. imaging of head and neck bone metabolism , head and neck soft - tissue perfusion , and brain perfusion was performed as previously described 21,22 . immediately after the injection of either 5 mg / kg ( n=10 ) or 10 mg / kg ( n=9 ) l - dopa / benserazide , rats were placed into the center of a cage with a top unit equipped with light - emitting diodes and a charge - coupled device camera ( phenotyper ; noldus information technology , wageningen , the netherlands ; open - field dimensions , 454556 cm ) .
durations and frequencies of motor ( ambulatory activity , sitting without any movement , grooming ) and exploratory behaviors ( rearing , head and shoulder motility while the animal is sitting or standing ) were rated by one of the investigators ( s.n . ) in blocks of 5 min each for a total of 30 min using ethovision xt ( noldus information technology ) .
contingent on the behavioral trials , animals were anesthetized and administered [ i]fp - cit as described above .
imaging data were evaluated using the multi - purpose - imaging - tool ( mpi - tool v3.29 ; advanced tomo vision gmbh , kerpen , germany ) as previously described 21,22 .
briefly , target and reference regions were identified using sets of fusion images ( [ tc]dpd-[tc]tetrofosmin , [ tc]dpd-[tc]hmpao , [ tc]dpd-[i]ifp - cit ) , allowing the identification of extracerebral anatomical landmarks such as cranium , orbitae , and harderian glands and the localization of the respective regions relative to the sites of specific accumulation of metabolic and perfusion markers ( fig .
striatal target and cerebellar reference regions were defined using the regional activity maxima ( fig .
thereby , maximum striatal count rates ( counts / pixel ) were determined on coronal slices by defining a circular region covering an area of 1.5 mm , which comprised a total of 11 pixels . on the same slices used for the determination of maximum striatal count rates , reference count rates ( counts / pixel ) were obtained in an elliptic region ( area , 7 mm comprising a total of 53 pixels ) 15 mm posterior to the frontal cortex corresponding anatomically to the rat cerebellum .
a template of striatal and cerebellar regions was positioned on the individual images by visual inspection without changing their shape or size . left and right striatal radioactivity concentrations were averaged . for baseline and treatment conditions , the equilibrium ratio of the distribution volumes of the specifically and the nonspecifically bound compartment [ v3=vt ( striatum)/vt ( cerebellum)1 ] was computed as an estimate for the binding potential 27 . ( a ) identification of the rat striatum and cerebellum relative to anatomical landmarks .
the left column shows the rat head [ tc]dpd scan ( top ) , the rat head [ tc]tetrofosmin scan ( middle ) , and the superimposition of bone metabolism and perfusion scans ( below ) . as these tracers
brain barrier , their distribution allows the identification of the orbitae ( 1 ) , cranium ( 2 ) , and harderian glands ( 3 ) .
the middle column shows the rat head [ tc]dpd scan ( top ) , the rat head [ tc]hmpao scan ( middle ) , and the superimposition of bone metabolism and brain perfusion scans ( below ) .
the fusion of [ tc]dpd and [ tc]hmpao images allows the alignment of the cranium ( 2 ) and the cerebral [ tc]hmpao accumulation and thus permits the identification of the cerebellum ( 4 ) relative to the rat cranium .
the right column shows the rat head [ tc]dpd scan ( top ) , the rat head [ i]fp - cit scan ( middle ) , and the superimposition of [ tc]dpd and [ i]fp - cit images ( below ) . the fusion image allows the identification of the rat striatum ( 5 ) relative to the orbitae ( 1 ) and the cranium ( 2 ) as visualized by [ tc]dpd , and to the sites of extracerebral [ i]-fp - cit accumulations corresponding to the sites of extracerebral [ tc]tetrofosmin accumulations in the harderian glands ( 3 ) .
furthermore , the cerebellum ( 4 ) may be identified relative to the cranium ( 2 ) and to sites of cerebellar ( 4 ) hmpao accumulations .
( b ) template consisting of cerebellar ( 4 ) and striatal ( 5 ) regions of interest .
thereby , in each pretreatment condition , v3 values as well as cerebellar count rates were found to be normally distributed ( 0.313p0.989 ) .
striatal v3 values and cerebellar radioactivity count rates obtained at baseline , after benserazide , and after 5 or 10 mg / kg l - dopa / benserazide were compared with one - way analyses of variance ( anovas ) for repeated measures ( holm sidak pairwise multiple comparison , =0.05 ) .
in addition , striatal v3 values and cerebellar radioactivity count rates were compared between baseline and the respective pretreatment condition ( benserazide , 5 mg / kg l - dopa / benserazide , 10 mg / kg l - dopa / benserazide ) with the paired t - test ( two tailed , =0.05 ) . moreover ,
striatal v3 values obtained in l - dopa / benserazide - treated animals subjected to behavioral analysis were compared between l - dopa doses using the independent t - test ( two tailed , =0.05 ) .
distributions of behavioral data ( duration and frequency of ambulatory activity , sitting , rearing , head and shoulder motility , and grooming in minutes 15 , 610 , 1115 , 1620 , 2125 , 2630 , and 130 ) were assessed with the nonparametric kolmogorov
thereby , the majority of behavioral parameters were normally distributed in each treatment group ( 0.052p1 ) .
no normal distribution could be ascertained for rearing duration ( p=0.032 ) , rearing frequency ( p=0.038 ) , and grooming frequency ( p=0.047 ) during 2530 min after pretreatment with 5 mg / kg l - dopa / benserazide and for grooming frequency during 2530 min after pretreatment with 10 mg / kg l - dopa / benserazide ( p=0.045 ) . for either treatment group , v3 values and normally and
not normally distributed behavioral data were correlated using the pearson product moment correlation coefficient ( =0.05 ) and the spearman rank correlation ( coefficient =0.05 ) , respectively .
a total of 41 adult male wistar rats ( tva , heinrich - heine university , dsseldorf , germany ) weighing 39852 g ( meansd ) underwent dat imaging studies at baseline , after treatment with the aadc inhibitor benserazide , and/or after treatment with either 5 or 10 mg / kg l - dopa plus benserazide . out of these rats ,
19 were subjected to behavioral measurements after administration of 5 or 10 mg / kg l - dopa with subsequent assessment of dat binding .
on three other rats , images of bone metabolism , soft - tissue perfusion , and brain perfusion were acquired with tc - labeled tracers to delineate the striatal and cerebellar target regions as previously published 21,22 .
the study was approved by the regional authority ; it was carried out in accordance with the principles of laboratory animal care ( nih publication no .
86 - 23 , revised 1985 ) and the german law on the protection of animals .
the small animal tomograph ( tierspect ) that was used in the study has been described in detail elsewhere 23 .
tomographic resolutions ( full - width at half - maximum ) amount to 3.4 and 2.8 mm for i and tc , respectively .
sensitivities are 16 cps / mbq ( i ) and 22 cps / mbq ( tc ) . in the present study , a low - energy ultrahigh - resolution parallel - hole collimator ( 3710.2 mm )
data were acquired in a 128128 matrix with a pixel width and a slice thickness of 0.664 mm .
reconstruction was performed using an iterative ordered - subset expectation maximization algorithm ( three iterations , four subsets / iteration ) .
an attenuation correction of 0.11 /cm was implemented for both i and tc , assuming a uniformly attenuating medium .
dat binding was assessed after challenge with 5 mg / kg l - dopa methylester ( sigma - aldrich , taufkirchen , germany ; n=15 ) plus benserazide ( sigma - aldrich ; dose , 10 mg / kg ) or 10 mg / kg l - dopa methylester plus benserazide ( n=16 ) . of the 15 rats pretreated with 5 mg / kg l - dopa / benserazide , all animals underwent dat imaging at baseline ; six animals
of the 16 rats pretreated with 10 mg / kg l - dopa / benserazide , 14 animals were scanned at baseline , whereas 13 animals additionally underwent dat imaging after benserazide .
baseline measurements and measurements after pharmacological challenges were performed in randomized order , and were 117 days apart .
challenges with benserazide ( concentration , 10 mg / ml ; vehicle , 0.9% saline ) and l - dopa ( concentrations , 5 , 10 mg / ml ; vehicle , 0.9% saline ) plus benserazide ( concentration , 10 mg / ml ; vehicle , 0.9% saline ) were applied intraperitoneally 30 min before radioligand application , as previous investigations in rats had shown that maximum striatal da concentrations are reached at 30 min after intraperitoneal application of l - dopa and remain stable for 2 h 24 . in the l - dopa / benserazide condition , l - dopa and benserazide were administered simultaneously as previous investigations had shown that pretreatment with benserazide up to 3 h before l - dopa administration did not alter motor responses to l - dopa compared with concomitant application with the latter compound 25 .
thirty minutes after l - dopa / benserazide or benserazide application , animals received intraperitoneal injections of 0.9 ml / kg ketamine hydrochloride ( ketavet ; pharmacia gmbh , erlangen , germany ; concentration , 100 mg / ml ) and 0.4 ml / kg xylazine hydrochloride ( rompun ; bayer , leverkusen , germany ; concentration , 2% ) . into the lateral tail vein , 233 mbq [ i]fp - cit ( datscan ; ge healthcare , munich , germany ; concentration range , 0.070.13
g / ml , specific activity range , 2.54.510 bq / mmol at reference time ) was injected using a winged infusion needle set .
the tube was rinsed with 1 ml 0.9% saline amounting to a total injection volume of 1.3 ml . as previous investigations on rats
had shown that equilibrium of [ i]fp - cit binding is reached at 2 h post injection with the ratio of specific to nonspecific striatal uptake remaining stable through the following 4 h 26 , spect measurements were started 2 h after radioligand application .
imaging data were recorded in a step - and - shoot mode over a circular orbit in angular steps of 6 ( 60 projections , 60 s / projection ) using a 65 mm radius of rotation .
the 15% energy window was centered on the 159 kev gamma photopeak of i. imaging of head and neck bone metabolism , head and neck soft - tissue perfusion , and brain perfusion was performed as previously described 21,22 .
immediately after the injection of either 5 mg / kg ( n=10 ) or 10 mg / kg ( n=9 ) l - dopa / benserazide , rats were placed into the center of a cage with a top unit equipped with light - emitting diodes and a charge - coupled device camera ( phenotyper ; noldus information technology , wageningen , the netherlands ; open - field dimensions , 454556 cm ) .
durations and frequencies of motor ( ambulatory activity , sitting without any movement , grooming ) and exploratory behaviors ( rearing , head and shoulder motility while the animal is sitting or standing ) were rated by one of the investigators ( s.n . ) in blocks of 5 min each for a total of 30 min using ethovision xt ( noldus information technology ) .
contingent on the behavioral trials , animals were anesthetized and administered [ i]fp - cit as described above .
imaging data were evaluated using the multi - purpose - imaging - tool ( mpi - tool v3.29 ; advanced tomo vision gmbh , kerpen , germany ) as previously described 21,22 .
briefly , target and reference regions were identified using sets of fusion images ( [ tc]dpd-[tc]tetrofosmin , [ tc]dpd-[tc]hmpao , [ tc]dpd-[i]ifp - cit ) , allowing the identification of extracerebral anatomical landmarks such as cranium , orbitae , and harderian glands and the localization of the respective regions relative to the sites of specific accumulation of metabolic and perfusion markers ( fig .
on coronal slices , striatal target and cerebellar reference regions were defined using the regional activity maxima ( fig .
thereby , maximum striatal count rates ( counts / pixel ) were determined on coronal slices by defining a circular region covering an area of 1.5 mm , which comprised a total of 11 pixels . on the same slices used for the determination of maximum striatal count rates , reference count rates ( counts / pixel ) were obtained in an elliptic region ( area , 7 mm comprising a total of 53 pixels ) 15 mm posterior to the frontal cortex corresponding anatomically to the rat cerebellum .
a template of striatal and cerebellar regions was positioned on the individual images by visual inspection without changing their shape or size . left and right striatal radioactivity concentrations were averaged . for baseline and treatment conditions , the equilibrium ratio of the distribution volumes of the specifically and the nonspecifically bound compartment [ v3=vt ( striatum)/vt ( cerebellum)1 ] was computed as an estimate for the binding potential 27 .
the left column shows the rat head [ tc]dpd scan ( top ) , the rat head [ tc]tetrofosmin scan ( middle ) , and the superimposition of bone metabolism and perfusion scans ( below ) . as these tracers
brain barrier , their distribution allows the identification of the orbitae ( 1 ) , cranium ( 2 ) , and harderian glands ( 3 ) .
the middle column shows the rat head [ tc]dpd scan ( top ) , the rat head [ tc]hmpao scan ( middle ) , and the superimposition of bone metabolism and brain perfusion scans ( below ) .
the fusion of [ tc]dpd and [ tc]hmpao images allows the alignment of the cranium ( 2 ) and the cerebral [ tc]hmpao accumulation and thus permits the identification of the cerebellum ( 4 ) relative to the rat cranium .
the right column shows the rat head [ tc]dpd scan ( top ) , the rat head [ i]fp - cit scan ( middle ) , and the superimposition of [ tc]dpd and [ i]fp - cit images ( below ) . the fusion image allows the identification of the rat striatum ( 5 ) relative to the orbitae ( 1 ) and the cranium ( 2 ) as visualized by [ tc]dpd , and to the sites of extracerebral [ i]-fp - cit accumulations corresponding to the sites of extracerebral [ tc]tetrofosmin accumulations in the harderian glands ( 3 ) .
furthermore , the cerebellum ( 4 ) may be identified relative to the cranium ( 2 ) and to sites of cerebellar ( 4 ) hmpao accumulations .
( b ) template consisting of cerebellar ( 4 ) and striatal ( 5 ) regions of interest .
distributions of binding data were assessed with the nonparametric kolmogorov smirnov test ( =0.05 ) .
thereby , in each pretreatment condition , v3 values as well as cerebellar count rates were found to be normally distributed ( 0.313p0.989 ) .
striatal v3 values and cerebellar radioactivity count rates obtained at baseline , after benserazide , and after 5 or 10 mg / kg l - dopa / benserazide were compared with one - way analyses of variance ( anovas ) for repeated measures ( holm sidak pairwise multiple comparison , =0.05 ) .
in addition , striatal v3 values and cerebellar radioactivity count rates were compared between baseline and the respective pretreatment condition ( benserazide , 5 mg / kg l - dopa / benserazide , 10 mg / kg l - dopa / benserazide ) with the paired t - test ( two tailed , =0.05 ) . moreover , striatal v3 values obtained in l - dopa / benserazide - treated animals subjected to behavioral analysis were compared between l - dopa doses using the independent t - test ( two tailed , =0.05 ) .
distributions of behavioral data ( duration and frequency of ambulatory activity , sitting , rearing , head and shoulder motility , and grooming in minutes 15 , 610 , 1115 , 1620 , 2125 , 2630 , and 130 ) were assessed with the nonparametric kolmogorov
thereby , the majority of behavioral parameters were normally distributed in each treatment group ( 0.052p1 ) .
no normal distribution could be ascertained for rearing duration ( p=0.032 ) , rearing frequency ( p=0.038 ) , and grooming frequency ( p=0.047 ) during 2530 min after pretreatment with 5 mg / kg l - dopa / benserazide and for grooming frequency during 2530 min after pretreatment with 10 mg / kg l - dopa / benserazide ( p=0.045 ) . for either treatment group , v3 values and normally and
not normally distributed behavioral data were correlated using the pearson product moment correlation coefficient ( =0.05 ) and the spearman rank correlation ( coefficient =0.05 ) , respectively .
distributions of binding data were assessed with the nonparametric kolmogorov smirnov test ( =0.05 ) .
thereby , in each pretreatment condition , v3 values as well as cerebellar count rates were found to be normally distributed ( 0.313p0.989 ) .
striatal v3 values and cerebellar radioactivity count rates obtained at baseline , after benserazide , and after 5 or 10 mg / kg l - dopa / benserazide were compared with one - way analyses of variance ( anovas ) for repeated measures ( holm sidak pairwise multiple comparison , =0.05 ) .
in addition , striatal v3 values and cerebellar radioactivity count rates were compared between baseline and the respective pretreatment condition ( benserazide , 5 mg / kg l - dopa / benserazide , 10 mg / kg l - dopa / benserazide ) with the paired t - test ( two tailed , =0.05 ) . moreover , striatal v3 values obtained in l - dopa / benserazide - treated animals subjected to behavioral analysis were compared between l - dopa doses using the independent t - test ( two tailed , =0.05 ) .
distributions of behavioral data ( duration and frequency of ambulatory activity , sitting , rearing , head and shoulder motility , and grooming in minutes 15 , 610 , 1115 , 1620 , 2125 , 2630 , and 130 ) were assessed with the nonparametric kolmogorov
thereby , the majority of behavioral parameters were normally distributed in each treatment group ( 0.052p1 ) .
no normal distribution could be ascertained for rearing duration ( p=0.032 ) , rearing frequency ( p=0.038 ) , and grooming frequency ( p=0.047 ) during 2530 min after pretreatment with 5 mg / kg l - dopa / benserazide and for grooming frequency during 2530 min after pretreatment with 10 mg / kg l - dopa / benserazide ( p=0.045 ) . for either treatment group , v3 values and normally and
not normally distributed behavioral data were correlated using the pearson product moment correlation coefficient ( =0.05 ) and the spearman rank correlation ( coefficient =0.05 ) , respectively .
figures 2a and 3a show characteristic images of [ i]fp - cit accumulations on coronal slices at baseline and after challenge with 5 mg / kg l - dopa / benserazide , 10 mg / kg l - dopa / benserazide , and benserazide alone .
striatal [ i]fp - cit accumulations are markedly reduced following pretreatment with both 5 mg / kg ( fig .
coronal [ i]fp - cit images of the same rat head at baseline , after pretreatment with 5 mg / kg l - dopa / benserazide , and after treatment with 10 mg / kg benserazide .
all images show v3 values ; it is understood that the calculation of v3 is only valid for regions of specific radioligand binding such as the rat striatum .
calculations were performed using matlab ( version 4.2c or version 6 ; the mathworks inc . , novi , michigan , usa ) .
( b ) striatal equilibrium ratios ( v3 ) at baseline , after 5 mg / kg l - dopa / benserazide , and after 10 mg / kg benserazide . rendered are means and sem .
dat , dopamine transporter ; [ i]fp - cit , [ i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ; l - dopa , l-3,4-dihydroxyphenylalanine .
( a ) coronal [ i]fp - cit images of the same rat head at baseline , after pretreatment with 10 mg / kg l - dopa / benserazide , and after treatment with 10 mg / kg benserazide .
all images show v3 values ; it is understood that the calculation of v3 is valid only for regions of specific radioligand binding such as the rat striatum .
calculations were performed using matlab ( version 4.2c or version 6 ; the mathworks inc . ) .
( b ) striatal equilibrium ratios ( v3 ) at baseline , after 10 mg / kg l - dopa / benserazide , and after 10 mg / kg benserazide . rendered are means and sem .
dat , dopamine transporter ; [ i]fp - cit , [ i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ; l - dopa , l-3,4-dihydroxyphenylalanine . after 5 and 10 mg /
kg l - dopa / benserazide , cerebellar radioactivity concentrations ( data not shown ) amounted to 812253 counts / pixel ( meansd ) and 665117 counts / pixel , respectively . at baseline ,
cerebellar radioactivity concentrations of 693231 counts / pixel ( 5 mg / kg l - dopa / benserazide ) and 656173 counts / pixel ( 10 mg / kg l - dopa / benserazide ) were obtained , whereas after benserazide treatment cerebellar radioactivity concentrations were 797208 counts / pixel ( 5 mg / kg l - dopa / benserazide ) and 621178 counts / pixel ( 10 mg / kg l - dopa / benserazide ) .
one - way anovas revealed no significant differences between treatments ( 5 mg / kg l - dopa / benserazide , p=0.289 ; 10 mg / kg l - dopa / benserazide , p=0.901 ) .
baseline v3 amounted to 1.400.42 ( 5 mg / kg l - dopa / benserazide ) and 1.690.44 ( 10 mg / kg l - dopa / benserazide ) , whereas after benserazide v3 values of 1.170.40 ( 5 mg / kg l - dopa / benserazide ) and 1.620.47 ( 10 mg / kg l - dopa / benserazide ) were obtained .
one - way anovas revealed significant differences between treatments ( 5 mg / kg l - dopa / benserazide , p=0.004 ; 10 mg / kg l - dopa / benserazide , p=0.005 ) .
pairwise multiple comparison procedures yielded p values of 0.001 for both v3 at baseline versus v3 after 5 mg / kg l - dopa / benserazide and for v3 at baseline versus v3 after 10 mg / kg l - dopa / benserazide . similarly
, significant differences were obtained between baseline and 5 mg / kg and 10 mg / kg l - dopa / benserazide ( p=0.001 and 0.003 , respectively ) using paired t tests . if the outlier in the lower dose group ( fig .
2b ) is excluded , the difference from baseline remains significant ( paired t - test ; p<0.001 ) .
there were no differences between baseline and benserazide conditions or between benserazide and 10 mg / kg l - dopa / benserazide ( paired t tests ; 0.127p0.384 ) .
for the comparison between benserazide and 5 mg / kg l - dopa / benserazide , a p of 0.051 was obtained .
there were no between - group differences of baseline v3 ( paired t tests ; p=0.081 ) .
however , v3 significantly differed between l - dopa treatment groups ( paired t tests ; p=0.005 ) . also in l - dopa / benserazide - treated animals subjected to behavioral analysis , striatal v3 values were significantly different between groups ( 5 mg / kg l - dopa / benserazide , 0.900.24 ; 10 mg / kg l - dopa / benserazide , 1.160.30 ; p=0.047 ) .
after 5 mg / kg l - dopa / benserazide , striatal v3 correlated negatively with rearing duration during 15 min ( r=0.7 , p=0.02 ) and sitting frequency during 615 min ( r=0.71 , p=0.02 ) . after 10 mg / kg l - dopa / benserazide , striatal v3 correlated positively with the durations of ambulatory activity during 130 min ( r=0.67 , p=0.05 ) and head and shoulder motility during 1115 min ( r=0.66 , p=0.05 ) , whereas negative correlations were observed with sitting duration during 130 min ( r=0.068 , p=0.045 ) and 1115 min ( r=0.75 , p=0.020 ; data not shown ) .
figures 2a and 3a show characteristic images of [ i]fp - cit accumulations on coronal slices at baseline and after challenge with 5 mg / kg l - dopa / benserazide , 10 mg / kg l - dopa / benserazide , and benserazide alone .
striatal [ i]fp - cit accumulations are markedly reduced following pretreatment with both 5 mg / kg ( fig .
coronal [ i]fp - cit images of the same rat head at baseline , after pretreatment with 5 mg / kg l - dopa / benserazide , and after treatment with 10 mg / kg benserazide .
all images show v3 values ; it is understood that the calculation of v3 is only valid for regions of specific radioligand binding such as the rat striatum .
calculations were performed using matlab ( version 4.2c or version 6 ; the mathworks inc . , novi , michigan , usa ) .
( b ) striatal equilibrium ratios ( v3 ) at baseline , after 5 mg / kg l - dopa / benserazide , and after 10 mg / kg benserazide . rendered are means and sem .
dat , dopamine transporter ; [ i]fp - cit , [ i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ; l - dopa , l-3,4-dihydroxyphenylalanine .
( a ) coronal [ i]fp - cit images of the same rat head at baseline , after pretreatment with 10 mg / kg l - dopa / benserazide , and after treatment with 10 mg / kg benserazide .
all images show v3 values ; it is understood that the calculation of v3 is valid only for regions of specific radioligand binding such as the rat striatum .
calculations were performed using matlab ( version 4.2c or version 6 ; the mathworks inc . ) .
( b ) striatal equilibrium ratios ( v3 ) at baseline , after 10 mg / kg l - dopa / benserazide , and after 10 mg / kg benserazide . rendered are means and sem .
dat , dopamine transporter ; [ i]fp - cit , [ i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ; l - dopa , l-3,4-dihydroxyphenylalanine . after 5 and 10 mg /
kg l - dopa / benserazide , cerebellar radioactivity concentrations ( data not shown ) amounted to 812253 counts / pixel ( meansd ) and 665117 counts / pixel , respectively . at baseline ,
cerebellar radioactivity concentrations of 693231 counts / pixel ( 5 mg / kg l - dopa / benserazide ) and 656173 counts / pixel ( 10 mg / kg l - dopa / benserazide ) were obtained , whereas after benserazide treatment cerebellar radioactivity concentrations were 797208 counts / pixel ( 5 mg / kg l - dopa / benserazide ) and 621178 counts / pixel ( 10 mg / kg l - dopa / benserazide ) .
one - way anovas revealed no significant differences between treatments ( 5 mg / kg l - dopa / benserazide , p=0.289 ; 10 mg / kg l - dopa / benserazide , p=0.901 ) .
baseline v3 amounted to 1.400.42 ( 5 mg / kg l - dopa / benserazide ) and 1.690.44 ( 10 mg / kg l - dopa / benserazide ) , whereas after benserazide v3 values of 1.170.40 ( 5 mg / kg l - dopa / benserazide ) and 1.620.47 ( 10 mg / kg l - dopa / benserazide ) were obtained .
one - way anovas revealed significant differences between treatments ( 5 mg / kg l - dopa / benserazide , p=0.004 ; 10 mg / kg l - dopa / benserazide , p=0.005 ) .
pairwise multiple comparison procedures yielded p values of 0.001 for both v3 at baseline versus v3 after 5 mg / kg l - dopa / benserazide and for v3 at baseline versus v3 after 10 mg / kg l - dopa / benserazide . similarly
, significant differences were obtained between baseline and 5 mg / kg and 10 mg / kg l - dopa / benserazide ( p=0.001 and 0.003 , respectively ) using paired t tests . if the outlier in the lower dose group ( fig .
2b ) is excluded , the difference from baseline remains significant ( paired t - test ; p<0.001 ) .
there were no differences between baseline and benserazide conditions or between benserazide and 10 mg / kg l - dopa / benserazide ( paired t tests ; 0.127p0.384 ) .
for the comparison between benserazide and 5 mg / kg l - dopa / benserazide , a p of 0.051 was obtained .
there were no between - group differences of baseline v3 ( paired t tests ; p=0.081 ) .
however , v3 significantly differed between l - dopa treatment groups ( paired t tests ; p=0.005 ) . also in l - dopa / benserazide - treated animals subjected to behavioral analysis , striatal v3 values were significantly different between groups ( 5 mg / kg l - dopa / benserazide , 0.900.24 ; 10 mg / kg l - dopa / benserazide , 1.160.30 ; p=0.047 ) .
after 5 mg / kg l - dopa / benserazide , striatal v3 correlated negatively with rearing duration during 15 min ( r=0.7 , p=0.02 ) and sitting frequency during 615 min ( r=0.71 , p=0.02 ) . after 10 mg / kg l - dopa / benserazide , striatal v3 correlated positively with the durations of ambulatory activity during 130 min ( r=0.67 , p=0.05 ) and head and shoulder motility during 1115 min ( r=0.66 , p=0.05 ) , whereas negative correlations were observed with sitting duration during 130 min ( r=0.068 , p=0.045 ) and 1115 min ( r=0.75 , p=0.020 ; data not shown ) .
challenge with 5 and 10 mg / kg l - dopa / benserazide led to mean reductions in dat binding by 34 and 20% , respectively , relative to baseline .
the 21% reduction in dat binding after 5 mg / kg l - dopa / benserazide relative to benserazide - only treatment was marginally significant .
cerebellar radioactivity concentrations did not differ between baseline and challenges , yielding evidence that under the present experimental conditions no confounding effects were exerted on radioligand accumulation for example , by affecting cerebral perfusion . as l - dopa acts by increasing
28 ) , the reduction in dat binding may be interpreted in terms of competition between [ i]fp - cit and endogenous da .
moreover , they confirm our findings of reduced dat binding after application of 25 mg / kg l - dopa without joint administration of an aadc inhibitor 29 .
they are not consistent , however , with the results of schillaci et al .
8 , nurmi et al .
11 , contin et al .
12 , and the parkinson study group 9,10 obtained in humans and with the findings reported by laruelle et al .
15 and fernagut et al .
16 in nonhuman primates .
as mentioned above , only two in - vivo investigations on dat binding have been conducted in rats contingent on pretreatment with l - dopa ; thereby , dresel et al .
14 determined dat binding in healthy animals after acute challenge with 125 and 150 mg / kg l - dopa , whereas sossi et al .
17 assessed dat binding in 6-ohda - lesioned rats after long - term treatment with 10 mg / kg l - dopa .
findings obtained in both studies are qualitatively consistent with the results of the present investigation . however , in the study by sossi et al .
17 , chronic application of the same dose ( 10 mg / kg l - dopa ) led to a 7% reduction in dat binding , whereas in the present investigation a much higher decrease of 20% was observed .
this may be accounted for by the differences in methodologies : whereas in our study dat binding was measured after acute treatment with l - dopa , sossi et al .
17 administered l - dopa chronically over 4 weeks , which may have induced compensatory mechanisms including a reduction in da release over the course of time .
moreover , in the study by dresel et al .
14 , application of the much higher doses of 125 and 150 mg / kg l - dopa reduced dat binding by 19 and 59% , whereas in our study pretreatment with 5 and 10 mg / kg decreased dat binding by 34 and 20% , respectively , relative to baseline .
these observations imply that doses of l - dopa in the therapeutic range may be sufficient to markedly increase synaptic da .
17 observed a decrease in dat binding in the intact contralateral striatum of rats with unilateral nigrostriatal 6-ohda lesions , but an increase in dat binding was observed on the lesioned side .
it may be conjectured that the lesion - induced decrease in striatal da concentration elevated ipsilateral dat binding relative to the contralateral side because of reduced competition with radioligand molecules .
this conclusion is supported by our own result of decreased dat binding in healthy animals , and also by the fact that in studies on lesioned nonhuman primates dat binding was not reduced upon pretreatment with l - dopa 15,16 .
it appears that the number of dat binding sites after l - dopa depends on the extent of nigrostriatal degeneration in two respects : the process of neurodegeneration , first , leads to a progressive reduction of dat binding sites and , second , diminishes the capacity to synthesize and release da contingent on administration of the precursor molecule . as a consequence , the reduction in radioligand binding after treatment with l - dopa
this is underlined by the fact that parkinsonian patients who displayed a significant decrease in dat binding after long - term treatment with l - dopa had a unified parkinson s disease rating scale score of 23.96.0 13 , whereas in those investigations in which no alterations in dat binding were observed unified parkinson s disease rating scale scores were higher , amounting to 35.111.8 8 , 31.048.0 11 , 25.312.0 12 , 30.611.4 9 , and 28.013.0 10 .
this implies that disease severity must be taken into account in in - vivo investigations assessing the effect of daergic treatments on constituents of the daergic synapse .
this not only pertains to parkinson s disease but also to other disorders affecting the daergic system , including schizophrenia , depression , and anxiety disorders ( for reviews , see nikolaus and colleagues 13 ) . in the present study ,
absolute v3 values differed significantly between animals administered 5 and 10 mg / kg l - dopa / benserazide .
this was also the case in l - dopa / benserazide - treated animals subjected to behavioral analysis .
these results indicate a biphasic response with a higher effect on dat after the lower dose of l - dopa .
biphasic regulatory actions of l - dopa on da and other neurotransmitters including noradrenaline and acetylcholine are widely known ( for review , see misu et al .
30 ) ; in vitro , nanomolar concentrations of l - dopa were repeatedly found to facilitate da release , whereas micromolar concentrations exerted inhibitory effects 28,3133 . in vivo ,
a comparison across studies provides less consistent results , with reports of either similar efficacy of 6 mg / kg 34 and 12 mg / kg l - dopa 35 , or increased efficacy of either 6 mg / kg 36 or 25 mg / kg l - dopa 37 relative to the 12 mg / kg dose 35 .
these findings , however , are not comparable to our results , as the challenging doses between 6 and 25 mg / kg were applied to 6-ohda - lesioned rats , and contingent on chronic administration of 525 mg / kg l - dopa injected once or twice daily over 1014 days 3437 .
therefore , further studies over a wider dose range and , in particular , after long - term treatment with l - dopa will help to elucidate the regulatory mechanisms underlying da synthesis and release with respect to applied dose and duration of treatment .
the latter may be of special interest given the high incidence of dyskinesias , motor fluctuations , and
wearing off phenomena in parkinsonian patients under chronic l - dopa therapy ( for review , see cenci et al .
the observed biphasic action of l - dopa raises the question as to by which mechanisms the disparate effects on da efflux and dat binding are exerted . apparently , in the present study , 5 mg / kg l - dopa increased synaptic da levels relative to the 10 mg / kg dose , leading to increased competition between endogenous da and the exogenous radioligand for dat binding sites
. da release is modulated by a negative feedback loop , which is established by da acting upon presynaptic terminal autoreceptors of the inhibitory d2/3 receptor subtype ( for reviews , see langer 39 ) .
l - dopa at the time of radioligand application were lower compared with the 5 mg / kg dose , because , in the 30 min time span between injection of l - dopa and application of [ i]fp - cit , the higher dose may have promoted the release of da in concentrations sufficient to activate feedback inhibition at the presynaptic terminal .
28 , who observed a blockade of da release after micromolar relative to nanomolar concentrations of l - dopa mediated by d2 autoreceptor binding .
28 , for instance , attributed the abolition of da efflux to direct action of l - dopa on presynaptic d2 receptor binding sites rather than to feedback inhibition exerted by newly released da molecules .
in addition , after nanomolar concentrations of l - dopa , da release was facilitated by interaction with excitatory presynaptic -adrenoreceptors 28 .
further , previous studies have shown that l - dopa may inhibit acetylcholine efflux 40 and facilitate the release of both glutamate 41 and -amino acid butyric acid ( 42 ) .
interaction of l - dopa with other neurotransmitter systems also includes l - dopa decarboxylation in striatal serotonin ( 5-ht ) neurons 43 with subsequent da storage in presynaptic vesicles 44 and its release into the synaptic cleft 45 .
as da neurons are modulated by all of these neurotransmitters ( ach 46 , glutamate 47 , -amino acid butyric acid 48 , and 5-ht 49 ) , further investigations are needed to clarify which synaptic site of which neurotransmitter system is challenged by which dose of l - dopa and whether da levels or the concentrations of its precursor are relevant for the induction of either forward inhibition or activation of the presynaptic feedback loop . to our knowledge , we present the first in - vivo evidence on the association between dat binding and parameters of motor and exploratory behavior . thereby , in the group administered 5 mg / kg , lower dat binding predicted longer rearing behavior during 15 min and higher sitting frequency during 1115 min . in the group administered 10 mg / kg ,
lower dat binding predicted decreased head and shoulder motility during 1115 min and less ambulation and more sitting throughout the trial .
these results indicate an association between striatal dat and motor and exploratory parameters , which appear to be both behavior - specific and time - dependent . in addition , our results may reflect an inter - relation with endogenous da : as l - dopa increased da concentrations ( and decreased dat binding ) , it may be that higher da levels ( reflected by lower dat binding ) decreased behavioral activity , whereas lower da levels ( reflected by higher dat binding ) had the opposite effects
. further investigations with a higher number of animals are required to thoroughly assess behavioral parameters together with dat binding over a wider range of l - dopa doses . moreover , as motor behavior in adult rats has been related to striatal d2 receptor function 50 our studies in the near future will be complemented with the joint assessment of behavioral parameters and d2 receptor after challenge with l - dopa .
two limitations of our study must be taken into account : first , previous investigations have shown that radioactivity concentrations in vivo may be underestimated because of partial - volume effects 51 .
. the latter may be the case in more rostral striatal regions , whereas the former hampers radioactivity quantification in thin structures like caudal striatal portions . as neither a partial - volume nor
a spill - over correction was performed in our study , quantification of striatal radioactivity counts may have been confounded relative to cerebellar radioactivity counts , as this structure due to its largeness and caudal localization is subject to neither effect . moreover , as no exact anatomical coregistration was possible , it can not be ruled out that positioning of regions of interest and reference regions did not slightly vary between animals or even between treatment conditions in the same animal .
second , [ tc]dpd , [ tc]tetrofosmin , and [ tc]hmpao scans were performed on rats other than those scanned with [ i]fp - cit . to minimize intersubject variation ,
further , rats were positioned in a custom - built head holder ( institute of medicine , research center jlich , jlich , germany ) to ensure reproducible positioning of the animals within the field of view .
notably , notwithstanding these limitations , quantitative data obtained with our small animal tomograph are conclusive in that they confirm both qualitatively and quantitatively the findings of l - dopa - induced reduction of dat binding in both humans 13 and rats 14 . it may be argued that , in view of the limited spatial resolution of small animal spect , ex - vivo autoradiography or immunohistochemistry might represent more suitable methods for the assessment of dat after pharmacological challenges .
however , the use of ex - vivo methods precludes the performance of more than one trial on a single animal , whereas in - vivo approaches such as small animal spect permit the use of each rat as its own control . as a consequence
, data do not have to be pooled from several animals and tested for significance between groups ; instead , the possible influence of intersubject variation may be reduced by the performance of pre post comparisons . besides , due to inherent methodological problems such as variations in slice thickness or cellular destruction in the cryosections ( quenching ) also ex - vivo methods are not supposed to yield error - free results , apart from the fact that it is debatable , in as much as findings obtained ex vivo on frozen brain slices may reflect the conditions , functions , and mechanisms in the brain of a living animal . in the present investigation , l - dopa was administered together with the aadc inhibitor benserazide .
previous findings obtained with in - vivo microdialysis have shown that a single administration of l - dopa ( 50 mg / kg ) with vehicle pretreatment instead of application of benserazide increase extracellular da by 560% 24 . furthermore , although the combination of l - dopa and benserazide led to a higher increase in extracellular da levels compared with l - dopa ( 50 mg / kg ) alone , the sole application of benserazide ( 50 mg / kg ) also induced a pronounced elevation in striatal da 19 .
therefore , it is likely that even the relatively low dose of benserazide ( 10 mg / kg ) applied in the present study contributed to our findings obtained after 5 and 10 mg / kg l - dopa by increasing extracellular da concentrations .
this is supported by the fact that in the present investigation benserazide led to a although nonsignificant reduction in dat binding relative to baseline .
moreover , dat binding after both 5 and 10 mg / kg l - dopa / benserazide was decreased relative to benserazide - only treatment , with the difference between 5 mg / kg l - dopa / benserazide and the benserazide - only condition exhibiting a marginal significance . besides , in a previous investigation , pretreatment with 25 mg / kg l - dopa without joint application of benserazide reduced [ i]fp - cit binding to the striatal dat by 20% 29 .
additional investigations on dat binding after varying doses of benserazide should be conducted to shed further light on this matter .
in addition , studies on 6-ohda - lesioned rats should be conducted to gauge the effects of l - dopa with and without benserazide in relation to unlesioned animals .
this is important as parkinsonian patients under chronic l - dopa therapy receive concomitant treatment with some aadc inhibitor , and effects on the outcome of dat - spect may be due to the concurring actions of both types of compounds .
taken together , findings yield evidence that therapeutic doses of l - dopa can notably reduce [ i]fp - cit binding to the dat .
results indicate a biphasic response with a higher effect on dat after the lower dose of l - dopa .
the reduction in dat binding may be interpreted in terms of competition between [ i]fp - cit and endogenous da .
moreover , there is evidence of an association between striatal dat and motor and exploratory parameters , which appears to be both behavior - specific and time - dependent .
further in - vivo investigations on normal rats and on parkinsonian lesion models after chronic l - dopa may be helpful in clarifying whether , and under which circumstances , the present findings have implications for the performance of dat - spect in neurologic and psychiatric patients receiving daergic medications .
| purposethe effect of clinical l-3,4-dihydroxyphenylalanine ( l - dopa ) doses on the binding of [ 123i]n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)nortropane ( [ 123i]fp - cit ) to the rat dopamine transporter ( dat ) was investigated using small animal single - photon emission computed tomography.materials and methodsdat binding was measured at baseline , after challenge with the aromatic l - amino acid decarboxylase inhibitor benserazide , and after challenge with either 5 or 10 mg / kg l - dopa plus benserazide . for baseline and challenges , striatal equilibrium ratios ( v3 )
were computed as an estimation of the binding potential . moreover ,
striatal v3 values were correlated with parameters of motor and exploratory behavior.resultsv3 differed significantly between baseline and either dose of l - dopa / benserazide .
moreover , v3 differed significantly between l - dopa treatment groups .
after 5 mg / kg l - dopa / benserazide , dat binding was inversely correlated with sitting duration ( 15 min ) and sitting frequency ( 1015 min ) .
after 10 mg / kg l - dopa / benserazide , an inverse correlation was found between dat binding and sitting duration ( 130 min ) , whereas dat binding and duration of ambulatory activity ( 130 min ) as well as head and shoulder motility ( 1015 min ) exhibited a positive correlation.conclusionchallenge with 5 and 10 mg / kg l - dopa / benserazide led to mean reductions in dat binding by 34 and 20% , respectively .
results indicate a biphasic response with a higher effect on dat after the lower dose of l - dopa .
the reduction in dat binding may be interpreted in terms of competition between [ 123i]fp - cit and endogenous dopamine .
moreover , there is preliminary evidence of an association between striatal dat and motor and exploratory parameters . |
about 50% of patients with a diagnosis of non - small cell lung carcinoma exhibit metastatic disease at the time of diagnosis .
the preferential sites of extrapulmonary spread are the lymph nodes , liver , brain , adrenal glands , and bones .
a 51-year - old smoker was diagnosed with a poorly differentiated non - small cell lung carcinoma of the right lower lobe in stage ct2a
he received front - line therapy with cisplatin - pemetrexed . a ct scan after 4 cycles revealed a partial response in the lung and a complete response in the brain .
after 2 cycles , locoregional progression was observed , with the extrapulmonary disease still controlled .
after 2 cycles , the patient complained of abdominal pain and reported melena , yet without signs of intestinal obstruction .
biological data revealed severe aregenerative anaemia ( haemoglobin concentration at 6.9 g / dl , reticulocyte count at 120,000/mm ) and iron deficiency signs consisting of low serum ferritin , iron , and transferrin saturation .
the stool examination for occult blood was strongly positive , while gastric fiberscopy and colonoscopy results were normal .
abdominal ct with contrast medium enhancement showed an intussusception of the small bowel ( fig 1 ) .
we then decided to explore the small bowel by means of capsule endoscopy , which revealed several secondary lesions , including one stenosing the first jejunal loop and another one ulcerated and bleeding ( fig 2 ; online suppl . videos 1 and 2 ; for all online suppl .
a double - balloon enteroscopy was performed thereafter for lesion biopsy , confirming the metastatic localisation of a poorly differentiated lung carcinoma . on account of the patient 's poor performance status ,
surgical treatment was not considered , with only symptomatic treatment performed using several blood transfusions and iron supplementation . at
last , the patient died 20 months following the cancer diagnosis and 5 months after discovering the intestinal metastasis .
improvement in metastatic lung cancer management has led to extending survival , and we are thus faced with an increasing number of uncommon metastatic sites .
gastrointestinal metastases are considered to be rare , usually occurring in advanced disease stages , and associated with poor prognosis . due to their mostly asymptomatic presentation ,
were observed in 12% of patients , with the small bowel as the most common site of metastatic involvement .
gastric and duodenal metastases likely cause abdominal pain , haematemesis , as well as chronic bleeding resulting in anaemia .
small bowel involvement often leads to acute abdomen , and may be associated with various clinical complications such as perforation , obstruction , or haemorrhage .
clinical cases of gastrointestinal metastases from lung cancer have been reported , with diagnosis systematically delayed . as illustrated in our case report ,
although conventional imaging revealed jejunal intussusception , the correct diagnosis of metastatic intestinal involvement was only brought upon by capsule endoscopy .
this non - invasive procedure thus represents a valuable method contributing to a rapid and detailed diagnosis while reducing underdiagnosis .
this strategy allows for obtaining a therapeutic effect in patients with solitary gastrointestinal metastases , and a favourable palliative effect on survival in those with preoperatively diagnosed metastases .
we reported a case of small bowel metastases from primary lung cancer revealed by abdominal pain and severe recurrent anaemia .
capsule endoscopy allowed for an aetiologic diagnosis to be obtained , and should thus be considered in lung cancer patients complaining of abdominal symptoms , which may indeed be related to gastrointestinal metastases .
written informed consent was obtained from the patient for publication of this case report and any accompanying images .
| gastrointestinal metastases from lung cancer are rare and usually asymptomatic .
we report a case of small bowel metastases from primary lung cancer revealed by abdominal pain and severe recurrent anaemia .
the diagnosis was obtained with capsule endoscopy .
this non - invasive procedure thus represents a valuable method contributing to a rapid and detailed diagnosis while reducing underdiagnosis , and it should thus be considered for lung cancer patients complaining of abdominal symptoms , which may indeed be related to gastrointestinal metastases . |
nowadays , one - dimensional nanostructural materials ( nanorods , nanowires , nanobelts and nanotubes ) have attracted heightened attention because of their potential applications in nanodevices and functional materials .
continuous effort has been devoted to the investigation on the crystalline phases of the layered transition metal dichalcogenides , because of their unusual crystal structure and unique physical and chemical properties . in analogy to graphite , nanoparticles of many inorganic compounds such as mos2 ,
ws2 are not stable against folding , and can adopt nanotubular and fullerene - like structures . as transition metal sulfides , mos2 has been the subject of significant research for applications including nonaqueous lithium batteries , catalytic hydride sulfurization of petroleum and wear resistance . for each of these applications ,
the important processes occur either at the surface or at the exposed edges of the mos2 layers .
mos2 is a layered semiconductor ( band gap = 1.2 ev , indirect ) that resists oxidation even in moist air at temperatures up to 85 c .
recent studies have suggested that reducing the size of the mos2 crystals can improve their lubrication properties in bearings , o - rings or other heavy wear applications .
inorganic syntheses using organic surfactants are successfully applied for the preparation of various nanostructural materials .
various methods of preparation includes chemical vapor deposition , sol gel processing , spray pyrolysis , co - precipitation , sonochemical synthesis , metathesis reactions , two step electrochemical synthesis etc are having been in practice [ 9 - 12 ] .
tenne and co - workers first reported the production of fullerene - like mos2 nanotubes via the gas phase reaction between moo3x and h2s in a reducing atmosphere at elevated temperature ( 8001000 c ) .
rao and co - workers obtained mos2 nanotubes by simple heating mos3 in a stream of hydrogen under high temperature .
synthesized fibers and tubules of mos2 by the thermal decomposition of ammonium thiomolybdate precursors at 450 c .
mos2 nanotubes were made by subliming 2h mos2 powder in presence of h2s at 1300 c .
recently mos2 nanorods were synthesized using -moo3 nanorods with a mixture of h2s h2 ( 15 vol% of h2s ) for 4 h at several temperatures .
however , almost all the methods to obtain mos2 nanotubes and nanorods utilize high temperature gas - solid reaction under reducing gas atmosphere .
synthesized mos2 nanorods and nanospheres by hydrothermal method using ammonium molybdate with na2s in presence of nh2oh hcl as oxidizing agent .
hydrothermal synthesis is becoming popular for environmental reason , since water is used as reaction solvent than organics .
this method has been widely used to prepare nanostructures due to its simplicity , high efficiency and low cost . in this article , as a part of our recent efforts on the investigation of 1-d materials , we report a simple low temperature hydrothermal method for preparation of mos2 nanofiber bundles with morphology control .
we examined the effect of precursors ( ammonium molybdate , sodium sulphide and citric acid ) concentration , duration and the temperature on the morphology of the product .
the hydrothermal process was carried out like our previous report to prepare mos2 nanofiber bundles . in a typical synthesis of mos2 nanofibers , sodium sulfide and h2s gas
were used as sulfur sources in order to investigate the source effect on the morphology of the product . in the first trail
1.235 g ammonium heptamolybdate tetrahydrate ( ( nh4)6mo7o24 4h2o ) was put into 25 ml distilled water under continuous stirring .
after 10 min , 0.5 g citric acid monohydrate ( c6h8o7 h2o ) was added to the above solution . to this solution mixture , 0.312 g sodium sulfide crystals were added .
the initial greenish black color was changed to red after 1h stirring on a magnetic stirrer . in the subsequent trail ,
0.5 g ammonium heptamolybdate tetrahydrate was put into 25 ml distilled water under continuous stirring .
gas is passed in to the warmed acidified with dilute hcl solution for about 58 min .
the sealed 25 ml teflon lined autoclaves containing above solution mixture were placed for hydrothermal treatment at 180 c for several hours .
the autoclaves were cooled and the resulted black solid was retrieved from the solution by centrifugation , washed with distilled water followed by ethanol to remove the ions possibly remaining in the end product , and finally dried in air .
powder x - ray diffraction data were recorded on philips xpert pro x - ray diffractometer with graphite monochromatized cu k radiation ( ( = 1.541 ) .
the fourier transform infrared spectrum of the samples was collected using nicollet ftir spectrometer . scanning electron micrograph images were taken with jeol ( jsm840 a ) scanning electron microscope .
x - ray photoelectron spectroscopy was carried out on an esca-3 mark ii x - ray photoelectron spectrometer , ( vg scientific , uk ) using al k radiation ( 1486.6 ev ) as the exciting source .
the morphology of the samples was observed using ( h-800em japan hitachi ) transmission electron microscope ( tem ) equipped with eds ( kevex sigma tm quasar , usa ) to measure the elements contained in the samples .
the hydrothermal process was carried out like our previous report to prepare mos2 nanofiber bundles . in a typical synthesis of mos2 nanofibers , sodium sulfide and h2s gas
were used as sulfur sources in order to investigate the source effect on the morphology of the product . in the first trail
1.235 g ammonium heptamolybdate tetrahydrate ( ( nh4)6mo7o24 4h2o ) was put into 25 ml distilled water under continuous stirring .
after 10 min , 0.5 g citric acid monohydrate ( c6h8o7 h2o ) was added to the above solution . to this solution mixture , 0.312 g sodium sulfide crystals were added .
the initial greenish black color was changed to red after 1h stirring on a magnetic stirrer . in the subsequent trail ,
0.5 g ammonium heptamolybdate tetrahydrate was put into 25 ml distilled water under continuous stirring .
gas is passed in to the warmed acidified with dilute hcl solution for about 58 min .
the sealed 25 ml teflon lined autoclaves containing above solution mixture were placed for hydrothermal treatment at 180 c for several hours .
the autoclaves were cooled and the resulted black solid was retrieved from the solution by centrifugation , washed with distilled water followed by ethanol to remove the ions possibly remaining in the end product , and finally dried in air .
powder x - ray diffraction data were recorded on philips xpert pro x - ray diffractometer with graphite monochromatized cu k radiation ( ( = 1.541 ) .
the fourier transform infrared spectrum of the samples was collected using nicollet ftir spectrometer . scanning electron micrograph images were taken with jeol ( jsm840 a ) scanning electron microscope .
x - ray photoelectron spectroscopy was carried out on an esca-3 mark ii x - ray photoelectron spectrometer , ( vg scientific , uk ) using al k radiation ( 1486.6 ev ) as the exciting source .
the morphology of the samples was observed using ( h-800em japan hitachi ) transmission electron microscope ( tem ) equipped with eds ( kevex sigma tm quasar , usa ) to measure the elements contained in the samples .
figure 1a , b shows the pxrd patterns of the samples prepared using sodium sulfide and h2 as sulfur sources in acidic medium at 180 c for 48 and 24 h , respectively .
it shows only a broad , weak envelope beginning at 2 = 30 and continuing out above 60 , the two maxima approximately located at the 100 and 110 positions of bulk 2h mos2 .
the pxrd patterns resemble that of the single - layer mos2 prepared by exfoliation method .
all the diffractions can be readily indexed as hexagonal 2h mos2 [ space group p63/mmc ( 194 ) ] with lattice constants a = 3.16116 and c = 12.2985 , identical to the reported data in the jcpds cards ( 371492 ) . at 180 c
the absence of the 002diffraction at 2 = 14.4 suggests that absence of stacking , thus the sample should be an aggregate of single sheets of mos2 .
powder xrd pattern of mos2 nanofiber bundles prepared at ( a ) 180 c for 48 h using na2s as sulfur source with 0.5 g citric acid .
( c ) 180 c for 48 h using na2s as sulfur source without citric acid .
( d ) 180 c for 48 h using na2s as sulfur source with 1 g citric acid chemical analysis using eds indicates the presence of mo and s ( elements of mos2 ) , and no other elements existed as observed in fig .
furthermore , the quantification of the peaks shows that the atomic ratio of s to mo is 1.92 , which is very close to the stoichiometric mos2 . from the ir spectrum ( fig .
3 ) , it is observed that the band at 480 cm corresponds to as(mo
o vibrations and they indicate that oxygen is present in similar types of coordination as in moo3 .
we assign the band at 941 cm to the as(mo o ) vibrations of terminal ( mo = o ) group and 693 cm to (mo o ) vibrations .
the frequency of the vibration is shifted to the lower wavenumber compare to its standard value in crystalline moo3 , due to the presence of reduced mo centers and sulfur as a ligand .
eds pattern of mos2 nanofiber bundles prepared at 180 c for 48 h ftir spectrum of mos2 nanofiber bundles prepared at 180 c for 48 h x - ray photoelectron spectra of mo(3d ) and s(2p ) core level regions of as - prepared nanoparticles are shown in fig .
mo(3d ) peaks in as - prepared nanoparticles were deconvoluted into sets of spin - orbit doublet .
accordingly , the high intense mo(3d5/2 ) peaks at 229.1 and low intense mo(3d3/2 ) peak at 232.1 ev could be attributed to mos2 . whereas lower binding energy shoulder peak at 226.25 ev is because of s(2s ) .
xps pattern of mos2 nanofiber bundles prepared at 180 c for 48 h xps data of mo(3d ) and s(2p ) whm = full width half maximum figure 4b shows s(2p ) doublet peak ( 2p3/2 and 2p1/2 ) at 161.25 and 162.94 ev respectively in which 2p3/2 and 2p1/2 orbital peaks coincide with each other .
it is to be noted that the peak positions of s(2p ) and mo(3d5/2 ) for mos2 is in good agreement with he results on mos2 .
the chemical shifts of mo(3d5/2 ) and s(2p ) in mos2 nanoparticle is + 1.6 and 2.2 ev in comparison to elemental mo and s .
therefore there occurs some partial electron transfer from molybdenum to sulfur . as a result ,
molybdenum becomes positively charged as shown by the increase in binding energy and sulfur becomes negatively charged as shown by the decrease in binding energy .
mos2nanofiber of about 120300 nm in diameter and 2025 m in length are identified from sem images ( fig .
they are well aligned and occur in - group of bundles with very high aspect ratio .
it can be seen that the nanofiber self assembled into bundles and the nanofiber in a bundles had the same growth direction .
the morphology and structure of individual mos2 nanorods have been characterized in further detail using tem , which are shown in fig .
6 . it can be seen that the nanorods are straight and uniform along their entire length and thickness of about up to 300 nm .
figure 6d is the high resolution tem image of the mos2 nanorods and this image shows solid nanorods are uniform in thickness .
sem images of mos2 nanofiber bundles prepared at 180 c for 48 h tem images of mos2 nanofiber bundles prepared at 180 c for 48 h the reduction of transition metal ions by surfactant is useful for the production of ultra fine particles of metal sulfides . unfortunately , molybdic compound reduction chemistry is quite complex and the nature of the products strongly depends on the reaction conditions . for example , afanasiev et al . show that the reduction of aqueous ammonium molybdate at different temperatures and conditions yields binary sulfides such as mos5.6 , mos6 , and mos3 as well as binary oxides such as moo3 and moo2 .
in fact , the mechanism differs markedly in aqueous and in nonaqueous solutions . in order to disclose the transition process from ( nh4)6mo7o24 4h2o to mos2 ,
the role of citric acid [ 30 - 32 ] on phase of products was investigated .
the possible reaction mechanism can be written as
( 1 ) step 1 is a sulfureted reaction , in which an intermediate phase ( nh4)2mos4 is produced , and step 2 is a reduction reaction under hydrothermal conditions .
thus by the addition of 1 g of citric acid or without citric acid , sulfureted reaction and reduction reaction do not react completely so that the products consist of much moo3 phase as shown in the fig .
1c , d. therefore during the formation of mos2 nanofiber bundles , citric acid plays a very prominent role .
figure 7a , b gives the sem images of the product prepared with and without citric acid .
sem images of mos2 nanofiber bundles prepared at ( a ) 180 c for 48 h with 1 g citric acid and ( b ) without citric acid the morphology of the product prepared at 180 c for 24 h ( fig .
8) using h2 resembles with the morphology of the product prepared at 180 c for 48 h ( fig .
the thickness of the fiber is found to be about 300 nm and length is about 12 m . from the sem images , we observed that the length of all fibers is found to be same with sharp ends having voids in each mos2 bundles .
sem images of mos2 nanofiber bundles prepared at 180 c for 24 h using h2s as sulfur source the possible reaction mechanism can be written when h2s as sulfur source is(2)(3 ) step 3 is a reduction reaction , in which an intermediate phase mocl4 is produced , and step 4 is a sulfureted reaction under hydrothermal conditions .
figure 9a , b shows the sem images of mos2 prepared at 180 c for 16 and 20 h. when the duration is increased to 48 h , nanorods ( fig .
the thickness of the nanorods is found to be in the range of 200600 nm .
sem images of mos2 nanofiber bundles prepared at 180 c h using h2s for ( a ) 16 h ( b ) 20 h and ( c ) 48 h
nanofiber of mos2bundles with 150300 nm in diameters and 2025 m in length have been successfully synthesized by a simple low temperature hydrothermal method .
pxrd pattern indicated the amorphous mos2was made up of single layer and layer stacking had not taken place .
the nanofiber self assembled into bundles and the nanofiber bundle had the same growth direction .
sem study reveals the formation of bundles of well - aligned mos2nanofiber with very high aspect ratio .
since the mos2nanomaterials are often very difficult to prepare and the search for more simple routine is quite valuable with regard to energy - saving and less pollution . it can be demonstrated that the optimum conditions for preparing mos2nanofiber are at 180 c for 48 h using citric acid and 180 c for 24 h by passing h2s in presence of hcl . under these conditions
the author g. t. chandrappa thankful to the department of science and technology , nsti phase - ii , new delhi , government of india for financial support to carryout this research work .
we also thank the central facility , dept . of physics and dept . of metallurgy , | mos2nanofiber bundles have been prepared by hydrothermal method using ammonium molybdate with sulfur source in acidic medium and maintained at 180 c for several hours .
the obtained black crystalline products are characterized by powder x - ray diffraction ( pxrd ) , fourier transform infrared spectrometer ( ftir ) , x - ray photoelectron spectroscopy ( xps ) , scanning electron microscopy ( sem ) and transmission electron microscopy ( tem ) .
the pxrd pattern of the sample can be readily indexed as hexagonal 2h - mos2 .
ftir spectrum of the mos2shows the band at 480 cm1corresponds to the as(mo - s ) .
sem / tem images of the samples exhibit that the mos2nanofiber exist in bundles of 120300 nm in diameter and 2025 m in length .
the effects of temperature , duration and other experimental parameters on the morphology of the products are investigated . |
as a small molecule probe , aptamers are composed of short , single - stranded oligonucleotides ( rna or ssdna ) ranging from 30 to 60 bases . similar to protein antibodies on the basis of their three - dimensional structures , aptamers specifically recognize and bind to their targets with high sensitivity and specificity .
these targets can include small molecules , macromolecules , proteins , viruses , cells , and tissues .
however , in contrast to antibodies , oligonucleotide aptamers are easily generated through chemical synthesis , and can be conveniently modified with a variety of functional molecules for different purposes .
therefore , aptamers have been widely studied for potential biomedical applications as a chemical antibody and , more importantly , for developing new clinical applications that can not be performed with current protein antibody technology .
metastasis or dissemination of primary tumor is the major cause of mortality in cancer patients .
recent studies have demonstrated that circulating tumor cells ( ctcs ) in the bloodstream are key factors in the establishment of metastatic tumors .
in addition , the number of ctcs present in whole blood is directly associated with cancer progression , recurrence , survival rate , and prognosis . therefore , accurate detection of ctcs will provide critical information for the proper management of cancer patients .
antibody technology has been widely used for a variety of biomedical applications since it was first developed .
however , the conjugated fluorochrome is constantly emitting a fluorescent signal , regardless of the status of whether the antibody is free or binding to cells , leading to a high level of background noise and potential off - target signals from normal blood cells .
therefore , the current antibody - mediated assays require multiple steps to isolate ctcs from normal blood cells and eliminate potential off - target signals . repeated washes to remove excess free fluorochrome - conjugated antibody from the assays are also required .
notably , these multi - step assays are time - consuming and , more importantly , lead to loss of the blood sample and damage to ctcs , which adversely impacts assay accuracy . to overcome these technical obstacles in antibody - mediated assays , a new reporter system that can selectively highlight only tumor cells without generation of background noise
for point - of - care purposes , an ideal ctc assay should be ( i ) simple ( one - step reaction ) , ( ii ) high throughput ( complete in minutes ) , and ( iii ) accurate ( no loss of blood sample and no damage of tumor cells through the assay process ) .
however , no such assay could be developed using current antibody - mediated technology as described above . to this end
, we designed a unique aptamer - reporter , which is composed of an oligonucleotide sequence specific for a tumor cell biomarker conjugated with a reporter of paired fluorochrome - quencher molecules .
the underlining hypothesis is that the intact aptamer - reporter is optically silent in the absence of tumor cells ( figure 1a ) , but can be activated by tumor cells to emit fluorescence .
as showed in figure 1b , in assays containing patient blood the oligonucleotide sequence will selectively target ctcs through interaction with a specific surface biomarker , which triggers internalization of the aptamer - reporter through a natural cellular process and , subsequently , into cell lysosomes that are rich in different enzymes .
this internalization mechanism has been successfully used for intracellular delivery of chemotherapeutic drugs for targeted therapy by a newly fda - approved antibody - drug - conjugate , brentuximab .
rapid degradation of the aptamer - reporter by lysosomal nucleases results in separation of the paired fluorochrome - quencher molecules , the released fluorochrome becoming optically active and emitting bright fluorescent signals exclusively within the targeted tumor cells .
notably , because it is controlled by a specific aptamer - biomarker interaction and an intracellular activation switch , the aptamer - reporter will generate no background noise or off - target signals from normal blood cells and thus , the assay can be carried out in one - step , load - then - read ( figure 1c ) . for the initial proof - of - concept studies ,
the aptamer - reporter was formulated by conjugating a 39-mer rna sequence specific for the cd30 biomarker with a fluorochrome cy3 compound and a bhq2 at the 5- and 3-ends , respectively ( figure 1a and figure 2a , left ) . due to covalent chemical conjugation ,
aptamer sequence is able to hold fluorochrome and quencher molecules close to each other and , thus , the bhq2 molecule will quench fluorescent signals emitted from the cy3 compound on the same aptamer .
a control aptamer was also synthesized by conjugating the same sequence with the fluorochrome cy3 compound alone ( figure 2a , right ) .
first , to validate the optical - activatable property , the aptamer - reporter was incubated with fresh lysates of cultured karpas 299 tumor cells of human alcl at room temperature as a cellular enzyme source .
incubation with cell lysates activated the aptamer - reporter and triggered a rapid increase in florescent signals in a time - dependent fashion ( figure 2b ) .
in contrast , cell lysates had no effect on the control aptamer which constantly emitted high levels of fluorescent signals . to rule out a potential effect due to conformation change , the aptamer - reporter was treated under different ph conditions , from ph 4.0 to physiological ph 7.4 , at room temperature for 30 minutes ( figure 2c ) .
notably , cell lysosomes have a low ph environment ( ph 45 ) which can cause denaturing of the aptamer sequence .
change in ph had no effect on fluorescent emission of the aptamer - reporter or the control aptamer .
these findings indicated that the formed aptamer - reporter can be optically activated by cell lysates , but not affected by the low ph - induced structure change . to further strengthen these observations ,
the control and aptamer - reporter were also incubated in human serum alone or in serum containing fresh cell lysates under .
kinetic analysis of fluorescence revealed that the aptamer - reporter was stable up to 90 minutes in human serum with minimal background , and was rapidly activated by the addition of cell lysates ( figure 2d ) .
to study tumor cell - induced activation , the aptamer - reporter was simply added into a culture of karpas 299 cells , which express high levels of the cd30 biomarker on the cell surfaces .
as shown in figure 2e ( upper panel ) , the aptamer - reporter was rapidly optically activated by the tumor cells and , thus , highlighted the tumor cells .
the fluorescence was initially observed at 10 minutes postincubation and lasted up to 120 minutes , with no background noise .
quantified fluorescent signals of tumor cells at different time points were calculated and summarized in graph in figure 2e .
subsequently , to validate intracellular optical activation , cells were treated with the aptamer - reporter at room temperature for 30 minutes and cell membranes were then stained with the green fluorescent dye , alexa 488 .
fluorescent signals derived from the aptamer - reporters ( red ) and prestained cell membranes ( green ) were recorded separately .
merged images confirmed that the aptamer - reporter emitted fluorescent signals exclusively confined within the targeted cells .
moreover , to detect intracellular distribution , the cells were treated with the aptamer - reporter and cell lysosomes were then stained with the fluorescent dye , lyso - id .
merging of confocal microscopic images revealed the colocation of fluorescent signals from the aptamer - reporter ( red ) and prestained lysosomes ( green ) within the same cells ( figure 2 g ) .
these findings confirmed that the aptamer - reporter could be optically activated and emit fluorescent signals exclusively within the targeted cells . to validate assay specificity ,
multiple cultured cells were incubated with the aptamer - reporter at room temperature for 30 minutes , and directly examined under fluorescent microscope .
as shown in figure 3a , the aptamer - reporter was activated by the tumor cells that express the cd30 biomarker and , thus , highlighted the tumor cells of sudhl-1 , hdlm2 , and kmh2 cells , but remained optically silent in control cultures of maver-1 , u937 , and ca46 cells that do not express cd30 , with no background noise .
additional studies with carcinoma tumor cells and normal peripheral cells are summarized in table 1 . to assess sensitivity ,
a cell mixture was made by diluting karpas 299 tumor cells at ratios from 1/10 to 1/10,000 in u937 control cells , which were prestained with green fluorescence ( figure 3b ) .
the cell mixtures were incubated with the aptamer - reporter , and fluorescent signals derived from the aptamer - reporters ( red ) and prestained control cells ( green ) were directly recorded .
merged images revealed that the aptamer - reporters were selectively activated and highlighted by cd30-expressing tumor cells , but did not react to control cells in the cell mixtures .
taken together , these findings indicate that the aptamer - reporter was both specific and sensitive for detection of tumor cells . for preclinical evaluation , peripheral blood and marrow aspirate were collected from patients with alcl tumors under a protocol approved by the irb for the houston methodist research institute .
diagnosis of alcl was made by tissue biopsy , and cd30 expression on lymphoma cells was confirmed by immunohistochemical staining of tumor tissues with anti - cd30 antibody ( figure 4a ) .
to detect ctcs of alcl , whole blood samples were added into a 12-well plate ( 40 million total blood cells / well ) , which was preloaded with the aptamer - reporter ( 5
after incubation for 30 minutes at room temperature , plates were directly examined under a microscope .
blood cells were well preserved for morphologic identification , including intact rbc , white blood cells , and possibly rare tumor cells that could not be distinguished ( figure 4b ) .
however , the ctcs in whole blood samples were selectively highlighted by the aptamer - reporter with bright fluorescence and without background noise or off - target signals from normal blood ( figure 4b , right panels ) .
similarly , patient marrow aspirate was also added into the assays , and ctcs were selectively highlighted by the aptamer - reporter as detected by fluorescent microscopy ( figure 4c ) .
currently , antibodies have been used as the gold standard probe for tumor cell detection in the clinical setting .
double - staining of ctcs were performed to confirm specificity of the aptamer - reporter .
patient marrow cells were initially probed by the fitc - conjugated anti - cd30 antibody .
after removal of free antibody from the reaction , the cells were then treated with the aptamer - reporter and directly examined under a fluorescent microscope .
cell signals derived from antibody ( green ) and the aptamer - reporters ( red ) were recorded separately ( figure 4d ) . merged images revealed that the ctcs in patient marrow were double - stained by both antibody and the aptamer - reporters , confirming specificity of the one - step assay .
notably , the aptamer and antibody did not compete for the same binding site(s ) on the same tumor cells .
additional blood specimens from different patients with alcl tumor were also analyzed , and ctcs were specifically highlighted and detected with a high signal - background noise ratio ( figure 4e , upper panel ) . to validate assay capacity ,
a high - concentration blood sample was loaded into a 12-well plate ( 120 million blood cells / well ) to form multiple layers of cells ( figure 4e , lower panel ) . interestingly ,
cellular fluorescent signals were easily detected , although the highlighted ctcs were overlapped by normal blood cells and could not be observed under a light microscope .
as a normal control , whole blood samples from healthy donors were tested under the same assay and showed no off - target signals ( figure 4f ) .
for point - of - care purposes , an ideal ctc assay should be ( i ) simple ( one - step reaction ) , ( ii ) high throughput ( complete in minutes ) , and ( iii ) accurate ( no loss of blood sample and no damage of tumor cells through the assay process ) .
however , no such assay could be developed using current antibody - mediated technology as described above . to this end
, we designed a unique aptamer - reporter , which is composed of an oligonucleotide sequence specific for a tumor cell biomarker conjugated with a reporter of paired fluorochrome - quencher molecules .
the underlining hypothesis is that the intact aptamer - reporter is optically silent in the absence of tumor cells ( figure 1a ) , but can be activated by tumor cells to emit fluorescence .
as showed in figure 1b , in assays containing patient blood the oligonucleotide sequence will selectively target ctcs through interaction with a specific surface biomarker , which triggers internalization of the aptamer - reporter through a natural cellular process and , subsequently , into cell lysosomes that are rich in different enzymes .
this internalization mechanism has been successfully used for intracellular delivery of chemotherapeutic drugs for targeted therapy by a newly fda - approved antibody - drug - conjugate , brentuximab .
rapid degradation of the aptamer - reporter by lysosomal nucleases results in separation of the paired fluorochrome - quencher molecules , the released fluorochrome becoming optically active and emitting bright fluorescent signals exclusively within the targeted tumor cells .
notably , because it is controlled by a specific aptamer - biomarker interaction and an intracellular activation switch , the aptamer - reporter will generate no background noise or off - target signals from normal blood cells and thus , the assay can be carried out in one - step , load - then - read ( figure 1c ) .
for the initial proof - of - concept studies , the aptamer - reporter was formulated by conjugating a 39-mer rna sequence specific for the cd30 biomarker with a fluorochrome cy3 compound and a bhq2 at the 5- and 3-ends , respectively ( figure 1a and figure 2a , left ) . due to covalent chemical conjugation ,
aptamer sequence is able to hold fluorochrome and quencher molecules close to each other and , thus , the bhq2 molecule will quench fluorescent signals emitted from the cy3 compound on the same aptamer .
a control aptamer was also synthesized by conjugating the same sequence with the fluorochrome cy3 compound alone ( figure 2a , right ) .
first , to validate the optical - activatable property , the aptamer - reporter was incubated with fresh lysates of cultured karpas 299 tumor cells of human alcl at room temperature as a cellular enzyme source .
incubation with cell lysates activated the aptamer - reporter and triggered a rapid increase in florescent signals in a time - dependent fashion ( figure 2b ) .
in contrast , cell lysates had no effect on the control aptamer which constantly emitted high levels of fluorescent signals . to rule out a potential effect due to conformation change , the aptamer - reporter was treated under different ph conditions , from ph 4.0 to physiological ph 7.4 , at room temperature for 30 minutes ( figure 2c ) .
notably , cell lysosomes have a low ph environment ( ph 45 ) which can cause denaturing of the aptamer sequence .
change in ph had no effect on fluorescent emission of the aptamer - reporter or the control aptamer .
these findings indicated that the formed aptamer - reporter can be optically activated by cell lysates , but not affected by the low ph - induced structure change . to further strengthen these observations ,
the control and aptamer - reporter were also incubated in human serum alone or in serum containing fresh cell lysates under
. kinetic analysis of fluorescence revealed that the aptamer - reporter was stable up to 90 minutes in human serum with minimal background , and was rapidly activated by the addition of cell lysates ( figure 2d ) . to study
tumor cell - induced activation , the aptamer - reporter was simply added into a culture of karpas 299 cells , which express high levels of the cd30 biomarker on the cell surfaces .
cells were then directly examined under fluorescent microscope . as shown in figure 2e ( upper panel ) , the aptamer - reporter was rapidly optically activated by the tumor cells and , thus , highlighted the tumor cells .
the fluorescence was initially observed at 10 minutes postincubation and lasted up to 120 minutes , with no background noise .
quantified fluorescent signals of tumor cells at different time points were calculated and summarized in graph in figure 2e .
subsequently , to validate intracellular optical activation , cells were treated with the aptamer - reporter at room temperature for 30 minutes and cell membranes were then stained with the green fluorescent dye , alexa 488 .
fluorescent signals derived from the aptamer - reporters ( red ) and prestained cell membranes ( green ) were recorded separately .
merged images confirmed that the aptamer - reporter emitted fluorescent signals exclusively confined within the targeted cells .
moreover , to detect intracellular distribution , the cells were treated with the aptamer - reporter and cell lysosomes were then stained with the fluorescent dye , lyso - id .
merging of confocal microscopic images revealed the colocation of fluorescent signals from the aptamer - reporter ( red ) and prestained lysosomes ( green ) within the same cells ( figure 2 g ) .
these findings confirmed that the aptamer - reporter could be optically activated and emit fluorescent signals exclusively within the targeted cells .
to validate assay specificity , multiple cultured cells were incubated with the aptamer - reporter at room temperature for 30 minutes , and directly examined under fluorescent microscope .
as shown in figure 3a , the aptamer - reporter was activated by the tumor cells that express the cd30 biomarker and , thus , highlighted the tumor cells of sudhl-1 , hdlm2 , and kmh2 cells , but remained optically silent in control cultures of maver-1 , u937 , and ca46 cells that do not express cd30 , with no background noise .
additional studies with carcinoma tumor cells and normal peripheral cells are summarized in table 1 . to assess sensitivity ,
a cell mixture was made by diluting karpas 299 tumor cells at ratios from 1/10 to 1/10,000 in u937 control cells , which were prestained with green fluorescence ( figure 3b ) .
the cell mixtures were incubated with the aptamer - reporter , and fluorescent signals derived from the aptamer - reporters ( red ) and prestained control cells ( green ) were directly recorded .
merged images revealed that the aptamer - reporters were selectively activated and highlighted by cd30-expressing tumor cells , but did not react to control cells in the cell mixtures .
taken together , these findings indicate that the aptamer - reporter was both specific and sensitive for detection of tumor cells . for preclinical evaluation , peripheral blood and marrow aspirate were collected from patients with alcl tumors under a protocol approved by the irb for the houston methodist research institute .
diagnosis of alcl was made by tissue biopsy , and cd30 expression on lymphoma cells was confirmed by immunohistochemical staining of tumor tissues with anti - cd30 antibody ( figure 4a ) .
to detect ctcs of alcl , whole blood samples were added into a 12-well plate ( 40 million total blood cells / well ) , which was preloaded with the aptamer - reporter ( 5
after incubation for 30 minutes at room temperature , plates were directly examined under a microscope .
blood cells were well preserved for morphologic identification , including intact rbc , white blood cells , and possibly rare tumor cells that could not be distinguished ( figure 4b ) . however
, the ctcs in whole blood samples were selectively highlighted by the aptamer - reporter with bright fluorescence and without background noise or off - target signals from normal blood ( figure 4b , right panels ) .
similarly , patient marrow aspirate was also added into the assays , and ctcs were selectively highlighted by the aptamer - reporter as detected by fluorescent microscopy ( figure 4c ) .
currently , antibodies have been used as the gold standard probe for tumor cell detection in the clinical setting .
double - staining of ctcs were performed to confirm specificity of the aptamer - reporter .
patient marrow cells were initially probed by the fitc - conjugated anti - cd30 antibody .
after removal of free antibody from the reaction , the cells were then treated with the aptamer - reporter and directly examined under a fluorescent microscope .
cell signals derived from antibody ( green ) and the aptamer - reporters ( red ) were recorded separately ( figure 4d ) . merged images revealed that the ctcs in patient marrow were double - stained by both antibody and the aptamer - reporters , confirming specificity of the one - step assay .
notably , the aptamer and antibody did not compete for the same binding site(s ) on the same tumor cells .
additional blood specimens from different patients with alcl tumor were also analyzed , and ctcs were specifically highlighted and detected with a high signal - background noise ratio ( figure 4e , upper panel ) . to validate assay capacity ,
a high - concentration blood sample was loaded into a 12-well plate ( 120 million blood cells / well ) to form multiple layers of cells ( figure 4e , lower panel ) .
interestingly , cellular fluorescent signals were easily detected , although the highlighted ctcs were overlapped by normal blood cells and could not be observed under a light microscope . as a normal control ,
whole blood samples from healthy donors were tested under the same assay and showed no off - target signals ( figure 4f ) .
it is generally believed that there are only a scant number of detectable ctcs in patient blood , although the actual number ranges are not yet fully known .
a recent study demonstrated that the average number of ctcs ranges from a mean of 79 to 196 cells per ml in whole blood specimens of patients with different types of cancers , which is much higher than the cut - off cell number ( 5 cells / ml ) detected by current commercial tests .
in addition , employing the cell search profile kit could detect more than 30-fold of the median ctc counts that are identified by the clinically used cell search epithelial kit in the same blood samples of patients with breast or lung cancers .
these findings indicate the presence of much more ctcs in patient blood than previously thought , suggesting the possibility that , with a sensitive detection approach , the current standard numeration of ctcs could be achieved using smaller blood samples .
since our one - step assay will eliminate any potential loss of the blood sample and damage of tumor cells during the assay process , it will be able to detect the actual number of ctcs , and thus , require a much smaller blood sample .
notably , although the number of ctcs present in blood may vary among different types of tumors and different stages of disease , our assay scale can simply be justified by employing different - sized plates as clinically indicated with 96- , 24- , 12- , 6- , and single - well(s ) to meet specific clinical needs .
in addition , the load - then - read technology provides the possibility of performing high - throughput screening assays for rapid detection of ctcs .
moreover , good preservation of intact ctcs in this one - step assay will allow us to collect viable ctcs for further analysis of cellular protein and/or gene expression profiles at a single - cell level . to eliminate background noise in whole - blood samples and enable intracellular highlighting of tumor cells ,
the one - step assay was designed by taking advantage of tumor cell internalization of the aptamer - reporter through a natural biological cell process , which is triggered by specific binding of aptamer sequences to surface cd30 receptors on tumor cells ( figure 1b ) .
interestingly , a similar internalization mechanism is employed for intracellular delivery of chemotherapeutic drug by an antibody - drug - conjugate , brentuximab , a newly fda - approved , targeted therapeutic specific for cd30-expressing lymphomas .
thus , it is considerable that , in addition to the potential for diagnostic applications , aptamers can also be used as a chemical antibody for targeted therapy . in summary ,
our study introduced a unique tumor cell - activatable aptamer - reporter technology and demonstrated a simple , one - step assay for detection of ctcs in patient whole blood .
since this technology improves upon the existing antibody - driven methods , we believe it holds a high potential in early identification of patients who are at a higher risk for developing tumor metastases due to their elevated levels of systemic ctcs .
reagents and cells . in the proof of principal studies , a 39-mer rna - based aptamer sequence , previously shown to specifically bind cd30-expressing tumor cells , was used .
a novel tumor cell - activatable aptamer - reporter was then chemically synthesized ( bio - synthesis , lewisville , tx ) by conjugating a fluorochrome cy3 together with a black hole quencher 2 ( bhq2 ) molecule at the opposing ends of the aptamer sequence as follows : cy3 - 5-rgrarururcrarurarurgrgrgrurgrgrgrarurcrgrgrgrarargrgrgrcrurarurgrararurcrg-3-bhq2 .
a control aptamer probe with the same sequence was conjugated with a fluorochrome cy3 compound at the 5 end .
alexa fluor 488 conjugates for cell membrane staining were purchased from invitrogen ( grand island , ny ) and lyso - id green detection kit for lysosome staining was purchased from enzo life sciences ( farmingdale , ny ) . the fitc - conjugated anti - human cd30 antibody was purchased from bd biosciences ( franklin lakes , nj ) .
cancer cells lines , including human anaplastic large cell lymphoma ( alcl ) cells ( karpas 299 and sudhl-1 cell lines from mark raffeld at nih ) , hodgkin lymphoma cells ( hdlm2 and kmh2 cell lines from barbara savoldo , baylor college of medicine , houston , tx ) , b cell lymphoma cells ( mino and maver-1 cell lines from atcc ) , and leukemia cells ( u937 cell line from atcc ) were cultured in rpmi 1640 medium supplemented with 10% fbs , 100 u / ml penicillin , and 100 g / ml streptomycin at 37 c under the atmosphere of 5% co2 and 95% humidity .
fresh tumor cell lysates were prepared to confirm the cell - activatable property of the formed aptamer - reporter .
briefly , cultured karpas 299 cells were harvested , washed twice , and suspended in pbs ( 5 10/ml ) .
cells were frozen at 80 c , thawed at room temperature twice , and centrifuged at 12,000 g for 20 minutes at 4 c . finally , 100 l supernatants of cell lysates were collected and added into wells of a 96-well black wall plate , which was preloaded with the aptamer - reporter ( 5
after incubation at room temperature , fluorescence signals of the assays were kinetically quantified by a synergy h4 microplate reader ( biotek , winooski , vt ) at different time points , as described .
each condition was tested in triplicate and experiments were repeated more than three times with similar results .
similarly , the control aptamer , containing the same sequence and conjugated with the fluorochrome cy3 compound alone , was tested under the same treatment conditions and change in fluorescent signals of the control group was kinetically monitored as described above . to rule out the effects of low ph conditions , which is seen in cell lysosomes and
might result in denature of the aptamer sequence 3d - structure , the aptamer - reporter ( 5
nmol / l final concentration ) was added into pbs with phs 4.0 , 5.0 , 6.0 , 7.0 , and 7.4 in a 96-well black wall plate .
after incubation at room temperature for 30 minutes , fluorescence of the assays was quantified by a synergy h4 microplate reader .
each condition was tested in triplicate and experiments were repeated more than three times with similar results .
finally , the aptamer - reporter ( 5 nmol / l final concentration ) were incubated in 100 l human serum ( atlanta biological , lawrenceville , ga , usa ) in a 96-well black wall plate and changes in fluorescence were quantified as described above . in control group , 100 l of cell lysate supernatants
each condition was tested in triplicate and experiments were repeated more than three times with similar results .
one - step assay and tumor cell detection . to validate the tumor cell - induced activation , 100 l of cultured karpas 299 cells ( 5 10/ml ) were preseeded in a 96-well black wall plate in pbs , and 5 l of the aptamer - reporter
was then added to each well at 5 nmol / l final concentration . without any additional preparation steps , the plates were directly examined under an olympus ix81 fluorescent microscope and cell fluorescent signals were kinetically imaged using a tritc filter under 547 nm of peak excitation wavelength and 572 nm of peak emission wavelength . for control purposes ,
cells were also treated with the control aptamer that was conjugated with fluorochrome cy3 compound alone under the same condition .
fluorescent images were recorded using the same exposure time ( 300 microseconds ) at different time points post treatment as described in figures .
each experiment with triple wells was repeated at least three times with similar results . for quantification , intensity of fluorescent signals from individual tumor cells
a total of 90 cells from triplet wells ( 30 cells / well ) at each time point were randomly gated and recorded . to get the background value of fluorescent intensity , 10 random noncellular locations with an average area of cell sizes were gated and calculated similarly .
the mean fluorescent intensity of tumor cells was derived after background signals were deducted , calculated with standard deviation , and shown in graph with sd error bars .
cultured karpas 299 cells were treated with the aptamer - reporter at room temperature for 30 minutes .
after washing once with pbs , the cells were suspended in pbs at room temperature , stained with concanavalin a conjugated to alexa fluor 488 ( 1 mg / ml ) for 2 minutes , fixed with 4% formaldehyde for 10 minutes , washed with pbs , and suspended in pbs / glycerin ( weight / weight ratio of 1:1 ) .
finally , 15 l of cell solution was loaded onto glass slides and examined by a confocal microscope to determine intracellular localization of the aptamer - reporter ( fitc filter for alexa
for lysosomal detection , karpas 299 cells were treated with the aptamer - reporter as described above .
after washing once with pbs , cells were stained with lyso - id green dye ( 1:1,000 dilution ) at room temperature for 1 hour , fixed with 4% formaldehyde for 10 minutes , washed with pbs , and then suspended in pbs / glycerin . finally , 15 l of cell solution was loaded onto glass slides and examined by a confocal microscope as described above to determined possible colocalization of aptamer - reporters with the lysosomes . specificity and sensitivity validation .
cd30-expressing cells ( karpas 299 , sudhl-1 , hdlm2 , and kmh2 ) and cd30-negative control cells ( maver-1 , u937 , and ca46 ) were tested to confirm specificity of the aptamer - reporter .
the cells were incubated with the aptamer - reporter for 30 minutes at room temperature and the fluorescence was detected as described above .
additional cell lines ( breast , prostate , and colon carcinomas ) and peripheral blood collected from healthy donors were also tested and are listed in table 1 . to test sensitivity of the aptamer - reporter
, cell mixtures were prepared from cd30-expressing karpas 299 cells and cd30-negative u937 control cells . for tracking purposes ,
u937 cells were stained with concanavalin a conjugated to alexa fluor 488 and karpas 299 cells were added in 1:10 to 1:10,000 ratios , as indicated .
subsequently , cell mixtures were added into 96-well black wall plates ( 2 10 cells / well ) that were preloaded with the aptamer - reporting system ( 5 nmol / l final concentration ) . after 30 minutes incubation at room temperature , cells were examined by a fluorescent microscope ( fitc filter for alexa fluor 488 to detect u937 cells , tritc filter for cy3 derived from aptamer - reporters ) .
detection of ctcs in patient whole blood and marrow aspirate samples in a single - step reaction . under an approved irb protocol ,
peripheral blood and bone marrow samples were collected from patients ( n = 6 ) with stage iv alcl tumors diagnosed by a histological evaluation of h&e - stained tumor tissue and immunohistochemical demonstration of cd30 expression on tumor cells .
ten blood and marrow samples collected from healthy donors were used as a control group . following cell counting , whole blood and marrow aspirate specimens were added into wells of a 12-well plate that contained the aptamer - reporter ( 5
nmol / l final concentration ) for a total of 40 million blood or marrow cells / well .
after incubation at room temperature for 30 minutes , single - step labeled ctcs were directly detected by a fluorescent microscope . for a morphological evaluation ,
ctc images were also obtained with a light microscope within the same view fields . to confirm specificity of the aptamer - reporter , marrow cells were double - stained with fitc - conjugated anti - human cd30 antibody and the aptamer - reporter .
briefly , bone marrow cells were diluted in 1 ml pbs and incubated with fitc - conjugated anti - human cd30 antibody ( bd biosciences , san jose , ca ) at room temperature for 30 minutes . to eliminate unbound antibody and minimize damage to ctcs ,
the reaction supernatant containing some red blood cells ( rbcs ) was gently removed by a pipette .
the remaining cells were then suspended in 200 l pbs and added into well plates that were preloaded with the aptamer - reporter at a 5 nmol / l final concentration .
after 30 minutes incubation at room temperature , plates were directly examined by a fluorescent microscope and cell fluorescence ( green : antibody , red : aptamer - reporter ) recorded separately . merged images showed that ctcs in patient marrow were double - stained , confirming specificity of the aptamer - reporter . to test the effect of blood concentration
, a total of 120 million blood cells from patients diagnosed with alcl were added into 12-well plates to form multiple cell layers .
after 30 minutes incubation with the aptamer - reporter , plates were examined by light and fluorescent microscopes to detect ctc fluorescence as described above .
merged images indicated that the assay was able to detect ctcs in high - concentration blood samples , even when ctcs overlapped with normal blood cells and could not be observed under a light microscope . to validate these results , whole blood samples from healthy donors
were also tested in the single - step assay , which revealed no false - positive or off - target staining in the normal blood cells . | the current antibody - mediated numeration assays of circulating tumor cells ( ctcs ) require multiple steps and are time - consuming . to overcome these technical limitations ,
a cancer cell - activatable aptamer - reporter was formulated by conjugating a biomarker - specific aptamer sequence with paired fluorochrome - quencher molecules .
in contrast to the antibody probes , the intact aptamer - reporter was optically silent in the absence of cells of interest . however , when used in an assay , the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker , which triggered internalization of the aptamer - reporter and , subsequently , into cell lysosomes . rapid lysosomal degradation of the aptamer - reporter resulted in separation of the paired fluorochrome - quencher molecules .
the released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells , with no background noise in the assay .
thus , the assays could be completed in a single step within minutes . by using this one - step assay , ctcs in whole blood and marrow aspirate samples of patients with lymphoma tumors
were selectively highlighted and rapidly detected with no off - target signals from background blood cells .
the development of the cancer cell - activatable aptamer - reporter system allows for the possibility of a simple and robust point - of - care test for ctc detection , which is currently unavailable . |
endodontically treated teeth , with a large amount of coronal tooth structure missing , frequently require the placement of a post inside the root canal , to retain a core for definitive restoration.[13 ] the choice of appropriate restoration for these teeth is influenced by strength and esthetics . depending on the clinical parameters ,
the choice may be a metal or an esthetic post and core restoration.[57 ] clinical studies have reported success rates of 95 to 99% for teeth restored with fiber - reinforced posts , with no occurrence of root fracture during the study periods . the most common cause of failure reported in these studies is not from the root fracture , as occurs with metal or cast posts , but rather from the pull out of the cement , post restoration assembly , as a result of lack of retention of the fiber posts .
the retention of fiber posts in the roots depends on the bond strength between the post material and a resin luting agent , as well as the bond strength between the resin luting agent and post space dentin.[1114 ] many in vitro studies have investigated various factors that influence this bond strength .
these factors include length , design , diameter , composition , surface treatment of the post , preparing the dentinal surface , the dentin bonding agent , luting cement , and polymerization mode .
the effect of cement type on the retention of the post in endodontically treated teeth has been investigated extensively and it has been found that resin cements can significantly increase the retention of posts and fracture - resistance of the teeth , compared to other cements.[1517 ] different resin luting agents and corresponding bonding systems have been proposed for cementing tooth - colored posts .
several in vitro studies have reported controversial results regarding bond strengths of different luting agents to endodontic posts and root canal dentin.[1926 ] selecting an appropriate adhesive and luting procedure for bonding posts to root dentin is an important challenge.[2729 ] according to adhesive dentistry , two strategies can be used to allow the bonding agents to react with the dentin .
some systems use etch - and - rinse with phosphoric acid to eliminate the smear layer and demineralize the underlying dentin .
this step is followed by application of a hydrophilic monomer that penetrates the conditioned dentin surface .
other systems use self - etching primers , with no rinsing step , which cause less aggressive demineralization and are followed by the application of an adhesive resin .
the bond strength of fiber posts to root canal dentin may be varied in different regions of the root canal .
this difference may arise from the difficulty of reaching the curing light to more apical parts of the root canal , difficulty of direct vision to the deeper regions of the root canal , and difficulty of moisture control and adhesive application in the apical region of the root canals .
the aim of the present study is to compare the push - out bond strength of a glass fiber reinforced composite post system , cemented with a self - etch and conventional resin cement systems , in different root canal regions .
the null hypotheses were : ( 1 ) self etching and conventional resin cements provide the same bond strength for glass fiber reinforced composite post , and ( 2 ) there are no differences between the bond strengths of different post space regions .
in this in vitro study , 20 human maxillary central incisors with 15 mm length ( from 1 mm above the cementoenamel junction to the apex ) , extracted for periodontal reasons , were selected .
the specimens were free of cracks , carious lesions , fractures , and resorption , with fully developed apices and without previous endodontic treatments , posts , or crowns .
they were cleaned off soft tissues and calculus , and placed in 2.5% sodium hypochlorite for two hours , for surface disinfection , and then stored in 0.1% nan3 solution until use .
the coronal part of each tooth was removed 1 mm above the cementoenamel junction with diamond disks ( ref.070 , d and z , berlin , germany ) , mounted on a dental lathe machine ( kavo polishing unit .
ewl 80 , leutkrich , germany ) at low speed , under constant water irrigation , to achieve a uniform length of 15 mm .
obturation was performed using ah26 ( dentsply caulk , milford , germany ) and gutta - percha ( aria dent , asia chemi teb co. , tehran , iran ) , with the vertical condensation method .
after completion of endodontic treatment , the coronal root canal openings were filled with a provisional restorative material ( gc caviton ; gc dental products corp . , tokyo , japan ) , and the teeth were stored in 100% humidity for one week at 37c , to allow the sealer to set .
after one week , the gutta - percha was removed from the coronal aspect of each root with a gates glidden drill # 3 ( dentsply - maillefer , ballaigues , switzerland ) leaving 4 mm gutta percha in the apices , to preserve the apical seal .
the post spaces were prepared to a depth of 10 mm with the appropriate drills ( fibio , anthogyr , sallanches , france ) .
post size 3 was tried , to ensure that the posts would reach the bottom of the post space .
all posts were marked at a distance of 10 mm from the apical end , and were cut to that size with diamond disks .
the shortened posts were cleaned with 70% ethanol for 60 seconds , rinsed with distilled water , and air dried . before the cementation procedures
the prepared roots were randomly divided into two groups of 10 specimens each for cementation procedures [ table 1 ] . in group 1
, the posts were luted with panavia f 2.0 ( kuraray medical , inc . , okayama , japan ) after conditioning the dentin with ed - primer ( a and b liquid mixture , kuraray ) for 60 seconds , using a microbrush .
the post space was gently air - dried and the excess primer was removed with paper points ( aria dent ) . in group 2 ,
variolink ii ( ivoclar vivadent , schaan , liechtenstein ) was used as the luting agent .
the canals were etched using 35% phosphoric acid ( ultra - etch , ultradent , south jordan , ut , usa ) for 15 seconds and rinsed with distilled water .
excess water was removed from the post spaces with a gentle stream of air and paper points .
the excite dsc ( ivoclar vivadent ) , dual - polymerizing , single - bottle bonding agent was applied for 10 seconds with a microbrush coated with chemical initiators and the excess bonding agent was removed with paper points and gently air - dried .
the bonding agent was polymerized with a halogen light unit , with 500-mw / cm intensity ( coltolux 50 , coltene , altstatten , switzerland ) , for 20 seconds , with the tip of the light unit directly in contact with the canal orifice .
for cementation of fiber posts , equal amounts of luting pastes ( panavia f 2.0 and variolink ii ) were mixed and applied onto the surface of the posts and into the root canals with a lentulo spiral instrument ( dentsply / maillefer ) .
the posts were inserted into the canal , to a full depth , by using gentle finger pressure , and the excess was immediately removed with a disposable brush . for panavia
f 2.0 the remaining cement around the post was protected with oxygen - inhibiting gel ( oxyguard ii , kuraray ) . for variolink
ii , after the initial chemical polymerization , light curing was performed for 60 seconds in such a way that the tip of the light unit was directly in contact with the coronal end of the posts .
the light output was monitored to ensure accurate light intensity before each exposure by using the light meter ( coltolux , coltene ) .
after the cementation procedures , all specimens were stored in sterile saline in a light - proof box for one week at 37c .
next , each root was sectioned perpendicular to the long axis with a diamond disk at low speed under constant distilled water cooling to create 3 mm - thick slices . in this manner
; from each root , three post / dentin sections ( coronal , middle , and apical ) were obtained .
due to the tapered design of the fiber posts , post diameters were measured on each surface of the post / dentin sections , using digital calipers ( electronic digital caliper , minova co , japan ) , with 0.01 mm accuracy .
the push - out test was performed by using the universal testing machine ( tlclo , dartec ltd . ,
stourbridge , england ) at a crosshead speed of 1 mm/ minute , using a pin ( diameter , 1.0 mm ) on the center of the apical aspect of the post surface in an apical - coronal direction , without stressing the surrounding post space walls . the peak force ( n ) required to extrude the post from the root slice was recorded . to express the bond strength in mpa ,
the load at failure ( n ) was divided by the area of the bonded interface , which was calculated with the following formula : where was the constant 3.14 , r1 was the coronal post radius , r2 was the apical post radius , and h was the thickness of the slice in millimeters .
the collected data were analyzed ( spss / pc 16.0 ; statistical package for the social sciences ( spss ) inc , chicago , ill ) using two - way analysis of variance ( anova ) and post hoc scheffe tests at p<0.05 levels of significance .
the mean push - out bond strength values ( mpa ) of the test groups in different root canal regions are shown in table 2 . the two - way anova showed no significant differences between the mean push - out bond strength values recorded for two experimental groups ( two types of cement systems ) and there was no interaction between the type of resin cement system and different root canal regions ( p=0.920 and p=0.731 , respectively ) [ table 3 ] .
otherwise , significant differences were observed among the bond strength values of the root dentin regions ( p<0.001 ) .
the post hoc scheffe test revealed that in both resin cement systems , there were significant differences between the coronal and apical regions [ table 4 ] .
descriptive statistics of push - out bond strength values ( mpa ) of two groups in different root canal regions scheffe test for push - out bond strength values ( mpa ) of two groups in different root canal regions
the results of this in vitro study support the first hypothesis that self - etching and conventional resin cements provide the same bond strength of a glass fiber reinforced composite post . however , the hypothesis that there are no differences between the bond strengths in different post space regions has been rejected . in the case of the first hypothesis ,
stated that the hybrid layer and resin tags that resulted from both bonding systems ( self - etch and total - etch ) were nearly the same , although the conditioning of the canal walls was different .
micromorphological investigations showed that etching the dentin with phosphoric acid ( ph2 ) ( as used in the etch - and - rinse systems ) completely dissolved the smear layer and exposed the tubule apertures , collagen fibrils , and interfibrillar spaces , while the use of self - etch bonding systems had a variable effect on the smear layer .
bitter et al . found a significant difference between these two types of luting agents when used as luting glass fiber reinforced composite posts on root canal dentin .
they noted that the hybrid layer appeared thinner with the self - etch system , than with the etch - and - rinse system .
they concluded that demineralization by phosphoric acid led to a deeper penetration of the adhesive than did a self - etch system , which could not completely penetrate the smear layer .
stated that a system with a self - etching primer and light - polymerized bonding agent provided a significantly higher bond strength to root canal dentin than the self - etching bonding agents and the strength of this bond was not dependent on the hybrid layer thickness .
in contrast , some authors stated that simplified self - etch and self - adhesive resin cements , such as panavia 21 , exhibited an etching potential insufficient ( even with a ph of 2.0 ) to dissolve the thick smear layers created in the post - space preparation , with slow - speed drills .
they stated that this fact yielded to the high shrinkage stress in the thin cement layer and the consequent opening of interfacial gaps might account for the relatively low push - out strength recorded for ed primer / panavia 21 as compared to the results obtained with the use of the total - etch system , excite dsc / variolink ii .
however , they proposed the use of self - adhesive resin cements for intra - canal posts , because of their simple luting procedure . in the case of the second hypothesis ,
our result was in agreement with the previous studies that reported higher bond strength for coronal dentin than values for the middle and apical sections of the root.[462333343840 ] according to akgungor and akkayan , the self - etching primer clearfil liner bond 2v did not show regional differences in post - space bonding , unlike the single bottle adhesive excite , which exhibited significantly lower strength at the apical level .
observed a higher bond strength in the coronal sections for excite / variolink ii , but similar values for relyx unicem .
it has been shown that tubule density is greater in the coronal and middle thirds than in the apical region of the root canal , and the diameter of the tubules decreases in the apical direction .
the difference in the number of tubules may explain why the strongest adhesion is achieved in the most coronal regions .
in the presence of a greater number of tubules per mma stronger bond will be expected , because the adhesion may be enhanced by the penetration of the resin into the tubules .
furthermore , it is shown that dentin hybridization is not uniform in the apical region of the root canal dentin and the lateral branches of the resin tags are not observed in the apical part of the interface post - adhesive system .
some methodological factors might have contributed to the discrepancies in bond strength values , such as , better accessibility of the coronal portion of the root canal , which made it easier to etch and apply the adhesive agents . in addition
, a reduction of light energy during transmission would decrease polymerization of the bonding agent at the medium and apical regions . however , some other studies found no significant differences in the bond strengths between the different regions of the bonded posts to root canal dentin . in this study ,
however , it was suggested that the amount of remaining coronal tooth structure played a major role in the longevity of the restoration of endodontically treated teeth . in the present study ,
the influences of fatigue loading and thermal cycling on the push - out bond strength of the glass fiber reinforced composite post system were not investigated .
within the limitations of this in vitro study , the following conclusions were drawn :
there was no significant difference between the push - out bond strengths of glass fiber reinforced composite posts for self - etching and conventional resin cementsthe coronal region of the root dentin showed a significantly higher bond strength of the glass fiber reinforced composite post than the apical region .
there was no significant difference between the push - out bond strengths of glass fiber reinforced composite posts for self - etching and conventional resin cements the coronal region of the root dentin showed a significantly higher bond strength of the glass fiber reinforced composite post than the apical region . | introduction : debonding is a common cause of failure encountered with fiber - reinforced composite ( frc ) posts , and usually occurs along the post space - dentin adhesive interface .
the aim of this in vitro study is to evaluate the regional push - out bond strength of a fiber - reinforced post system , using two resin cements.materials and methods : in this in vitro study 20 maxillary central incisors were decoronated and the roots were endodontically treated . following post space preparation ,
the roots were divided into two groups of 10 specimens each .
fiber - reinforced composite posts were cemented with two resin cement systems : ( a ) self - etch system ( panavia f2.0/ed - primer ii ) and ( b ) conventional system ( variolink ii / excite dsc ) .
three slices of each root , with a thickness of 3 mm , were prepared .
the push - out test was performed with a universal testing machine at a crosshead speed of 1 mm / minute , and bond strength values were calculated .
the data were analyzed with a two - way analysis of variance ( anova ) and scheffe tests ( =.05).results : there were no significant differences between the mean push - out bond strengths of two experimental groups ( panavia f : 12.595.44 , variolink ii : 12.494.52 mpa ) ( p=0.920 ) , but there were significant differences between the mean push out bond strengths of the root dentin regions ( p<0.001).conclusion : under the conditions of this study , there was no significant difference between the mean push out bond strength of self - etching and the conventional resin cement systems .
the coronal region of the root dentin showed a significantly higher bond strength than the apical region . |
women with breast cancer who have undergone standard medical treatment , i.e. , surgery ,
chemotherapy , and/or radiotherapy commonly experience pain as a consequence of these
interventions1 .
the anterior and/or
lateral region of the thorax , axilla , and upper limbs are the anatomic sites most often
affected by pain in such cases2 .
this pain
is generally chronic , lasting more than three months , and requires the attention of
different healthcare professionals , including a physiotherapist3 . for the assessment of pain experienced by breast cancer survivors ,
recent studies have
employed algometry4 , the numerical rating
scale5 , visual analog scale3 , catastrophizing scale6 and mcgill pain questionnaire7 . however , pain is a complex , multidimensional phenomenon , especially
for this group of patients .
its interpretation involves different clinical aspects ,
including a psychophysical approach that addresses functional , qualitative , subjective ,
emotional , motivational , and cultural components8 ,
9 .
information gleaned through studies categorize pain experienced by breast cancer survivors
can help healthcare professionals customize clinical approach and interventions , catering to
patients specific needs .
ferreira et al.10 found that most patients experienced constant daily pain that began
soon after the surgical procedure .
fabro et al.11 found that pain was more frequently experienced by young women
subjected to axillary lymph node dissection . according to de oliveira et al.7 ,
a large proportion of breast cancer
survivors categorize their pain as tenderness , aching ,
shooting , and
tiring - exhausting . considering its multidimensional nature , other relevant aspects of pain
still need to be characterized in this population of patients . by investigating the impact of pain among breast cancer survivors and their lives using the
brief pain inventory ( bpi ) , this study can help physiotherapists design pain management
strategies to address common signs and symptoms .
the present study s procedures were approved by the human research ethics committee of the
nursing school of ribeiro preto ( university of so paulo , ribeiro preto , sp , brazil ) under
process number 162/2007 . the study objective and procedures were explained to patients ;
those agreeing to participate signed a statement of informed consent .
thirty women enrolled at the breast cancer teaching , research and rehabilitation center of
the nursing school of ribeiro preto were recruited in this cross - sectional study .
inclusion
criteria were : being 3080 years of age , having received treatment for breast cancer
( surgery and complementary treatment ) at least 12 months before the study , and having
reported chronic treatment - related pain .
the exclusion criteria were receiving surgical or
complementary ( radiotherapy and chemotherapy ) treatment during the study period , recurrence
and/or metastasis , and a diagnosis of neurological disorder , osteoarticular disorder , and/or
fibromyalgia .
this
questionnaire was developed by cleeland and ryan12 and has been validated for use on the brazilian population13 .
the bpi is divided into a sensory
dimension and a reactive dimension ( interference of pain in the patient s life ) .
mood , walking
ability , normal work , relationships with others , sleep , and enjoyment of life .
the
interference of pain is scored on an 11-point scale , where 0 represents the absence of pain
or interference and 10 represents as bad as it can be .
only the reactive dimension was
employed , as the purpose was to evaluate the impact of pain on the daily lives of breast
cancer survivors .
a physiotherapist who was familiar with the bpi administered the
questionnaire in an interview format without time constraints in a private , well - lit ,
climate - controlled room . for data analyses ,
thus , the data were expressed in terms of
minimum , maximum , median , first quartile , and third quartile . the spss version 17.0
( chicago , il , usa ) was used for all statistical analyses .
the participants mean age was 55.6911.46 years ( range : 32.1879.30 years ) .
twenty - four of
the 30 participants had received chemotherapy and 23 had received radiotherapy .
fifteen had surgery on the right
side , fourteen had surgery on the left side , and one had bilateral surgery . moreover , all
except one had undergone axillary node clearance .
table 1table 1.central tendency and dispersion values for the breast cancer survivors responses
to the brief pain inventory itemsitemsminvmaxvfqttqtmdgeneral activities0.0010.000.004.250.50mood0.0010.001.007.255.00walking ability0.0010.000.004.250.00normal work0.0010.000.008.005.00relationships with others0.0010.000.005.000.00sleep0.0010.000.008.004.50enjoyment of life0.0010.000.006.503.00minv : minimum value ; maxv : maximum value ; fqt : first quartile ; tqt : third quartile ;
md : median displays the breast cancer survivors scores for items on the bpi .
the highest
median values were found for mood , normal work , and sleep .
minv : minimum value ; maxv : maximum value ; fqt : first quartile ; tqt : third quartile ;
md : median
in the present study , mood , normal work , and sleep , items of the bpi were aspects of
the breast cancer survivors lives that were most impacted by pain .
the importance of this
study lies in the need to understand how pain affects breast cancer patients so that
therapeutic approaches , based on the actual needs of women , can be established .
recent clinical trials conducted with breast cancer survivors have mainly investigated the
effect of physiotherapeutic programs on pain intensity and the pressure pain threshold .
cantarero - villanueva et al.3 found a
greater reduction in pain intensity in the neck , shoulders , and axillae following an aquatic
physical therapy program in comparison with standard care .
fernndez - lao et al.4 reported that a multidimensional program
involving strengthening exercises and massage as the major components was more effective in
terms of improving neck and shoulder pain and reducing widespread pressure hyperalgesia in
comparison to standard treatments .
silva et al.14 found that transcutaneous electrical neural stimulation promote
electrical modification in the parietal region and a decrease in pain in patients with
intercostobrachial pain after breast cancer surgery .
therefore , future studies should
consider the use of the bpi to allow better identification of the effects of
physiotherapeutic treatment regarding other dimensions of pain among cancer patients .
these findings underscore the importance of considering those aspects of living that are
compromised by pain when evaluating and developing treatment plans .
moreover ,
galiano - castillo et al.15 reported that
depressed mood is associated with the level of physical activity , perceived shoulder pain ,
fatigue , and muscle strain in breast cancer survivors .
ha and choi16 found that proprioceptive neuromuscular facilitation
exercises helped lower depression and anxiety rates in breast cancer survivors .
castro - snchez et al.17 found that manual
therapy effectively improved sleep quality in individuals with fibromyalgia syndrome .
similarly , eadie et al.18 found that a
physiotherapy program led to improved sleep quality in individuals with chronic low back
pain .
thus , based on these studies , physical therapy interventions can act on the
relationship between pain , mood , and sleep . regarding the influence of pain on work , we emphasize the importance of the
physiotherapist s role . according to phillips et al.19 , a physiotherapeutic program for improving the physical health
status of patients with pain
thus , any physiotherapeutic approach first requires a complete patient history that
identifies labor activities and activities of daily living to establish effective
interventions that promote physical autonomy .
an
investigation involving colorectal cancer survivors yielded similar results , with a moderate
influence of pain on mood , normal work , and sleep20 .
scarpi et al.21
used the bpi to assess pain in patients with bone metastases and found that pain
considerably interfered with walking ability . in the present study
, pain did not interfere
with walking among breast cancer survivors as much as it did in previous studies , probably
because breast cancer is related to trunk and upper limb impairments .
therefore , each type
of cancer involves different areas and aspects of pain that in turn influence various
aspects of life .
further , the study only
focused on the characterization of pain and no comparisons were made with a control group ,
nor were other statistical approaches used . in conclusion
, pain exerts a negative impact mainly on mood , normal work , and sleep among
breast cancer survivors . | [ purpose ] this study attempted to assess the impact of pain on the life of breast cancer
survivors using the brief pain inventory ( bpi ) . [ subjects and methods ] a cross - sectional
study was conducted .
participants comprised 30 women , aged 3080 years , who had received
treatment for breast cancer ( surgery and complementary treatment ) at least 12 months prior
to the study and had reported chronic pain related to the treatment procedures .
[ results ]
the highest scores were found for mood ( median : 5.00 points ; first quartile : 1.00
points ; third quartile : 7.25 points ) , normal work
( median : 5.00 points ; first quartile :
0.00 points ; third quartile : 8.00 points ) , and sleep
( median : 4.50 points , first
quartile : 0.00 points , third quartile : 8.00 points ) . [ conclusion ] pain exerts a negative
impact primarily on mood , normal work , and sleep among breast cancer survivors . |
alzheimer 's disease ( ad ) is an irreversible , progressive disorder with loss of cognitive function , particularly memory , judgment , reasoning , and perception .
the disease primarily affects parts of the association cortex and deep structures including the hippocampus and basal frontal nuclei through a number of pathophysiologic processes .
studies on visual perception are valuable for understanding the disease and shedding light on what the ad patients understand .
the commonly used neuropsychological tests for dementia , such as the mini - mental state examination ( mmse ) or the montreal cognitive assessment , do not evaluate visual - perceptual abilities in context judgment and information processing .
the objective of this study was to evaluate the potential utility of a complex picture test ( cpt ) to assess the existence of the associations between mmse and cpt and the degree of the decline in dementia .
the cpt we have developed involves images presented to the patients with a paradox they had not previously experienced .
this presents challenges to the capacity of the subjects to interpret a novel or a complex situation and explain it verbally .
the current study is part of a large population - based study , which was designed to assess the prevalence of different forms of dementia in a given population .
the participants of the study were randomly selected from the population - based sample within the lublin region of poland composed of 2,182,191 inhabitants in the year 2005 when the population was sampled . as a result of the project ,
patients with a previously established diagnosis of mild cognitive impairment ( mci ) or different forms of dementia took part in the study .
they were examined in the department of neurodegenerative diseases , institute of agricultural medicine , lublin , poland .
the study group was comprised of 14 patients with vascular dementia ( vad ) , 30 with ad , 6 patients with dementia in parkinson 's disease ( pdd ) , and 12 patients with mci .
the diagnoses were consistent with the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) criteria and national institute of neurologic and communicative disorders and stroke - ad and related disorders association ( nincds - adrda ) criteria for possible or probable ad .
the modified hachinski ischemic score was determined ; all the patients with vad had a composite score of 5 or more and a neuroimaging study consistent with vad .
ct or mri scans were obtained routinely for all patients and were consistent with the diagnoses .
patients included into the study had to be otherwise healthy and ambulatory , with vision and hearing sufficient for compliance with testing procedures .
laboratory test values had to be within normal limits or considered to be clinically insignificant by the physician .
patients were excluded if they had evidence of clinically significant gastrointestinal , renal , hepatic , endocrine , or other systemic disease , epilepsy , primary psychiatric diseases ( notably dsm - iv - defined depression ) , traumatic brain injury , newly treated hypothyroidism , or a known or suspected history ( within the past 10 years ) of alcoholism or drug abuse .
additional reasons for exclusion included evidence of neoplasm , insulin - dependent diabetes or diabetes not stabilized by diet or oral hypoglycemic agents , obstructive pulmonary disease or asthma , recent ( < 2 years ) hematologic / oncologic disorders , pernicious anemia , or vitamin b12 or folate deficiency .
the control group consisted of 30 healthy subjects whose scores on the mmse were in the range of 2830 .
controls were matched by age and gender and were subject to the same exclusions as the case group ( tables 1 , 2 ) .
bercal was carried out in accordance with the institute of agricultural medicine bioethical commission ( irb ) and after the protocol was evaluated and approved .
after complete description of the study protocol , written informed consent was obtained from the patients ( if possible ) , the caregivers , or the patient 's representative ( if applicable ) before beginning detailed screening activities . a test composed of 10 complex pictures
the pictures used in the study were of varying complexity , with each consisting of two prominent characteristics that contradicted each other ( table 3 ) .
all patients were able to see clearly , with vision from 20/20 to 20/40 with or without correction , and took the test with their best corrected vision in a well - lit room .
each picture was shown to the patients who were asked to describe it and to comment on the picture .
the picture was given to the patients , and they were able to hold it to view it in whatever position they preferred .
points were scored for naming the object in the picture , noticing the paradox , and commenting on the paradox appropriately after seeing the picture . naming the object in the picture and noticing
the paradox reflected our test points and were scored visual - spatial abilities , and commenting on the paradox appropriately reflected insight and judgment .
thus , the subjects could score 3 points for each picture , with a maximum of 30 points altogether .
the patients also received the mmse and the clock drawing test ( cdt ) following the procedure suggested by shah and the scoring system developed by manos as they combine simplicity , quantitative scoring , and further qualitative information relevant for clinical diagnosis . each patient
was first given a sheet of paper with a pre - drawn circle signifying the clock face and asked to complete it with appropriate numbers and clock hands indicating 11:10 ( the free drawing version ) .
then , the patient was given a complete , pre - drawn picture of the clock and instructed to copy it ( the copying version ) .
the free drawing version is assumed to examine language functions ( verbal comprehension of the command ) , memory functions ( recalling visual engrams stored in the long - term memory and recalling the instruction on the time on which the clock hands were to be set / stored in the short - term memory ) , and executive functions ( planning , sequencing , organizing ) , which generally correspond to the temporal and frontal lobe activities . the copying version is less dependent on language and memory function as it activates mainly perceptual and visual - spatial abilities associated with the parietal lobe control .
the scoring system involved the evaluation of the spatial correctness of the numbers written in the clock face and the appropriateness of the clock hands showing the indicated time .
one point was given for any of the numbers 1 , 2 , 4 , 5 , 7 , 8 , 10 , and 11 if they were placed in the correct area of the clock face .
one point was also given for the short hand indicating number 11 , and for the long hand indicating number 2 .
the possible score range is from 0 to 10 , with higher scores indicating better performance .
scores lower than 8 indicate an increased likelihood of intellectual impairment , and scores lower than 5 evidence severe intellectual impairment . additionally , the wais - r digit span subtest , which examines attention and short term memory ( the scores range from 08 ) , was implemented .
patients were asked to remember a series of 8 digits and repeat them to the examiner .
the associations between the scores on the cpt and the scores on the other tests were calculated as spearman 's rho correlation coefficients .
the results of the statistical tests were considered significant at a level of p < 0.05 .
the current study is part of a large population - based study , which was designed to assess the prevalence of different forms of dementia in a given population .
the participants of the study were randomly selected from the population - based sample within the lublin region of poland composed of 2,182,191 inhabitants in the year 2005 when the population was sampled . as a result of the project ,
patients with a previously established diagnosis of mild cognitive impairment ( mci ) or different forms of dementia took part in the study .
they were examined in the department of neurodegenerative diseases , institute of agricultural medicine , lublin , poland .
the study group was comprised of 14 patients with vascular dementia ( vad ) , 30 with ad , 6 patients with dementia in parkinson 's disease ( pdd ) , and 12 patients with mci .
the diagnoses were consistent with the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) criteria and national institute of neurologic and communicative disorders and stroke - ad and related disorders association ( nincds - adrda ) criteria for possible or probable ad .
the modified hachinski ischemic score was determined ; all the patients with vad had a composite score of 5 or more and a neuroimaging study consistent with vad .
ct or mri scans were obtained routinely for all patients and were consistent with the diagnoses .
patients included into the study had to be otherwise healthy and ambulatory , with vision and hearing sufficient for compliance with testing procedures .
laboratory test values had to be within normal limits or considered to be clinically insignificant by the physician .
patients were excluded if they had evidence of clinically significant gastrointestinal , renal , hepatic , endocrine , or other systemic disease , epilepsy , primary psychiatric diseases ( notably dsm - iv - defined depression ) , traumatic brain injury , newly treated hypothyroidism , or a known or suspected history ( within the past 10 years ) of alcoholism or drug abuse .
additional reasons for exclusion included evidence of neoplasm , insulin - dependent diabetes or diabetes not stabilized by diet or oral hypoglycemic agents , obstructive pulmonary disease or asthma , recent ( < 2 years ) hematologic / oncologic disorders , pernicious anemia , or vitamin b12 or folate deficiency .
the control group consisted of 30 healthy subjects whose scores on the mmse were in the range of 2830 .
controls were matched by age and gender and were subject to the same exclusions as the case group ( tables 1 , 2 ) .
bercal was carried out in accordance with the institute of agricultural medicine bioethical commission ( irb ) and after the protocol was evaluated and approved .
after complete description of the study protocol , written informed consent was obtained from the patients ( if possible ) , the caregivers , or the patient 's representative ( if applicable ) before beginning detailed screening activities .
the pictures used in the study were of varying complexity , with each consisting of two prominent characteristics that contradicted each other ( table 3 ) .
all patients were able to see clearly , with vision from 20/20 to 20/40 with or without correction , and took the test with their best corrected vision in a well - lit room .
each picture was shown to the patients who were asked to describe it and to comment on the picture .
the picture was given to the patients , and they were able to hold it to view it in whatever position they preferred .
points were scored for naming the object in the picture , noticing the paradox , and commenting on the paradox appropriately after seeing the picture . naming the object in the picture and noticing
the paradox reflected our test points and were scored visual - spatial abilities , and commenting on the paradox appropriately reflected insight and judgment .
thus , the subjects could score 3 points for each picture , with a maximum of 30 points altogether .
the patients also received the mmse and the clock drawing test ( cdt ) following the procedure suggested by shah and the scoring system developed by manos as they combine simplicity , quantitative scoring , and further qualitative information relevant for clinical diagnosis .
each patient was first given a sheet of paper with a pre - drawn circle signifying the clock face and asked to complete it with appropriate numbers and clock hands indicating 11:10 ( the free drawing version ) .
then , the patient was given a complete , pre - drawn picture of the clock and instructed to copy it ( the copying version ) .
the free drawing version is assumed to examine language functions ( verbal comprehension of the command ) , memory functions ( recalling visual engrams stored in the long - term memory and recalling the instruction on the time on which the clock hands were to be set / stored in the short - term memory ) , and executive functions ( planning , sequencing , organizing ) , which generally correspond to the temporal and frontal lobe activities .
the copying version is less dependent on language and memory function as it activates mainly perceptual and visual - spatial abilities associated with the parietal lobe control .
the scoring system involved the evaluation of the spatial correctness of the numbers written in the clock face and the appropriateness of the clock hands showing the indicated time .
one point was given for any of the numbers 1 , 2 , 4 , 5 , 7 , 8 , 10 , and 11 if they were placed in the correct area of the clock face .
one point was also given for the short hand indicating number 11 , and for the long hand indicating number 2 .
the possible score range is from 0 to 10 , with higher scores indicating better performance .
scores lower than 8 indicate an increased likelihood of intellectual impairment , and scores lower than 5 evidence severe intellectual impairment .
additionally , the wais - r digit span subtest , which examines attention and short term memory ( the scores range from 08 ) , was implemented .
patients were asked to remember a series of 8 digits and repeat them to the examiner .
the associations between the scores on the cpt and the scores on the other tests were calculated as spearman 's rho correlation coefficients .
the results of the statistical tests were considered significant at a level of p < 0.05 .
the cpt scores were significantly higher in the control group ( 29.5 1.4 ) than in the ad , vad , mci , and pdd groups ( 13.1 2.3 , 16.9 3.5 , 23.2 1.4 , and 23.6 3.4 , respectively ; p < 0.05 ) ( table 4 ) .
significant positive correlations between mmse , digit span , and cdt scores and cpt scores were observed ( rho 0.76 , 0.35 , and 0.56 , respectively ) .
this study proposes a new measure for the assessment of visual - perceptual deficits in ad patients .
the cpt significantly correlates with the mmse , the most widely used measurement for the assessment of the severity in ad . based on clinical experience
, it can be generalized that the greater the severity of ad , the deeper the impairment on this visual perception measure .
the results of the analysis of variance indicate that particular items on this new scale significantly differentiate between ad patients and controls .
there are many reasons why visual perceptions of complex pictures are compromised in ad patients .
turner 's study suggests that contrast sensitivity plays a significant role in the ability of ad patients to see pictures clearly .
even though all the subjects were tested for visual acuity , such testing is performed under high - contrast settings and thus does not take into account differences in contrast sensitivity .
it has been reported that increasing the stimulus contrast significantly increases cognitive performance in ad patients .
visual perception of complex pictures is compromised in ad because of the impaired function of the parietal - occipital cortex .
furthermore , visual - special recognition impairment in patients with ad usually accompanies but also impairment of judgment and information processing .
this set of dysfunctional abilities may influence many different aspects of everyday life and may create danger in certain situation such as , for example , driving and operating complicated tools or machinery .
the most common example of a potentially dangerous situation where the person relies on both the visual - spatial component of cognition and clear judgment would be driving .
there are data showing that driving crashes in patients with ad should be recognized as an important public health problem .
bringing in mind the above - mentioned data , it seems not surprising that the risk of car accidents is higher in patients with ad .
the risk is attributable not only to visual impairment but also to slowed mentation and attention .
since these attention mechanisms are impaired in ad , the ad patient 's ability to interpret a picture with two distracting features is compromised .
patients who can not properly scan the visual field and perceive a number made up by smaller parts ( table 3 ) may be unfit for driving as driving requires a large effective field of vision .
the tool which will closely address impaired visual functioning , judgment , and information processing may be potentially used to assess the ability of a patient to be involved in activities of a certain level of exposure to danger like the one involved in driving .
although the cpt has not been correlated with driving ability or with accidents , it may , however , be considered as a candidate for a tool in the assessment of safe driving abilities . another neurologic defect in ad that could impair
neurodegeneration of the retina itself is also found in ad , with structural damage of the optic nerve head and death of retinal ganglion cells . the nerve fiber layer in the retina of ad patients
the strong correlation between the cpt scores and mmse scores suggests that tests of visual perception could be used as a brief screening tool for dementia .
the mental status examination is often a stressful experience for the patient and family , and the cpt is valuable for providing temporary relief .
the images are shared with the family after they are seen by the patient , and the humor in many images ( such as the tiger rabbit , table 3 ) often lightens the seriousness of the moment .
it is also valuable for the family to see the extent of the patient 's impairment as denial of illness is often shared by the patients and their families and can interfere with compliance with advice concerning care and hazardous behaviors .
the aim of our study was to turn clinical , observation based phenomenon which is impaired complex pictures understanding and interpretation in demented patients to more structured , valid test .
the proposed tool requires , however , more extensive study in terms of population tested , statistics implemented , and comparison with existing screening as well as diagnostic tools .
we plan to examine a larger sample of people with dementia using the cpt tool .
| the newly developed complex picture test ( cpt ) examines main cognitive domains , especially perceptual and visual - spatial abilities .
the potential usefulness of the cpt in the assessment of dementia was evaluated .
patients with different forms of dementia [ 14 vascular dementia ( vad ) , 30 alzheimer 's disease ( ad ) , 6 parkinson 's disease dementia ( pdd ) ] , 12 subjects with mild cognitive impairment ( mci ) , and 30 matched controls were examined by the cpt , mini - mental state examination ( mmse ) , the wais - r digit span subtest , and the clock drawing test ( cdt ) .
the cpt consists of 10 complex pictures , each presenting characteristics paradoxically in opposition to each other .
the subjects scored points for naming the object and noticing the paradox .
one point was added for commenting on the paradox without prompting ( 3 points each picture / maximum of 30 ) .
cpt scores were significantly higher in the control group ( mean sd , 29.46 1.43 ) than in the ad , vad , mci , and pdd groups ( 13.1 2.3 , 16.9 3.5 , 23.2 1.4 , and 23.6 3.4 , respectively ; p < 0.05 ) .
significant positive correlations between mmse , digit span , cdt , and cpt scores were observed ( rho 0.76 , 0.35 , and 0.56 , respectively ) .
these data show perception of complex pictures being compromised in dementia .
the correlation between cpt scores and mmse scores suggests that tests may be used as a brief screening tool for dementia . |
all experiments were conducted in lupiro village in the kilombero valley of rural southern tanzania using commercially available irs formulations of pyrethroids and organophosphates , which were combined with existing binding agent ( ba ) products for extending insecticide durability on llins . in this area of southern tanzania , intense malaria transmission
funestus , which mediates rebounding ( 14 ) malaria transmission in this setting because it is physiologically resistant to pyrethroids , carbamates and organochlorines ( 27 ) .
arabiensis , which mediates resilient residual transmission ( 14 ) because it is physiologically resistant to pyrethroids ( 27 ) and also exhibits early exiting behavior that renders it robust to indoor control with llins and irs ( 18,28,29 ) .
all procedures were approved by the institutional review board of the ifakara health institute ( ihi / irb/342014 ) and the medical research coordination committee of the national institute for medical research ( nimr / hq / r.8a / vol ix/1903 ) .
we used 13 experimental huts of the ifakara design ( 24,29,30 ) and standard methods ( 31 ) to assess effects of llins , irs , and insecticide - treated wsebs .
four of these huts were randomly selected , and their inner wall and roof surfaces were sprayed with 2 g / m of a capsule suspension formulation of pirimiphos - methyl ( actellic 300cs ) by using standard programmatic application procedures ( 32 ) .
another 4 randomly selected huts were sprayed with 30 mg / m of the pyrethroid lambda - cyhalothrin , which was also in a capsule suspension formulation ( icon 10cs ) . both of these long - lasting , microencapsulated , insecticide formulations are manufactured by syngenta ag ( basel , switzerland ) for irs applications and are well characterized ( 3335 ) .
the remaining 5 huts were sprayed only with water to serve as negative controls . after spraying , 2 mattresses and intact permanet llins ( 100-denier polyester multifilament mesh with 156 holes / inch ,
surface - treated with 4555 mg / m of deltamethrin in a resin foundation ; vestergaard , lausanne , switzerland ) were installed in each hut .
eave baffles are incorporated into experimental hut designs to ensure that mosquitoes can enter through approximately half of the eave gaps between the wall and the roof but are then all either retained in the hut or forced into interception traps fitted to the remaining exit points ( 24,25 ) . in a conventional experimental hut study ,
those remaining exit points are windows and the remaining unbaffled half of eave gaps ( 24,25 ) . however , the purpose of this study was to evaluate wsebs as an insecticide delivery format .
therefore , all wseb treatments , except for the negative control , included eave baffles fitted to all eave gaps , with and without exit traps , and identically treated screens fitted over all windows ( table ; figure 1 ) .
treated wsebs were fitted in front of exit traps , which were fitted immediately outside the hut ( 24 ) , so that any mosquito attempting to exit through any eave gap or window would be forced into contact with these insecticidal netting barriers ( figure 1 ) .
the only treatment without screens over the windows or eave baffles over the half of the eave gaps with exit traps immediately outside were the negative control ( table ) .
these controls had untreated eave baffles fitted only to the half of the eave spaces lacking exit traps , thus enabling mosquitoes to enter and exit .
the 2 partial negative controls had screens fitted over the windows and baffles fitted to all eave gaps , regardless of whether they acted as entry or exit points for mosquitoes , but were not treated with any insecticides ( table ) .
one partial negative control was treated with the noninsecticidal ba that syngenta ag includes along with lambda - cyhalothrin ( the same icon 10cs formulation we used for irs ) in their icon maxx product to extend its active life on polyester netting ( 36 ) . the first insecticidal wseb treatment ( table )
was this same long - lasting icon maxx product , this time including both ba and lambda - cyhalothrin ( 36 ) .
although the manufacturer - recommended dose of lambda - cyhalothrin on netting treated with the icon maxx product ( 55 mg / m ) is somewhat higher than that used for irs ( 30 mg / m ) , it is similar to that for deltamethrin on permanet llins used in this study ( 45 - 55 mg / m ) .
wsebs treated with pirimiphos - methyl were assessed at 3 doses that were comparable with typical irs application rates per square meter treated ( table ) .
these 3 pirimiphos - methyl doses were also assessed as a co - treatment with ba to potentially extend insecticide life , with and without lambda - cyhalothrin as a complementary second insecticide from a different chemical class ( table ) .
lambda - cyhalothrin was chosen , despite being a pyrethroid to which both vector species in the study area are resistant ( 27 ) , to assess the potential of such combinations to select for restored pyrethroid susceptibility ( 37 ) .
conceptually , this approach relies on selectively reducing mortality rates for insects that are susceptible to its lethal mode of action and responsive to its irritant / repellent effects on mosquito behavior ( 37 ) .
the mathematical modeling study that motivated assessment of this combination assumed that these 2 pyrethroid susceptibility and responsiveness phenotypes , and presumably their underlying genotypes , are closely associated and therefore co - selected ( 37 ) .
although all exit traps on eaves and windows were made of teflon - coated fiberglass mesh ( 24 ) , all eave baffles and window screens were made of 100-denier polyester netting ( a to z textile mills , arusha , tanzania ) of the kind typically used for bed nets .
all wsebs were treated by soaking in aqueous suspensions of the insecticides , ba , or both and then drying in the shade . to execute the experimental design of this study , duplicate
sets of the 13 detachable , movable wseb treatments ( table ) were rotated nightly through the 13 huts over two 26-day rounds of experimental replication ( technical appendix 1 ) during december 5 , 2015february 1 , 2016 .
each night , 2 men ( volunteers ) slept under the 2 llins inside each hut from 7:00 pm to 7:00 am .
these men then collected all mosquitoes inside the hut by using a prokopak aspirator ( john w. hock co. , gainesville , fl , usa ) ( 38 ) and those inside the exit traps by using a mouth aspirator ( 24 ) .
dead mosquitoes were then sorted taxonomically , classified by sex and abdominal status , and counted .
specimens collected alive were maintained in a field insectary for 24 h before separating live and dead specimens for sorting , classification , and counting . a random sample of 242 specimens from the an .
each pair of men remained assigned to a fixed experimental hut throughout the study so that variability associated with these volunteers and the huts could be analyzed as a single , consistent source of variance .
after mosquito collection each morning , each pair of men was responsible only for installing the set of wsebs assigned to their hut that evening and for removing that set from the hut it had been fitted to the previous night .
all volunteers used a fresh pair of gloves each morning and were not allowed to handle any wsebs other than those to be used in their hut that night .
all wseb sets were labeled and stored in labeled buckets during transfer between huts and the 13-day storage period of each 26-day replication cycle ( technical appendix 1 ) .
all field data were collected on hard copies of the adult field collection ( ed1 ) and sample sorting ( ss3 ) forms , recently described for informatically robust collection of entomologic data ( 40 ) . to ensure rigid compliance with the experimental design ,
all attributes defined by it were prefilled into the forms ( technical appendix 1 ) .
all statistical analysis was accomplished by using generalized linear mixed models with a binomial distribution and logit link function for the binary mosquito death outcome and fitted by using r version 3.2.1 ( https://www.r-project.org/ ) .
wseb treatments were included as categorical independent variables , and hut and night were included as random effects .
a total of 1,318 specimens from the an . funestus group and 5,842 from the an .
all of the 176 specimens that were successfully amplified ( 73% of the 242 specimens from this complex ) were identified as an .
all wesbs , other than the negative control , clearly retained mosquitoes within the huts , because this is where most ( > 90% ) were collected , rather than in exit traps . when used alone , most ( 8/10 )
funestus mosquitoes as did irs alone with the same insecticide formulations ( figure 2 , panel a ) .
mortality rates for lambda - cyhalothrin plus ba - treated wsebs alone were indistinguishable from those for lambda - cyhalothrin irs ( p = 0.363 ) .
the only exceptions among the 10 wseb treatments were the highest pirimiphos - methyl dose plus ba and the intermediate pirimiphos - methyl dose plus lambda - cyhalothrin and ba .
effect of window screens and eave baffles treated with 13 combinations of insecticides and binding agents on malaria vector mosquito mortality rates inside experimental huts , tanzania .
huts were previously sprayed with 1 of 3 alternative indoor residual spraying regimens ( technical appendix 1 ) and occupied by 2 volunteers sleeping under pyrethroid - treated , long - lasting insecticidal nets .
estimated mean mortality rates and 95% cis , as well as statistical contrasts between the most relevant treatment pairs , are indicated in technical appendix 2 ) .
funestus mosquitoes than irs with lambda - cyhalothrin alone ; a similar but nonsignificant pattern was observed for comparisons of the same wseb treatments alone with pirimiphos - methyl irs alone ( figure 2 ; technical appendix 2 ) . nonetheless , mortality rates for pirimiphos - methyl treated wsebs alone were consistently high ( figure 2 , panel a ) , regardless of treatment dose ( p>0.156 ) , and were statistically indistinguishable from pirimiphos - methyl irs alone ( p>0.713 ) , even though the lowest wseb dose per unit area treated was only half that for irs .
although all combinations of pirimiphos - methyl treated wsebs with pirimiphos - methyl irs resulted in higher mortality rates than pirimiphos - methyl irs alone or pirimiphos - methyl treated wsebs alone , none of these differences were significant ( p>0.080 ) because too few mosquitoes survived either treated wsebs alone or irs alone .
overall , insecticide - treated wsebs either matched or were superior to irs when used against an .
wsebs treated with lambda - cyhalothrin plus ba showed similar mortality rates as irs with the same lambda - cyhalothrin formulation ( p = 0.345 ) .
wsebs treated with the lowest dose of pirimiphos - methyl showed similar mortality rates for an .
arabiensis mosquitoes as irs with twice as much pirimiphos - methyl per square meter treated ( p = 0.419 ) . however , increasing the pirimiphos - methyl treatment dose from 1 to 2 or 4 g / m increased the mortality rate for wsebs ( odds ratio [ or ] 2.10 , 95% ci 1.163.79 , p = 0.0139 ; and 2.34 , 95% ci 1.284.26 , p = 0.0055 , respectively ) , although there was no difference between intermediate and high doses ( p = 0.758 ) .
arabiensis mosquitoes ( or 5.9 , 95% ci 1.4 24.3 , p = 0.0145 ; 10.8 , 95% ci 1.674.8 , p = 0.0157 , respectively ) than irs , even though the intermediate pirimiphos - methyl dose was the same as for irs per square meter treated .
arabiensis mosquito mortality rates for the lowest wseb dose ( or 4.8 , 95% ci 1.515.5 , p = 0.0081 ) , which was half that of irs per unit area treated
. however , supplementary pirimiphos - methyl irs did not increase mortality rates when wsebs were treated with the same dose as irs ( p = 0.748 ) or twice that dose ( p = 0.429 ) . adding ba had no effect on the mortality rates for pirimiphos - methyl treated wsebs for an .
arabiensis ( p = 0.424 ) mosquitoes . supplementing organophosphate pirimiphos - methyl plus ba treatment with the irritant pyrethroid lambda - cyhalothrin as a second active ingredient reduced an .
funestus mosquito mortality rates for wsebs ( or 0.64 , 95% ci 0.460.89 , p = 0.0076 ) , presumably because the irritant properties of lambda - cyhalothrin reduced mosquito contact times with co - treated wsebs , and therefore exposure to both insecticides .
when used alone , most ( 8/10 ) wseb treatments that included insecticides killed similarly high proportions of an .
funestus mosquitoes as did irs alone with the same insecticide formulations ( figure 2 , panel a ) .
mortality rates for lambda - cyhalothrin plus ba - treated wsebs alone were indistinguishable from those for lambda - cyhalothrin irs ( p = 0.363 ) .
the only exceptions among the 10 wseb treatments were the highest pirimiphos - methyl dose plus ba and the intermediate pirimiphos - methyl dose plus lambda - cyhalothrin and ba .
effect of window screens and eave baffles treated with 13 combinations of insecticides and binding agents on malaria vector mosquito mortality rates inside experimental huts , tanzania .
huts were previously sprayed with 1 of 3 alternative indoor residual spraying regimens ( technical appendix 1 ) and occupied by 2 volunteers sleeping under pyrethroid - treated , long - lasting insecticidal nets .
mortality rates and 95% cis , as well as statistical contrasts between the most relevant treatment pairs , are indicated in technical appendix 2 ) .
funestus mosquitoes than irs with lambda - cyhalothrin alone ; a similar but nonsignificant pattern was observed for comparisons of the same wseb treatments alone with pirimiphos - methyl irs alone ( figure 2 ; technical appendix 2 ) . nonetheless , mortality rates for pirimiphos - methyl treated wsebs alone were consistently high ( figure 2 , panel a ) , regardless of treatment dose ( p>0.156 ) , and were statistically indistinguishable from pirimiphos - methyl irs alone ( p>0.713 ) , even though the lowest wseb dose per unit area treated was only half that for irs .
although all combinations of pirimiphos - methyl treated wsebs with pirimiphos - methyl irs resulted in higher mortality rates than pirimiphos - methyl irs alone or pirimiphos - methyl treated wsebs alone , none of these differences were significant ( p>0.080 ) because too few mosquitoes survived either treated wsebs alone or irs alone .
overall , insecticide - treated wsebs either matched or were superior to irs when used against an .
wsebs treated with lambda - cyhalothrin plus ba showed similar mortality rates as irs with the same lambda - cyhalothrin formulation ( p = 0.345 ) .
wsebs treated with the lowest dose of pirimiphos - methyl showed similar mortality rates for an .
arabiensis mosquitoes as irs with twice as much pirimiphos - methyl per square meter treated ( p = 0.419 ) . however , increasing the pirimiphos - methyl treatment dose from 1 to 2 or 4 g / m increased the mortality rate for wsebs ( odds ratio [ or ] 2.10 , 95% ci 1.163.79 , p = 0.0139 ; and 2.34 , 95% ci 1.284.26 , p = 0.0055 , respectively ) , although there was no difference between intermediate and high doses ( p = 0.758 ) .
arabiensis mosquitoes ( or 5.9 , 95% ci 1.4 24.3 , p = 0.0145 ; 10.8 , 95% ci 1.674.8 , p = 0.0157 , respectively ) than irs , even though the intermediate pirimiphos - methyl dose was the same as for irs per square meter treated . supplementing pirimiphos - methyl
arabiensis mosquito mortality rates for the lowest wseb dose ( or 4.8 , 95% ci 1.515.5 , p = 0.0081 ) , which was half that of irs per unit area treated
. however , supplementary pirimiphos - methyl irs did not increase mortality rates when wsebs were treated with the same dose as irs ( p = 0.748 ) or twice that dose ( p = 0.429 ) .
adding ba had no effect on the mortality rates for pirimiphos - methyl treated wsebs for an .
arabiensis ( p = 0.424 ) mosquitoes . supplementing organophosphate pirimiphos - methyl plus ba treatment with the irritant pyrethroid lambda - cyhalothrin as a second active ingredient reduced an .
funestus mosquito mortality rates for wsebs ( or 0.64 , 95% ci 0.460.89 , p = 0.0076 ) , presumably because the irritant properties of lambda - cyhalothrin reduced mosquito contact times with co - treated wsebs , and therefore exposure to both insecticides .
therefore , the most striking advantage of wsebs is that they reduced the surface area treated per hut by > 5-fold .
furthermore , co - application with existing bas that already extend durability of pyrethroids on llins ( 36 ) for as much as 3 years ( 41 ) suggests new opportunities for reducing reapplication frequency by up to 6-fold , relative to irs .
these wsebs are an experimental prototype that were evaluated in the necessarily homogenous and controlled environment of experimental huts .
this short - term efficacy study did not address key issues regarding potential effectiveness and cost - effectiveness of wsebs under programmatic operational conditions .
it is encouraging that a set of these wsebs for these experimental huts , specifically designed to match the dimensions of local houses ( 24 ) , required only 11 m of netting to manufacture , similar to a typical llin .
however , this netting had to be carefully hand - tailored with hooks and velcro to enable easy daily removal and reinstallation in experimental huts , at a manufacturing labor cost of $ 47 per set .
more practical and affordable formats for operational use in a diversity of house designs must be developed and rigorously evaluated before wsebs could be considered for routine , programmatic deployment by national programs .
nevertheless , the potential of this approach merits consideration , even if only speculatively at this early stage .
it takes almost an entire 833-ml bottle of the 0.3 g / ml pirimiphos - methyl formulation used here , costing $24 , to treat 1 typical rural house in tanzania twice a year with irs at the ideal recommended dose of 2 g / m . in comparison , a house of equivalent size with wsebs installed could be treated with the same insecticide at the same dose per square meter of treated netting for only $ 2.15 .
although greater quantities of ba might be required than applied here ( 42 ) , it could extend the life of pirimiphos - methyl on netting to the same extent as for lambda - cyhalothrin on llins that are approved for 3 years of use .
if ba - treated wsebs were similarly durable , they could provide up to 3 years of protection for only $ 0.72 per year in recurrent insecticide procurement costs . because scale - up nationally in tanzania would cost only $ 4.8 million for insecticide procurement
, a combination of 3 similarly expensive complementary insecticides would be affordable to the national program at a cost of < $ 15 million annually .
corresponding global costs would be < $ 1.2 billion annually for such a triple combination .
changing deployment format for existing irs formulations could also eliminate the need to apply them in potentially hazardous aerosol form .
although handling insecticides is always associated with some risks , and protective clothing , eyewear , and a breathing apparatus might be required , wsebs may be impregnated by simply dipping them in an aqueous suspension , similarly to bed nets .
therefore , wseb deployment formats might enable national programs to develop and manage their vector control platforms more flexibly than when using irs .
although these insecticide cost estimates are entirely speculative , assume that ba will be equally efficacious for extending longevity of pirimiphos - methyl , and do not consider costs of netting installation or maintenance , they outline the potential economic benefits that could be accrued by optimizing wseb deployment formats , netting materials , and treatment formulations .
in addition , such reduced insecticide requirements might make rational resistance management ( 8) feasible and affordable with existing budgets and off - the - shelf insecticide products .
the observation that supplementing pirimiphos - methyl treated wsebs with the irritant pyrethroid lambda - cyhalothrin reduced mortality rates for an . funestus mosquitoes , which were strongly resistant to pyrethroids but not organophosphates ( 27 ) , suggests that wsebs could be used in an affordable format with which to field - test the theory that such combinations might select for restored pyrethroid susceptibility ( 37 ) .
the underlying assumption of this hypothesis is that physiologic susceptibility and behavioral responsiveness to pyrethroids are genetically linked , so that insecticide combinations , such as the lc - pm mixture used here , would selectively kill insects that are both resistant and non - responsive to pyrethroids .
the case for assuming that physiologic susceptibility and behavioral responsiveness are at least phenotypically associated has recently been strengthened by laboratory studies of culex quinquefasciatus mosquitoes , which demonstrated that 4 pyrethroid - resistant field populations were all less responsive to the irritant properties of permethrin than a fully susceptible laboratory colony ( 43 ) .
these empirical studies ( 43 ) also suggest grounds for optimism regarding the recent theory that combining recently developed , low - technology emanators for airborne pyrethroid vapor ( 44,45 ) with complementary nonpyrethroid indoor control measures , such as irs , wsebs , or alternative technologies , such as eave tubes ( 4648 ) and entry traps ( 49 ) , could coselect for evolutionarily stabilized restoration of physiologic susceptibility and behavioral responsiveness to pyrethroids generally ( 50 ) .
also , both mathematical models predicting restoration of these preferred traits ( 37,50 ) , by definition , merely illustrate the plausibility of these hypotheses in mathematically explicit terms .
alternatively , selection for physiologic resistance to both insecticides might be exacerbated by reducing contact exposure to sublethal levels .
although potential benefits and risks of combining irritant pyrethroids with nonirritant insecticides from complementary classes remain to be satisfactorily assessed , our results suggest that wsebs might be a potentially scalable delivery format with which to test these hypotheses empirically through large - scale field studies .
twenty - six day schedule applied to complete 1 full replicate of evaluation for duplicates of the 13 treatments of window screens and eave baffles by rotating them through all of the 13 presprayed experimental huts for study of control of malaria vector mosquitoes by insecticide - treated combinations of window screens and eave baffles , tanzania .
estimated mortality rates of both malaria vector species in houses with each of the 39 combinations of treatments for indoor residual spraying and window screens and eave baffles , as well all practically relevant statistical contrasts between them , tanzania . | we assessed window screens and eave baffles ( wsebs ) , which enable mosquitoes to enter but not exit houses , as an alternative to indoor residual spraying ( irs ) for malaria vector control .
wsebs treated with water , the pyrethroid lambda - cyhalothrin , or the organophosphate pirimiphos - methyl , with and without a binding agent for increasing insecticide persistence on netting , were compared with irs in experimental huts . compared with irs containing the same insecticide ,
wsebs killed similar proportions of anopheles funestus mosquitoes that were resistant to pyrethroids , carbamates and organochlorines and greater proportions of pyrethroid - resistant , early exiting an .
arabiensis mosquitoes .
wsebs with pirimiphos - methyl killed greater proportions of both vectors than lambda - cyhalothrin or lambda - cyhalothrin plus pirimiphos - methyl and were equally efficacious when combined with binding agent .
wsebs required far less insecticide than irs , and binding agents might enhance durability .
wsebs might enable affordable deployment of insecticide combinations to mitigate against physiologic insecticide resistance and improve control of behaviorally resistant , early exiting vectors . |
the data were collected as part of nationwide screening programs in 3 consecutive years in 2007 , 2008 and 2009 .
this program is regularly performed by the ministry of health and medical education and the ministry of education and training among all children entering elementary schools .
elementary education is mandatory in iran , thus the study population comprised all children entering public and private elementary schools .
the national data and safety monitoring board closely supervised the quality control and quality assurance of each survey .
training sessions were organized for health care providers who measured children 's weight and height according to standard protocols by using calibrated instruments.15 body mass index ( bmi ) was computed as weight in kilograms divided by the square of height in meters . in all surveys , we used the growth charts of the centers for disease control and prevention,16 which are in close agreement with iranian charts.17 overweight and obesity were considered as age- and gender specific bmi of 85 - 94 and 95 percentile curves , respectively.16 because of significant differences in the prevalence of overweight and obesity among adults in the northern , central and southern regions of iran , with similarities in the geographic and socio - demographic characteristics in each region,18 in this study we compared the corresponding figures for children .
after editing , the data were analyzed using the statistical package for social sciences statistical software package version 16:0 ( spss inc . ,
all analyses were initially stratified by gender , but as the differences were not significant , the results are presented for girls and boys in combination .
data were obtained from all 6-year - old children entering elementary schools in all 30 provinces in iran .
the study population comprised 2,600,065 children including 862,433 in 2007 , 782,244 in 2008 and 955,388 in 2009 .
of total children , 12.8% , 13.5% and 10.9% were overweight in 2007 , 2008 and 2009 , respectively ( p > 0.05 ) .
the corresponding figures for obesity were 3.4% , 3.5% and 3.4% , respectively ( p > 0.05 ) .
figure 1 presents the prevalence and trend of overweight and obesity in the three regions of the country . in all surveys ,
the prevalence of overweight was higher in southern region than in the other two regions .
p for trend was not significant for prevalence rates of overweight and obesity in none of the regions .
secular trends in prevalence of overweight and obesity in 6-year - old iranian children during 2007 - 2009 in a. northern , b. central and c. southern regions .
this study , presenting the national trend of about 2,600,000 children over three years revealed no significant difference of prevalence of overweight and obesity over a 3-year period ( 2007 - 2009 ) .
various health policies have been implemented to improve the nutritional status of iranian children from early life .
the focus of most of them has been to control underweight and micronutrient deficiency . as recent national studies showed considerably high prevalence of overweight and obesity in iranian adults1819 and school - aged children,20 in addition to the aforementioned policies , primordial and primary prevention of childhood overweight
have been considered in the program of health professionals , notably pediatricians , general physicians and child health care providers . moreover , in order to increase the public awareness about the importance of prevention and control of overweight in children , mass media programs were used as a tool to advocate for children 's healthy lifestyle and more specifically preventing child overweight .
given that small changes at the individual level may result in large benefits at the population level , such policies might have been effective , so that the childhood overweight and obesity rates have remained constant for at least three years .
however , as some regional studies revealed increasing trend of overweight and obesity in adults21 and school - aged children,22 more intensive interventions and legislations are necessary to tackle obesity in older ages . at a very young age ,
children 's lifestyle is more under control of parents and they can guide their children in making choices that will lead to healthier lives . by getting older ,
children may feel they have more power over their choice of foods and activity , and may adopt obesogenic lifestyle behaviors .
it is noteworthy to mention that in all three surveys , children living in the southern region had the highest prevalence of overweight .
this higher prevalence might be because of differences in socioeconomic background of this region , as well as ethnic differences .
in general , the southern provinces in iran have lower socioeconomic position than other provinces .
some studies in developing countries reported higher rates of childhood obesity in higher socioeconomic populations,12 whereas others reported it in lower socioeconomic class.2325 an increasing body of evidence showed that over the past years , a secular trend toward increase in obesity is more marked among children of lower than higher socioeconomic status , which might be because low - income households can affect a child 's tendency to gain weight by higher intake of energy - dense foods , lower intake of complex carbohydrate , fruits and vegetables.2629 moreover , regarding the ethnic differences in the prevalence and complications of childhood obesity,3031 and considering the popularity of arab ethnicity in southern iran , part of this difference might be explained by ethnicity .
future studies about lifestyle behaviors in this region might help to design more effective preventive and interventional programs .
in other viewpoint , although there was no significant increase in the prevalence of overweight and obesity in children at school entry , but in general , the prevalence of overweight is high at a level comparable to developed countries . because of the very large sample size of the study in 3 consecutive years , details on socio - economic background and lifestyle habits of the children under study could not be documented .
the main strengths of the study are its novelty in reporting national trends of childhood overweight from a developing country , and the nationwide coverage of all schoolentry children .
because of the very large sample size of the study in 3 consecutive years , details on socio - economic background and lifestyle habits of the children under study could not be documented .
the main strengths of the study are its novelty in reporting national trends of childhood overweight from a developing country , and the nationwide coverage of all schoolentry children .
because of the very large sample size of the study in 3 consecutive years , details on socio - economic background and lifestyle habits of the children under study could not be documented .
the main strengths of the study are its novelty in reporting national trends of childhood overweight from a developing country , and the nationwide coverage of all schoolentry children .
to the best of our knowledge , this study is the first of its kind in reporting the nationwide trend of overweight and obesity among young children living in a developing country .
it revealed a considerably high prevalence of overweight and obesity among young iranian children , but with a constant rate in three years .
the higher prevalence of overweight in southern region than in central and northern regions in all three surveys might be related to the lower socioeconomic position of this region . at a very young age ,
children 's lifestyle is more under control of parents and they can guide their children in making choices that will lead to healthier lives , primordial and primary prevention efforts against the overweight epidemic can be effective and shall be further strengthened .
| background : this study aimed to determine the secular trends in the national prevalence of overweight and obesity among 6-year - old iranian children , and to compare the results in northern , central and southern parts of the country.methods:the data were collected as part of a routine and mandatory national screening program on children entering elementary schools in 2007 , 2008 and 2009.results:the study population comprised 2,600,065 children including 862,433 in 2007 , 782,244 in 2008 and 955,388 in 2009 .
of total children 12.8% , 13.5% and 10.9% were overweight in 2007 , 2008 and 2009 , respectively ( p > 0.05 ) .
the corresponding figures for obesity were 3.4% , 3.5% and 3.4% , respectively ( p > 0.05 ) . in all surveys ,
the prevalence of overweight was higher in southern region than in the other two regions .
p for trend was not significant for prevalence rates of overweight and obesity in any of the regions.conclusions:to the best of our knowledge , this study was the first of its kind in presenting the nationwide trend of overweight and obesity in young children living in a developing country .
it showed a considerably high prevalence of overweight and obesity , but with a constant rate in three years .
the higher prevalence of overweight in southern region than in central and northern regions might be related to the lower socioeconomic position of this population . at a very young age ,
children 's lifestyle is more under control of parents .
primordial and primary prevention efforts against the overweight epidemic can be effective and shall be further strengthened . |
zoonotic visceral leishmaniasis ( zvl ) is a potentially fatal disease in human , primarily in children .
it is caused by leishmania infantum in which dogs , foxes , and jackals are the main reservoir hosts .
four species of the subgenus larrousius and one species of subgenus paraphlebotomus are considered as probable vector species in iran ( yaghoobi - ershadi 2012 ) .
the disease is endemic in many rural communities of 7 out of 31 provinces and also sporadically reported from other regions of the country ( yaghoobi - ershadi 2012 , zahraei - ramazani et al .
1993 ) in which some are suspected or proven vectors of zvl . in the old world
these include : phlebotomus longiductus in china , kazakhstan , kyrgyzstan , ukraine , and uzbekistan .
p. halepensis ranges in syrian arab republic , azerbaijan and georgia ( who 2010 ) .
it should be noted that p. ( adlerius ) arabicus has also reported as the vector of l. tropica in palestinian territories ( svobodova et al .
the subgenus adlerius is almost entirely palaearctic ( seyedi - rashti and nadim 1992 ) in various eurasian countries and is thought to have originated from central asia where biodiversity is most abundant ( lger and pesson 1987 ) .
females from about 20 species of the subgenus adlerius can transmit leishmania major ( sadlova et al . 2003 , volf and myskova 2007 ) , l. tropica ( jacobson et al . 2003 , sadlova et al . 2003 , svobodova et al .
2006a , volf and myskova 2007 ) , l. donovani ( zhang and leng 1997 , who 2010 ) and l. infantum ( killick - kendrick 1999 , volf and myskova 2007 , giorgobiani et al . 2012 ) .
there is no published morphological key for species identification that are generally recognized on the basis of male morphology ( lewis 1982 , seyedi - rashti and nadim 1992 ) .
these sand flies are found in diverse natural microhabitats , especially in mountainous areas , such as : cracks , crevices and around the nests and gatherings of the visceral leishmaniasis reservoirs ( artemiev 1973 ) .
because of the short sunny season in the mountains and highland provinces , the collection time of the sand flies is too short . prior to this study
, the systematic ( morphological and molecular ) and molecular studies of some medically significant arthropods such as aedes , anopheles , culex , mites , and sand flies have been studied by molecular methods . the high sensitivity and accuracy of the molecular methods related to dna in terms of morphological identification of species
these are undetectable such as females of the subgenus adlerius is important ( munsterman and conn 1997 , artemiev 1980 , artemiev and neronov 1984 ) . according to previous studies ,
the studies on genetic variation and molecular systematics of the subgenus adlerius sand flies present evidence of ecological differences between the group .
access to the genetic variation and molecular systematics of the subgenus adlerius , in addition to assisting the systematic delineation and their classification may offer evidence to interpret and explain the ecological differences among the species .
species knowledge , abundance , and distribution of males and females , and changes in their population are useful for all research programs in control of leishmaniasis .
the objective of this study was to determine the morphological and genotypic variations among the species of the subgenus adlerius in their distributional areas of iran .
iran is an arid land of 1.6 million km
, extending north to armenia , azerbaijan , turkmenistan , and the caspian sea , east to afghanistan and pakistan , south to the persian gulf and sea of oman , and bordered by turkey and iraq on the west .
mountains span the nation in the shape of a large v. between these ranges lies a high plateau where flowing water from the mountains disappears into desert sand .
the caspian sea littoral zone comprises the northern slopes of the alborz mountains and the caspian plain .
this is a narrow strip of land , forest covered , with mediterranean climate , with the average temperature ranging between 10 and 35 c and the average relative humidity between 70 and 100% .
the central plateau , situated between the alborz and zagros ranges , are very mountainous in the northwest where the ranges originate and becomes a lower desert in the east .
the climate is dry with average temperature between 0 and 40 c , with hot , dry summers and cold , snow - laden winters .
the persian gulf littoral and the khuzestan plain , to the south of the foothills of the zagros mountains , are characterized as a tropical climate ( fig .
was conducted from 40 north to 29 south latitude and 44 western to 62 eastern longitude respectively ( statistical culture of iran , 1990 , personal communication ) .
a detailed study of distribution of the subgenus adlerius species was performed in rural areas of 17 of 31 provinces including [ ( 1 ) azarbaijan - e - sharqi , ( 2 ) azarbaijan - e - gharbi , ( 3 ) ardabil , ( 4 ) kordestan , ( 5 ) ghom , ( 6 ) ilam , ( 7 ) lorestan , ( 8) khuzestan , ( 9 ) bushehr , ( 10 ) fars , ( 11 ) sistan va baluchestan , ( 12 ) kerman , ( 13 ) kohkiluyeh va boyer ahmad , ( 14 ) chahar mahal - o - bakhtiari , ( 15 ) esfahan , ( 16 ) markazi , and ( 17 ) khorasan - e - razavi ] ( fig .
the sand flies were collected using 200 sticky paper traps ( consisted of white sheets 1521 cm , coated with castor oil ) placed in different habitats and various biotypes .
this included areas outdoors , such as gardens , mountain caves , animal shelters , wall cracks , burrows , tree holes , under stones , and rocks in 34 villages suitable for sand fly breeding .
these were located in the plains and mountains areas within each province and sampling was conducted twice each year ( june and september ) from 20082010 .
sand flies were preserved in 96 % ethyl alcohol glass containers and were kept at 4 c in the fridge . the number of the trap sample , date , and location was recorded on the glass containers ( moin - vaziri et al .
identification , sand flies were mounted in puri s medium , which was manufactured in our leishmaniasis laboratory in iran and identified after 24 h ( yaghoobi - ershadi and javadian 1997 ) using the keys of theodor and mesghali ( 1964 ) , artemiev ( 1973 ) , and lewis ( 1982 ) .
morphological characters used for male identification of the subgenus adlerius included antennal segments , shape of the antennae and ascoid , number of hairs on coxite , length of style on ventral process , length of filament , length of pump , and sub - terminal tubercle of the aedeagus ( lewis 1982 ) .
the eight organs were measured for our morphometrical study included : ( 1 ) length of the third segment of antennae , ( 2 ) length of style , ( 3 ) length of epipharynx , ( 4 ) length of coxite , ( 5 ) width of style , ( 6 ) length of surstyle , ( 7 ) length of aedeagus and ( 8) number of hairs on coxite .
the morphometric measurements were conducted with an ocular micrometer and olympus microscope ( ch-2 ) ( fig .
organs measured in morphometrical studies of the subgenus adlerius in iran statistical analyses were used to determine if morphometric characters among the mounted specimens in each species were similar with the species found in different geographical areas .
we used spss - version 17.0 , and evaluated for differences with the t - test , anova , and kruskal - wallis test with an a priori level of significance set at p < 0.05 .
if statistical tests were significantly different , the post hoc - bonferroni test was used .
when the result of the kruskal - wallis test was significantly different , the mann - whitney test was used for comparing the results .
sections of the abdomen , wings , and legs of the sand flies used for dna extraction were stored in 1.5 ml sterile microtubes containing 96 % ethanol .
dna from 120 specimens were extracted , of which 50 were used for mtdna - pcr having 16 sequences ( table 1 ) .
the washed , dried body of each sand fly was frozen then defrosted with liquid nitrogen and homogenised and ground with a heavy cylindrical metal and shaking vigorously .
the content was washed with 300 ml lysis buffer [ 10 mm tris , 25 mm edta ( ph= 8) , 1 % sds , 25 mm nacl , 2% triton100 ] and boiled for 10 minutes .
after the ethanol precipitation , dna was dissolved in 15 l distilled water and stored at 20 c .
number of sand flies used in molecular studies a 25 l pcr reaction mixture consisted of 12.5 l premix 2[10 pcr buffer , mgcl , 10 mm dntps and dna taq polymerase ] , 1 l of cb3-pdr forward primer [ 5-ca ( y= t / c ) attcaacc ( w= a / t ) gaatgata-3 ] , 1 l of nin - pdr reverse primer [ 5-ggta ( y=
c / t ) ( w= a / t ) ttg cctc ga ( w = t / a ) ttcg ( t / a ) tatga-3 ] , 5.5 l ddh2o and 5l of sand fly genomic dna were used for amplifying of 550 bp .
the pcr amplification was carried out with the following thermal profile using a gene amp pcr system 2700 thermal cycle ( ab applied biosystems ) : 6 min . for initial denaturation at 95 c , 35 cycles of denaturation at 94 c for 45 sec .
, annealing at 55 c for 1 min . , extension at 72 c for 1 min . in this
study the chromatogram of the dna sequencing samples were edited by using bioedit software . the program clustal w ( thompson et al .
our sequences were compared with the sequences available in genbank by using blast available on , www.ncbi.nlm.gov/. the blast 2 sequences were used to find multiple local alignments and detect the best homologous between the female and the male specimens .
we measured the eight different morphological characters in all specimens of each adlerius species which captured by sticky traps from outdoors of the provinces , and then the similarity scores ( % ) have been identified by sequence comparing alignment pairwise . therefore
, if the more similarity score and less e - value were identified between the pairwise comparison , it showed that they are same species .
iran is an arid land of 1.6 million km
, extending north to armenia , azerbaijan , turkmenistan , and the caspian sea , east to afghanistan and pakistan , south to the persian gulf and sea of oman , and bordered by turkey and iraq on the west .
mountains span the nation in the shape of a large v. between these ranges lies a high plateau where flowing water from the mountains disappears into desert sand .
the caspian sea littoral zone comprises the northern slopes of the alborz mountains and the caspian plain .
this is a narrow strip of land , forest covered , with mediterranean climate , with the average temperature ranging between 10 and 35 c and the average relative humidity between 70 and 100% .
the central plateau , situated between the alborz and zagros ranges , are very mountainous in the northwest where the ranges originate and becomes a lower desert in the east .
the climate is dry with average temperature between 0 and 40 c , with hot , dry summers and cold , snow - laden winters .
the persian gulf littoral and the khuzestan plain , to the south of the foothills of the zagros mountains , are characterized as a tropical climate ( fig .
was conducted from 40 north to 29 south latitude and 44 western to 62 eastern longitude respectively ( statistical culture of iran , 1990 , personal communication ) .
a detailed study of distribution of the subgenus adlerius species was performed in rural areas of 17 of 31 provinces including [ ( 1 ) azarbaijan - e - sharqi , ( 2 ) azarbaijan - e - gharbi , ( 3 ) ardabil , ( 4 ) kordestan , ( 5 ) ghom , ( 6 ) ilam , ( 7 ) lorestan , ( 8) khuzestan , ( 9 ) bushehr , ( 10 ) fars , ( 11 ) sistan va baluchestan , ( 12 ) kerman , ( 13 ) kohkiluyeh va boyer ahmad , ( 14 ) chahar mahal - o - bakhtiari , ( 15 ) esfahan , ( 16 ) markazi , and ( 17 ) khorasan - e - razavi ] ( fig .
the sand flies were collected using 200 sticky paper traps ( consisted of white sheets 1521 cm , coated with castor oil ) placed in different habitats and various biotypes .
this included areas outdoors , such as gardens , mountain caves , animal shelters , wall cracks , burrows , tree holes , under stones , and rocks in 34 villages suitable for sand fly breeding .
these were located in the plains and mountains areas within each province and sampling was conducted twice each year ( june and september ) from 20082010 .
sand flies were preserved in 96 % ethyl alcohol glass containers and were kept at 4 c in the fridge . the number of the trap sample , date , and location was recorded on the glass containers ( moin - vaziri et al .
for species identification , sand flies were mounted in puri s medium , which was manufactured in our leishmaniasis laboratory in iran and identified after 24 h ( yaghoobi - ershadi and javadian 1997 ) using the keys of theodor and mesghali ( 1964 ) , artemiev ( 1973 ) , and lewis ( 1982 ) .
morphological characters used for male identification of the subgenus adlerius included antennal segments , shape of the antennae and ascoid , number of hairs on coxite , length of style on ventral process , length of filament , length of pump , and sub - terminal tubercle of the aedeagus ( lewis 1982 ) .
the eight organs were measured for our morphometrical study included : ( 1 ) length of the third segment of antennae , ( 2 ) length of style , ( 3 ) length of epipharynx , ( 4 ) length of coxite , ( 5 ) width of style , ( 6 ) length of surstyle , ( 7 ) length of aedeagus and ( 8) number of hairs on coxite .
the morphometric measurements were conducted with an ocular micrometer and olympus microscope ( ch-2 ) ( fig .
statistical analyses were used to determine if morphometric characters among the mounted specimens in each species were similar with the species found in different geographical areas .
we used spss - version 17.0 , and evaluated for differences with the t - test , anova , and kruskal - wallis test with an a priori level of significance set at p < 0.05 .
if statistical tests were significantly different , the post hoc - bonferroni test was used .
when the result of the kruskal - wallis test was significantly different , the mann - whitney test was used for comparing the results .
sections of the abdomen , wings , and legs of the sand flies used for dna extraction were stored in 1.5 ml sterile microtubes containing 96 % ethanol .
dna from 120 specimens were extracted , of which 50 were used for mtdna - pcr having 16 sequences ( table 1 ) .
the washed , dried body of each sand fly was frozen then defrosted with liquid nitrogen and homogenised and ground with a heavy cylindrical metal and shaking vigorously .
the content was washed with 300 ml lysis buffer [ 10 mm tris , 25 mm edta ( ph= 8) , 1 % sds , 25 mm nacl , 2% triton100 ] and boiled for 10 minutes .
after the ethanol precipitation , dna was dissolved in 15 l distilled water and stored at 20 c .
number of sand flies used in molecular studies a 25 l pcr reaction mixture consisted of 12.5 l premix 2[10 pcr buffer , mgcl , 10 mm dntps and dna taq polymerase ] , 1 l of cb3-pdr forward primer [ 5-ca ( y= t / c ) attcaacc ( w= a / t ) gaatgata-3 ] , 1 l of nin - pdr reverse primer [ 5-ggta ( y=
c / t ) ( w= a / t ) ttg cctc ga ( w = t / a ) ttcg ( t / a ) tatga-3 ] , 5.5 l ddh2o and 5l of sand fly genomic dna were used for amplifying of 550 bp .
the pcr amplification was carried out with the following thermal profile using a gene amp pcr system 2700 thermal cycle ( ab applied biosystems ) : 6 min . for initial denaturation at 95 c , 35 cycles of denaturation at 94 c for 45 sec . , annealing at 55 c for 1 min . ,
in this study the chromatogram of the dna sequencing samples were edited by using bioedit software .
our sequences were compared with the sequences available in genbank by using blast available on , www.ncbi.nlm.gov/. the blast 2 sequences were used to find multiple local alignments and detect the best homologous between the female and the male specimens .
we measured the eight different morphological characters in all specimens of each adlerius species which captured by sticky traps from outdoors of the provinces , and then the similarity scores ( % ) have been identified by sequence comparing alignment pairwise . therefore ,
if the more similarity score and less e - value were identified between the pairwise comparison , it showed that they are same species .
a total of 9,319 sand flies were collected , of which 3,847 ( 41.3% ) and 5,472 ( 58.7% ) were from the genus phlebotomus and sergentomyia , respectively . from the total number of sand flies of the genus phlebotomus , 167 ( 1.8% )
were from the subgenus adlerius [ 26 ( 15.57% ) were female and 141(84.43% ) were male ] .
of the males , phlebotomus ( adlerius ) brevis 8 ( 5.67% ) , p. ( adl . ) halepensis 66 ( 46.81% ) , p. ( adl . ) longiductus 30 ( 21.28% ) , p. ( adl . ) balcanicus 23 ( 16.31% ) , p. ( adl . ) kabulensis 13 ( 9.22% ) and p. ( adl . ) comatus 1(0.71% ) .
the p. ( adl . ) kabulensis artemiev , 1978 is a new record from iran . in case of p. ( adl . ) comatus which was reported by the authors as a new record by morphological characters from the country ( zahraei - ramazani et al .
3 ) . number of males of subgenus adlerius species collected from 17 provinces we updated the morphological key for the male subgenus adlerius species of iran as follows :
one ascoid on antennal segments 915 .
antenna 8 with two ascoids ..................... ( 2).-one ascoid on antennal segments 915 . antenna 8 with one ascoid .
antenna 6 and 7 with one ascoid or with one long and one short ........................................................ ( 6).coxite with 1427 hairs in group , rarely 29 ............................................................
( 3).-coxite with 29115 hairs in group , rarely 27 . part of 2785 hairs of group on distal half of coxite ..........................................
tubercle of aedegus 616 m from tip ..................... p. turanicus ( akhoundi et al . 2011).-part of hair - group on distal half of coxite .
tubercle of aedegus 1928 m from tip .............. p. brevis ( theodor and mesghali 1964).aedegus with rectangular subterminal notch ................ p. halepensis ( adler 1946).-aedegus with normal obtuse - angle subterminal notch .......................................................
sperm tubes are long ( 12001700 m ) ...................................................... p. longiductus ( theodor and mesghali 1964 ) ( also p.cf .
ventral process of style long ( about 20 m ........ ( 7).-coxite with 3085 group - hairs
antenna 6 and 7 with one ascoid or with one long and one short ........................................................ ( 6 ) .
part of 2785 hairs of group on distal half of coxite .......................................... ( 4 ) .
tubercle of aedegus 616 m from tip ..................... p. turanicus ( akhoundi et al . 2011 ) .
tubercle of aedegus 1928 m from tip .............. p. brevis ( theodor and mesghali 1964 ) . aedegus with rectangular subterminal notch ................ p. halepensis ( adler 1946 ) .
sperm tubes are long ( 12001700 m ) ...................................................... p. longiductus ( theodor and mesghali 1964 ) ( also p.cf .
coxite very wide , whole group of 125200 hairs on its basal half ................................................ p. comatus ( zahraei et al . 2013 ) .
-coxite narrow , part of hair - group on its distal half .................................... p. balcanicus ( theodor and mesghali 1964 ) ( also p.cf .
the male and female sand flies of subgenus adlerius were collected from 12 provinces and the male were collected from 11 out of 17 provinces .
the males were not collected from markazi , bushehr , kerman , kordestan , ghom , and kohkiluyeh va boyer ahmad .
the maximum number collection was from ardabil ( 52 specimens ) and the minimum number was from khuzestan and azerbaijan - e - gharbi ( 1 specimen ) provinces .
so for the statistical comparison of the 8 measureable morphological characters of the specimens of p. halepensis , we sorted them according to the four geographical regions ( north - west , north - east , south - west and south - east ) .
the p. halepensis specimens collected from three provinces ( ardabil , azerbaijan - e - sharqi and esfahan ) showed significant difference between the length of style and length of aedeagus .
the statistical analysis of eight morphological characters of 30 males of p. balcanicus specimens confirmed that there is no significant difference between the characters in the specimens of ardabil and azerbaijane - sharqi .
there is a significant difference between the lengths of the coxite of the p. kabulensis specimens in the three provinces ( esfahan , lorestan and ilam ) .
the post hoc - bonferroni test shows that the length of coxite of p. kabulensis specimens in lorestan province is significantly more than ilam .
the statistical analysis between the 7 specimens of p. brevis in chahar mahal - o - bakhtiari and 1 specimen in azarbaijan - e - gharbi did not confirm any significant difference .
the statistical analysis of 30 p. longiductus specimens in four geographical regions ( north - west , north - east , south - west and south - east ) confirmed that there are significant difference value between the length of surstyle , the length of aedeagus , the number of hairs on coxite , the length of coxite , and the length of styles ( p < 0.05 ) the pcr experiments amplified a fragment of 550 bp of the mitochondrial genome and it was successfully achieved for 6 species of the subgenus adlerius ( table 1 , 2 ) . these were registered on jx885982 to jx 885998 accession numbers in the genbank .
profile of sequenced species based on
cytb
gene in the subgenus
adlerius
in the pair wise comparison of the specimens , we focused on the genetic similarities of the specimens among each other and determined the level of such similarities of male and female specimens and identified the females among 6 male species .
the pair wise comparison in azv8 female specimens with nucleotides of six male species showed that the maximum similarity , i.e. 98% is found in p. comatus . in total ,
528 nucleotides were compared with these 2 sequences which show 8 ( 1.5% ) polymorphic sites and it is confirmed that they are 2 haplotypes ( table 3 ) .
comparison of
cytb gene nucleotide sequences in the subgenus
adlerius
species the pair wise comparison in males of the p. balcanicus species with nucleotides of the three female specimens shows that there are minimum 98% and maximum 99% similarities among the male and the shc11 female specimen .
it shows 22 ( 4.3% ) polymorphic sites and it confirms that there are 4 haplotypes ( table 3 ) .
the nucleotides comparison of the 5 males of the p. halepensis specimens with the ckhe1342 female specimen shows that there are minimum 90.1% and maximum 92.6% similarities between them .
the polymorphic sites is 46 ( 10% ) and the sequence comparison revealed that there are 6 haplotypes .
also for the two males of p. brevis , it shows 99% similarities with 2 ( 0.4% ) polymorphic sites and 2 haplotypes between these two sequenced specimens ( table 3 ) .
the sequence result of the length 550 bp of cytb gene shows that 5 male species : p. halepensis , p. brevis , p. kabulensis , p. comatus and p. balcanicus are in the areas of our study .
longiductus which are close to p. balcanicus and p. longiductus are on neighbor - joining tree ( fig .
the specimens of p. halepensis and p. brevis are in independent and different branches in the tree , but other species are close together in one branch .
p. comatus and the female azv8 specimen are in the same branch , which shows that they are the same species . also the branch of female shc11 specimen is near the branch of azv4 and azv5 , which shows that females of this species are close to these specimens .
the branches of two p. balcanicus specimens i.e. ash10 and azv11 are close together and p. kabulensis is in a different branch . the position in the phenetic tree of the 3 females species and the male species are correct and the similarities among the males and females confirm our above results ( fig .
neighbor - joining tree for dna haplotypes of cytb - mtdna of the subgenus adlerius sand flies species
a total of 9,319 sand flies were collected , of which 3,847 ( 41.3% ) and 5,472 ( 58.7% ) were from the genus phlebotomus and sergentomyia , respectively . from the total number of sand flies of the genus phlebotomus , 167 ( 1.8% )
were from the subgenus adlerius [ 26 ( 15.57% ) were female and 141(84.43% ) were male ] .
of the males , phlebotomus ( adlerius ) brevis 8 ( 5.67% ) , p. ( adl . ) halepensis 66 ( 46.81% ) , p. ( adl . ) longiductus 30 ( 21.28% ) , p. ( adl . ) balcanicus 23 ( 16.31% ) , p. ( adl . ) kabulensis 13 ( 9.22% ) and p. ( adl . ) comatus 1(0.71% ) .
the p. ( adl . ) kabulensis artemiev , 1978 is a new record from iran . in case of p. ( adl . ) comatus which was reported by the authors as a new record by morphological characters from the country ( zahraei - ramazani et al .
3 ) . number of males of subgenus adlerius species collected from 17 provinces we updated the morphological key for the male subgenus adlerius species of iran as follows :
one ascoid on antennal segments 915 .
antenna 8 with two ascoids ..................... ( 2).-one ascoid on antennal segments 915 . antenna 8 with one ascoid .
antenna 6 and 7 with one ascoid or with one long and one short ........................................................ ( 6).coxite with 1427 hairs in group , rarely 29 ............................................................
( 3).-coxite with 29115 hairs in group , rarely 27 . part of 2785 hairs of group on distal half of coxite ..........................................
tubercle of aedegus 616 m from tip ..................... p. turanicus ( akhoundi et al . 2011).-part of hair - group on distal half of coxite .
tubercle of aedegus 1928 m from tip .............. p. brevis ( theodor and mesghali 1964).aedegus with rectangular subterminal notch ................ p. halepensis ( adler 1946).-aedegus with normal obtuse - angle subterminal notch .......................................................
sperm tubes are long ( 12001700 m ) ...................................................... p. longiductus ( theodor and mesghali 1964 ) ( also p.cf .
ventral process of style long ( about 20 m ........ ( 7).-coxite with 3085 group - hairs
antenna 6 and 7 with one ascoid or with one long and one short ........................................................ ( 6 ) .
part of 2785 hairs of group on distal half of coxite .......................................... ( 4 ) .
tubercle of aedegus 616 m from tip ..................... p. turanicus ( akhoundi et al . 2011 ) .
tubercle of aedegus 1928 m from tip .............. p. brevis ( theodor and mesghali 1964 ) . aedegus with rectangular subterminal notch ................ p. halepensis ( adler 1946 ) .
sperm tubes are long ( 12001700 m ) ...................................................... p. longiductus ( theodor and mesghali 1964 ) ( also p.cf .
coxite very wide , whole group of 125200 hairs on its basal half ................................................ p. comatus ( zahraei et al . 2013 ) .
-coxite narrow , part of hair - group on its distal half .................................... p. balcanicus ( theodor and mesghali 1964 ) ( also p.cf .
the male and female sand flies of subgenus adlerius were collected from 12 provinces and the male were collected from 11 out of 17 provinces .
the males were not collected from markazi , bushehr , kerman , kordestan , ghom , and kohkiluyeh va boyer ahmad .
the maximum number collection was from ardabil ( 52 specimens ) and the minimum number was from khuzestan and azerbaijan - e - gharbi ( 1 specimen ) provinces .
so for the statistical comparison of the 8 measureable morphological characters of the specimens of p. halepensis , we sorted them according to the four geographical regions ( north - west , north - east , south - west and south - east ) .
the p. halepensis specimens collected from three provinces ( ardabil , azerbaijan - e - sharqi and esfahan ) showed significant difference between the length of style and length of aedeagus .
the statistical analysis of eight morphological characters of 30 males of p. balcanicus specimens confirmed that there is no significant difference between the characters in the specimens of ardabil and azerbaijane - sharqi .
there is a significant difference between the lengths of the coxite of the p. kabulensis specimens in the three provinces ( esfahan , lorestan and ilam ) .
the post hoc - bonferroni test shows that the length of coxite of p. kabulensis specimens in lorestan province is significantly more than ilam .
the statistical analysis between the 7 specimens of p. brevis in chahar mahal - o - bakhtiari and 1 specimen in azarbaijan - e - gharbi did not confirm any significant difference .
the statistical analysis of 30 p. longiductus specimens in four geographical regions ( north - west , north - east , south - west and south - east ) confirmed that there are significant difference value between the length of surstyle , the length of aedeagus , the number of hairs on coxite , the length of coxite , and the length of styles ( p < 0.05 )
the pcr experiments amplified a fragment of 550 bp of the mitochondrial genome and it was successfully achieved for 6 species of the subgenus adlerius ( table 1 , 2 ) . these were registered on jx885982 to jx 885998 accession numbers in the genbank .
profile of sequenced species based on
cytb
gene in the subgenus
adlerius
in the pair wise comparison of the specimens , we focused on the genetic similarities of the specimens among each other and determined the level of such similarities of male and female specimens and identified the females among 6 male species .
the pair wise comparison in azv8 female specimens with nucleotides of six male species showed that the maximum similarity , i.e. 98% is found in p. comatus . in total ,
528 nucleotides were compared with these 2 sequences which show 8 ( 1.5% ) polymorphic sites and it is confirmed that they are 2 haplotypes ( table 3 ) .
comparison of
cytb gene nucleotide sequences in the subgenus
adlerius
species the pair wise comparison in males of the p. balcanicus species with nucleotides of the three female specimens shows that there are minimum 98% and maximum 99% similarities among the male and the shc11 female specimen .
it shows 22 ( 4.3% ) polymorphic sites and it confirms that there are 4 haplotypes ( table 3 ) .
the nucleotides comparison of the 5 males of the p. halepensis specimens with the ckhe1342 female specimen shows that there are minimum 90.1% and maximum 92.6% similarities between them .
the polymorphic sites is 46 ( 10% ) and the sequence comparison revealed that there are 6 haplotypes . also for the two males of p. brevis , it shows 99% similarities with 2 ( 0.4% ) polymorphic sites and 2 haplotypes between these two sequenced specimens ( table 3 ) .
the sequence result of the length 550 bp of cytb gene shows that 5 male species : p. halepensis , p. brevis , p. kabulensis , p. comatus and p. balcanicus are in the areas of our study .
longiductus which are close to p. balcanicus and p. longiductus are on neighbor - joining tree ( fig .
the specimens of p. halepensis and p. brevis are in independent and different branches in the tree , but other species are close together in one branch .
p. comatus and the female azv8 specimen are in the same branch , which shows that they are the same species .
also the branch of female shc11 specimen is near the branch of azv4 and azv5 , which shows that females of this species are close to these specimens .
the branches of two p. balcanicus specimens i.e. ash10 and azv11 are close together and p. kabulensis is in a different branch .
the position in the phenetic tree of the 3 females species and the male species are correct and the similarities among the males and females confirm our above results ( fig .
neighbor - joining tree for dna haplotypes of cytb - mtdna of the subgenus adlerius sand flies species
but , the species involved have seldom been identified because despite the taxonomic efforts of artemiev ( 1980 ) and others .
the morphological characteristics do not distinguish between the females of adlerius species that are generally recognized on the basis of male morphology ( lewis 1982 , sadlova et al .
2013 ) and p. kabulensis during the present study has assisted to revise the morphological key .
the access to new molecular techniques and genetic information about these probable vectors ( artemiev 1973 , ardehali et al .
1995 ) and their systematics , may aid in resolving the problem of identification in morphological similarity , as well . we provide evidence to interpret and explain their morphology and ecology in detail .
the systematics of the phlebotomine sand flies due to the nature of their diagnostic characteristics is highly variable .
the latest revision of the genus phlebotomus sand flies in old world is included : ( 1 ) adlerius , ( 2 ) anaphlebotomus , ( 3 ) euphlebotomus , ( 4 ) kasaulius , ( 5 ) transphlebotomus , ( 6 ) synphlebotomus , ( 7 ) phlebotomus , ( 8) larroussius , ( 9 ) paraphlebotomus ( rispail and leger 1998 ) .
the upgma is one of the most popular methods in ecology for the classification of sampling units on the basis of their pairwise similarities in relevant descriptor variables ( such as species composition ) ( legendre and legendre 1998 ) . in bioinformatics ,
upgma is used for the creation of phenetic trees ( phenograms ) . in our molecular analysis ,
all of the cytb gene sequences confirm the monophyletic of the subgenus adlerius and the morphological identification of these six species are confirmed by the reliable morphological identification keys .
the haplotypes are 6 in this species . according to our morphological , statistical , and molecular findings , three specimens : azv2 , msh458 and arm754 which were collected from the north - west of iran , fall in one branch along with three p. halepensis of genbank . arm754 and
msh458 were captured from ardabil and azv2 was captured from azerbaijan - e - sharqi , two different areas . in the statistical analysis ,
the length of the style in p. halepensis specimens in ardabil province was significantly different from that of esfahan and azerbaijan - e - sharqi .
azv2 is in a different branch but is closer to msh458 and arm754 specimens . fj103 and ek77 are in one branch too . in the statistical analysis ,
the mean of length of style of ek77 is more than that of msh458 , arm754 and azv2 .
so in the upgma tree it shows that they are close to the fj103 specimen in fars province .
the fj103 and the three p. halepensis specimens from the genbank found in the north of iran are closer or these specimens in two different areas and are near to their phenetic characters . some geographical event such as desert winds in the central plateau ( fig .
the pairwise comparison in males of p. halepensis specimens with nucleotides of ckhe1342-female shows that there are minimum 90.1 % and maximum 92.6 % similarity among them . in statistical analysis ,
the mean of the length of labrum , surstyle , and aedeagus of the p. halepensis specimens in the south - east ( ckhe1342 ) of iran are different from the specimens of other geographical regions .
however , ckhe1342-female is in different branch because of the geographical distance between these specimens . in this study
all the p. halepensis specimens are on different branch and are in a larger group .
it also seems that this species has been separated from another species of adlerius group many years ago than the other adlerius species . according to the statistical analysis and molecular findings
the present study on molecular analysis of p. brevis population shows that one line - age with two haplotypes for cytb .
statistical analysis did not show any difference between them and the specimens from azerbaijan - e - gharbi .
along with them , another species i.e 306450786 was submitted to the genbank from iran which is in one independent branch .
it seems that p. brevis is not a common ancestor of the other adlerius but diverged early and shares one common ancestor with other species .
the second group of the phylogeny tree including lpm5 , azv11 , ash10 , azv5 , azv4 , azv10 and two females specimens : shc11 and azv8 show that cytb gene does not have sufficient resolution for them and indicates the morphological character overlapping .
the length of the branches in this group is short , which indicates that the changes among them are low .
this can also be said that the specimens of each species are not far genetically . in statistical analysis by using t - test , we did not find any difference between the specimens of p. balcanicus in ardabil and azerbaijan - e - sharqi provinces . in the molecular study
, the pairwise comparison in three males of this species with nucleotides of shc11-female specimen shows that there are minimum 98 % and maximum 99 % similarities among themselves .
also , it is confirmed that there are 4 haplotypes among the four sequenced specimens . in the present study , azv4 and azv5
however , in our upgma tree , we found that they are differents between p. balcanicus specimens and they are in an independent branch but closer to p. balcanicus branches .
the results of the molecular investigations indicate that according to the concept of the species they ( azv5 and azv4 ) can not be considered as a separate species . because the concept of a species is based on the reproductive isolation , while the existence of the intermediate forms and the same mitochondrial gene sequences in this study indicates that there is no reproductive isolation between azv5 morphotypes and azv4 morphotypes .
also , we can not assume these two specimens as subspecies because two subspecies can not be found as sympatric .
so , this indicates that these species e.g p. longiductus and p. balcanicus in iran , including their populations have morphological and genetic differences .
the similarity between azsh10 and azv11 is 97% and e - value between them is 0.0 .
the branches of the four haplotypes of p. balcanicus show that genetically they are not far . the similarity and e - value between the shc11 and azv5 are 99 % and 0.0 respectively .
the pairwise comparison of the nucleotides of cytb - mtdna gene in azv8 female specimen with nucleotides of six male species shows that the maximum similarity i.e 98% is found in azv10 .
so , these evidences confirm our results as both azv8 and azv10 are p. comatus . in the upgma tree ,
this indicates that many changes have occurred during the course of evolution of this species .
also this species seems a common ancestor for the other species in this group of the upgma tree .
the location and the number of hairs on coxite is the most important morphological characteristic feature for identifying and separating one adlerius species from the other . at times
it is difficult for the researchers as the numbers and locations of the hairs of different species are the same and overlap among themselves . in p. kabulensis ,
the hairs on coxite are 2750 and in p. longiductus the hairs on coxite are 5085 . if we collect a specimen which has 50 hairs on its coxite , we can not identify that it belongs to which one of the afore noted two species . also for p. balcanicus and p. longiductus , we encountered the same difficulty . in this case , the researchers use
it is mainly used in academic writings to indicate a reference to a contrasting finding or viewpoint ( it is often used instead of but see ) .
it can also appear occasionally in binomial nomenclature by placing before the species name to indicate that the species is not confirmed ( strunk and white 1979 ) .
balcanicus . in our study , these species have been confirmed by using both morphological and molecular methods .
but in our phenetic tree it is found that they are different from p. balcanicus and p. longiductus .
obviously , detailed studies for detection of genetic markers to identify the females of six adlerius species are essential .
also , further studies on morphology and genetic diversity of the populations of the subgenus adlerius will be of particular importance .
the positions of three female and males of subgenus adlerius in the upgma tree are correct and the similarities among them confirm our results .
the branches of each species are not genetically distinct that justify the overlapping morphological characters among them .
molecular sequencing of cytb - mtdna haplotypes can be used for female identification of different species of subgenus adlerius in iran . | background : female sand flies of subgenus adlerius are considered as probable vectors of visceral leishmaniasis in iran .
the objective of this study was to determine the morphological and genotypic variations in the populations of this subgenus in the country.methods:sand flies collected using sticky traps from 17 provinces during 20082010 .
the morphometric measurements were conducted with an ocular micrometer .
data was analyzed by spss .
the cytb gene was used to estimate population genetic diversity and identify the female specimens .
upgma phenetic tree was used for dna haplotypes of cytb gene.results:six species of subgenus adlerius identified from which one species , p. ( adlerius ) kabulensis , is new record .
the identification key is provided for males .
results revealed the molecular systematic in the species of subgenus adlerius and determine the relationship of three females of p. comatus , p. balcanicus and p. halepensis.conclusion:the positions of three females and the males in the upgma tree are correct and the similarities among them confirm our results .
the branches of each species are not genetically distinct which justify the overlapping morphological characters among them .
molecular sequencing of cytb - mtdna haplotypes can be used for female identification for different species of subgenus adlerius in iran . |
there has been an increase of prevalence of obesity which is associated with several adverse health problems , including type 2 diabetes , hyperlipidemia and hypertension .
the role obesity plays in the development of obesity - related disorders is complex and probably due to an interaction of genetic , nutritional and metabolic factors [ 1 , 2 ] .
a number of candidate genes have been implicated in the pathogenesis of obesity in humans .
tumor necrosis factor- ( tnf- ) is primarily secreted in adipocytes , and elevated levels of this cytokine have been linked to obesity and insulin resistance .
tnf- messenger rna levels in skeletal muscle and adipose tissue of humans are positively correlated with body mass index ( bmi ) , fat mass , and insulinemia and inversely correlated with lipoprotein lipase activity [ 35 ] .
the gene which is coded human tnf- is located on the short arm of chromosome 6 .
recently , attention has been focused on two nucleotide polymorphisms identified in the promoter region of the tnf- gene : g a substitutions at 308 and 238 respectively . in vitro experiments have demonstrated that the polymorphism 308 which is identified by ncoi restriction fragment length polymorphism ( rflp ) , increases transcriptional activation of the tnf- gene .
the potential functional impact of the polymorphism at position 238 of the tnf- gene has not yet been investigated in in vitro expression studies . however , the variant is located within a putative y box , which is a regulatory motif typical of the promoter region of major histocompatibility complex class ii genes .
the purpose of the present study is to determine whether the two mentioned tnf- promoter polymorphisms are associated with obesity in iranian population .
subjects were randomly selected from cohort tehran lipid and glucose study and adults were classified in three groups according to their bmi : bmi < 25 , 25 bmi < 30 , bmi 30 . as for 308 polymorphism , 202 adults ( 81 men , 121 women ) and 42 under-18 subjects ( 21 boys , 21 girls ) were placed in two groups according to their bmi : under and above 85th percentile as far as age and sex are concerned . and as for 238 polymorphism , 239 ( 78 men , 120 women ) and 41 under-18 subjects ( 17 boys , 24 girls ) were classified in two groups according to their bmi : under and above 85th percentile as far as age and sex are concerned .
bmi , waist to hip ratio , fat mass , and plasma concentration of glucose , serum levels of triglyceride , total cholesterol , and high - density lipoprotein ( hdl ) cholesterol were analyzed . in order to genotype the subjects ,
the standard salting - out proteinase k method was used to extract the germinal dna .
both tnf- promoter polymorphisms were detected using the rflp method after amplification of the sequences by polymerase chain reaction ( pcr ) .
a 107-bp fragment encompassing the 308 variant site was amplified from genomic dna by pcr using standard reagents , technique , and primers ( 5-agg caataggttttgaggggccat-3 ) and ( 5-tcctccctgctccgattccg-3 ) for the 308 variant and ( 5-atctggaggaagcggtagtg-3 ) and ( 5-agaagacccccctcggaacc-3 ) for the 238 variant allele . to detect 308
variant alleles the pcr product was digested with ncoi at 37c overnight , leaving a 107 bp fragment when the variant was present and products of 87 and 20 bp fragments for the normal allele visualized on 3% agarose gel . to detect the 238 variant alleles ,
the pcr product was digested with mspi at 37c for an hour , leaving a 152 bp fragment when the variant was present and products of 133 and 19 bp fragments for the normal allele visualized on 3% agarose gel .
the mean levels of all normally - distributed variants were tested by anova and the mean levels of all abnormally - distributed variants were tested by nonparametric kruskal
wallis h test and the data were presented as means sd of the differences .
the mean levels of all normally - distributed variants were tested by anova and the mean levels of all abnormally - distributed variants were tested by nonparametric kruskal
wallis h test and the data were presented as means sd of the differences .
the distribution of tnf- genotype for 308 site in under-18 subjects was as follows : gg ( 81% ) , ga ( 16.6% ) , and aa ( 2.4% ) .
in adults the following results were produced : gg ( 86.6% ) , ga ( 12.4% ) , and aa ( 1% ) .
as for the 238 site in under-18 subjects the results were : gg ( 80.5% ) , ga ( 19.5% ) , and aa ( 0% ) , and in adults the following results were recorded : gg ( 79.3% ) , ga ( 19.2% ) , and aa ( 1.5% ) .
clinical and biochemical data of participants , when classified in accordance to g / a polymorphisms at positions 308 and 238 of the tnf- gene promoter , are presented in table 1 ( 308 ) and table 2 ( 238 ) . to examine whether the variant alleles were more common in patients with obesity
, adults were classified in three groups according to their bmi : bmi < 25 , 25 bmi < 30 , bmi 30 and under-18 subjects were classified in two groups according to their bmi : under 85th percentile bmi and above 85th percentile as far as age and sex are concerned
table 3 ( 308 ) and table 4 ( 238).table 1clinical and biochemical data of subjects when classified in accordance to the genotype of the g / a polymorphisms at positions 308 of the tnf- gene promoterunder 18above 18traitgg ( 34)ga ( 7)aa ( 1)pgg ( 175)ga ( 25)aa ( 2)page ( years)12.51 2.814.21 2.2510.50.24043.34 15.6240.96 16.0451.25 1.760.591sex ( m / f)17/173/41/00.57270/10510/151/10.960bmi ( kg / m)19.73 3.8419.91 2.0726.710.17926.65 4.5527.33 4.8232.18 2.70.196fbs ( mg / dl)86.45 5.6287.35 6.5173.50.08889.00 22.1890.50 8.6687.5 7.070.865homa10.1 5.809.45
( mg / dl)158.57 2.44169.79 22.38178.000.421196.57 42.73197.00 47.91208.75 44.900.925hdl - c ( mg / dl)44.30 8.6043 7.34290.20842.26 9.6342.76 9.2031.75 13.780.295ldl - c ( mg / dl)95.96 20.75109.69 22.87113.400.241124.53 3.46118.52 43.83128.75 40.090.723tg ( mg / dl)94.44
39.0085.85 21.80178.500.074128.50 81.07142.50 154.54241.25 44.190.053whr0.83 0.050.82 0.070.980.0530.87 0.080.86 0.090.95 0.060.344sbp ( mmhg)101.31 8.46104.21 5.12970.579114.50 17.26116.50 11.02131.25 22.270.426dbp ( mmhg)67.22 5.8571.21 4.8757.50.05974.36 8.0975.34
8.9883 16.260.303table 2clinical and biochemical data subjects when classified in accordance to the genotype of the g / a polymorphisms at positions 238 of the tnf- gene promoterunder 18above 18traitgg ( 33)ga ( 8)aa ( 0)pgg ( 175)ga ( 38)aa ( 3)page ( years)13.24 2.9612.62 2.210.58542.18 15.0139.93 16.5042.50 21.070.585sex ( m / f)13/204/40.58997/6017/210.745bmi ( kg / m)20.12 3.5819.08 2.880.45426.40 4.3726.84 4.7123.37 3.120.415fbs ( mg / dl)87.63 6.2484.50 6.830.21889 41.6287.25 23.1389 13.470.766homa10.79 6.4210.28
3.030.82812.13 8.8510.15 9.1810.19 1.040.158cho ( mg / dl)162.18 23.99146.38 26.830.110199.08 40.46183.78 37.69214.17 92.590.090hdl - c ( mg / dl)43.83 7.6243.12 7.800.81642.85 9.0342.18 10.2541.66 4.190.904ldl - c ( mg / dl)100.08 19.5287.30 27.620.134125.92 34.11112.62 32.22148.07 83.890.052tg ( mg / dl)94.30 37.8580 30.230.328123.00 97.55145.25 57.70103.50 56.730.519whr0.82 0.040.8 0.110.4510.87 0.080.87 0.10.83 0.110.780sbp ( mmhg)100.42 8.51104.56 7.720.217115.14 15.20115.86 15.04103 21.650.757dbp ( mmhg)66.33
6.650.407table 3clinical and biochemical data of subjects when classified in accordance to bmi for polymorphisms at positions 308 of the tnf- gene promoterunder 18above 18traitgrade 1grade 2pgrade 1grade
2grade 3pbmi < 85%bmi > 85%bmi < 2525 bmi < 30bmi 30(n = 26)(n = 16)(n = 75)(n = 81)(n = 46)gg21(80.8%)13(81.2%)67(89.3%)71(87.7%)37(80.4%)ga5(19.2%)2(12.51%)8(10.7%)10(12.3%)7(15.2%)aa1(6.2%)2(4.3%)age ( years)12.88 2.7612.53 2.810.69236.37 16.6545.56 14.3649.85 11.460.000bmi ( kg / m)17.64 2.032.60 23.630.00022.27 1.9327.49 1.4232.90 3.370.000fbs ( mg / dl)86.34 6.2186.34 6.210.94887 21.0791 16.6691.50 26.020.001homa9.94
4.8010.22 6.270.87012.34 10.1812.47 413.4010.31 5.780.071cho ( mg / dl)155.9 22.13169.03 25.490.086181.50 42.11201.32 43.50213.51 36.660.000hdl - c ( mg / dl)43.19 7.7444.59 9.930.61242.53 9.1836.98 8.3745.62 11.340.005ldl - c ( mg / dl)96.12 19.71102.79 23.820.332113.77 36.47125.82 34.53136.71 32.790.002tg ( mg / dl)86.78 27.92108.37 49.330.077108 67.10154.50 117.21135.25 70.220.000whr0.81 0.050.86 0.060.0080.83 0.070.89 0.080.9 0.070.000sbp
( mmhg)100.78 8.14103.15 7.650.355111.50 11.78116 18119 17.750.000dbp ( mmhg)67.51 6.3667.87 5.520.85471.57 7.2375.51 8.1177.79 8.690.000table 4clinical and biochemical data of subjects when classified in accordance to bmi for polymorphisms at positions 238 of the tnf- gene promoterunder 18above 18traitgrade 1grade 2pgrade 1grade
2grade 3pbmi < 85%bmi > 85%bmi < 2525 bmi < 30bmi 30(n = 26)(n = 16)(n = 75((n = 81)(n = 46)gg21(80.8%)13(80%)65(81.2%)60(77.9%)32(78%)ga5(19.2%)2(20%)13(16.2%)16(20.8%)9(22%)aa2(2.5%)1(1.3%)age ( years)13.50 2.75 12.46 2.90 0.264 38 16.61 42.42 13.85 50.24 12.16 0.000bmi ( kg / m)18.05 2.28 23.15 2.63 0.00022.27 1.89 27.50 1.43 32.57 3.18 0.000fbs ( mg / dl)86.86 7.21 87.30 4.88 0.83787 54 89 26.03 92 15.04 0.011homa10.97 5.69 10.21 6.39 0.69611.32 8.92 12.14 9.73 10.44 6.73 0.385cho ( mg / dl)154.36 22.76 167.30 27.41 0.112186.30 41.59 200.50 42.15 208.26 34.04 0.011hdl - c ( mg / dl)42.19 6.31 46.30 8.99 0.09443.35 8.64 40.66 8.26 45.30 11.39
0.023ldl - c ( mg / dl)95.35 19.41 101.44 25.09 0.391117.18 36.66 126.27 34.73 131.60 30.38 0.071tg ( mg / dl)87.8 29.78 97.93 46.60 0.4110.50 69.11 144.50 111.46 135 69.81 0.000whr0.80 0.06 0.85 0.05 0.012 0.83 0.08 0.89 0.08 0.89 0.08 0.000sbp ( mmhg)99.4 8.42 104.4 7.71 0.067111.75 12.67 115 14.84 118.5 17.34 0.001dbp ( mmhg)66.94 5.68 67.30 5.45 0.84572.11
7.22 74.13 7.54 78.09 7.87 0.000 clinical and biochemical data of subjects when classified in accordance to the genotype of the g / a polymorphisms at positions 308 of the tnf- gene promoter clinical and biochemical data subjects when classified in accordance to the genotype of the g / a polymorphisms at positions 238 of the tnf- gene promoter clinical and biochemical data of subjects when classified in accordance to bmi for polymorphisms at positions 308 of the tnf- gene promoter clinical and biochemical data of subjects when classified in accordance to bmi for polymorphisms at positions 238 of the tnf- gene promoter
the purpose of the present study is to determine whether the g a polymorphisms of the tnf- gene promoter are associated with obesity and anthropometric parameters in cohort tehran lipid and glucose study .
there are no associations between g-308a and g-238a tnf- promoter polymorphisms and increases in bmi .
tnf- is one of the cytokines secreted from adipose tissue in obesity ; it can be a candidate gene to inspect obesity and related diseases .
obesity is a complex metabolic disorder with a strong genetic component and there are many candidate genes for obesity and its related phenotypes .
most of these genes are candidate because their mutations cause rare genetic syndromes affecting adipocytes differentiation .
recent interest has focused on the role of the tnf- gene in insulin resistance and obesity .
we therefore examined the association between the polymorphism of tnf- and obesity without metabolic disease .
it came as another study reported an association between this genetic marker and excessive fat accumulation in women .
moreover , a potential role for the tnf- locus in obesity is also supported by a linkage study , which reported a relationship between the tnf- locus and various global measures of adiposity in pima indians and european caucasians [ 11 , 12 ]
however , it is located within a consensus sequence for the transcription factor ap-2 in the promoter region of the gene . the g-308a mutation in the promoter region of tnf- acts in vitro as a much stronger transcriptional activator than the wild - type tnf- and it is suggested that a higher transcriptional activity would result in higher tnf- concentrations followed by decreased insulin sensitivity .
so this variant could play a role in the development of obesity and related phenotypes .
interestingly , obesity is known to be associated with increased levels of tnf- mrna and protein in fat tissue both in humans and in genetically obese or diabetic rodents . moreover
, a study has shown that the tnf- mrna level in adipose tissue are significantly correlated with the percentage of body fat in human obese subjects . a recent study , while confirming linkage between the tnf- locus and obesity in families of french - canadian origin , has also found a significant association between this locus and high blood pressure .
a significant positive correlation between serum tnf- concentration and systolic blood pressure has also been reported among individuals of an isolated native canadian population over a wide range of adiposity .
an italian population study suggests that the g-308a mutation of the tnf- gene is unlikely to play an important role in the development of obesity and its related metabolic abnormalities such as insulin resistance and dyslipidemia . as for the g-238a variant ,
a study of non - diabetic relatives and control subjects in britain showed that the 238 variant was associated with increased insulin sensitivity as estimated by two independent methods . a recent meta - analysis ( 2,106 cases and 2,920 controls ) of the rs361525 ( g-238a )
variant has not detected a significant association with type 2 diabetes , nor has the study of caucasian and african american subjects .
benedicte fontaine - bisson and co - workers suggest that the 238 g a and 308 g a polymorphisms of tnf- alter circulating free fatty acids and insulin resistance in obese subjects with type 2 diabetes mellitus . | tumor necrosis factor- ( tnf- ) , an adipokine , is produced in adipocytes , and the elevation of its levels has been linked to obesity and insulin resistance in some population . in this
study the relationship between tnf- promoter gene polymorphism and obesity in an iranian population has been studied .
subjects were randomly selected from tehran cohort lipid and glucose study .
adult participants placed in three groups according to their body mass index ( bmi ) : bmi < 25 , 25 bmi < 30 , bmi 30 and under-18 subjects placed in two groups , under 85th percentile bmi and above 85th percentile . finally , 244 persons were selected for g-308a and g-238a polymorphisms analysis .
the fbs , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , triglyceride , cholesterol levels and blood pressure and homa of all subjects were measured .
the polymorphism 308 and 238 were revealed by restriction fragment length polymorphism ( rflp ; ncoi and mspi ) after the promoter site was amplified by pcr .
the allele frequency of tnf- polymorphism was in the hardy weinberg equilibrium .
there was no relation between bmi and the frequency of this allele .
the fact that there is no association between g-308a and g-238a tnf- promoter polymorphisms and obesity probably shows that it is not an important risk factor for obesity and consequently for cardiovascular disease . |
lipoma is the most common benign neoplasm of mesenchymal origin , and it is composed of mature adipose tissue without cellular atypia .
though predominantly located in subcutaneous sites , they are also found intramuscularly , retroperitoneally , and in intraosseous locations .
osseous lipomas are very rare despite the large amount of fatty marrow in the adult .
they most commonly occur in the calcaneus and long bones of lower extremities . depending upon the location in relation to bone
, they may be intraosseous ( intramedullary and intracortical ) or juxtacortical ( parosteal and subperiosteal ) .
the occasional symptomatic case may be present with swelling and rarely with pain . in this article
, we are reporting a case of intraosseous lipoma of the mandible , which to the best of our knowledge is the fifth only reported case in anterior mandible .
also , significant is its presentation as a swelling , which is very rare for an intraosseous lipoma ( iol ) in the jaw bones .
a 15-year - old boy reported to our institution with a complaint of swelling in the right lower jaw of 2 years duration , associated with increasing difficulty in speech .
intraoral examination revealed a smooth , non tender , and bony hard , ovoid swelling of about 4 2.5 cm size , located towards the lingual aspect of mandible extending from 41 to 47 [ figure 1 ] .
the pre - operative view shows a smooth , ovoid swelling on the lingual aspect of the mandible extending from mesial of 41 to 47 a mandibular occlusal radiograph revealed cortical expansion with hazy radiopacity on the lingual surface of mandible [ figure 2 ] .
cross - sectional mandibular occlusal radiograph shows cortical expansion with hazy radiopacity on the lingual surface of mandible extending from 41 to 47 region the specimen was decalcified overnight in 8% nitric acid and routinely processed to obtain paraffin - embedded , 10 thick hematoxylin and eosin ( h and e ) stained slides .
microscopically , the tissue was composed of areas of mature adipocytes interspersed with few trabeculae of mature lamellated bone and prominent thin - walled , large caliber vascular spaces .
the major part of the lesion was surrounded by a continuous layer of cortical bone , which appeared to be thinned out [ figure 4 ] .
( h and e , stain ; 100 original magnification ) photomicrograph showing thinned out cortical bone present at the periphery of the lesion ( h and e , 100 original magnification ) based on these findings , a diagnosis of intraosseous lipoma was made .
iol is one of the rarest bone tumors accounting for approximately 0.1% of all benign primary bone tumors .
the first case of iol was described in 1880 by cornil and ranvier in the femur .
nearly every bone can be affected by this tumor , although favored sites are the calcaneus and the metaphysis of long bones .
these adipocyte tumors occur most commonly in the fourth decade with a slight male predilection .
symptoms , when present , are localized mild pain , swelling or rarely fracture of the affected bone . based on their location , osseous lipomas can be intraosseous ( intramedullary and intracortical ) and juxtacortical ( parosteal and subperiosteal ) .
the only two reported cases of intracortical lipoma were seen in the diaphysis of long bones , as well - demarcated radiolucent areas not communicating with the medullary cavity . a total of 24 cases of intramedullary lipomas have been reported in the jaw bones till date . of these , only four have occurred in the anterior mandible , our case thus being the fifth .
although clinical examination combined with computed tomography ( ct ) or magnetic resonance imaging ( mri ) can point towards iol , a definitive diagnosis requires microscopic examination .
microscopically , sheets of mature adipocytes without atypia and absence of hematopoietic elements are the essential features .
based on the level of involution , three stages of intraosseous lipomas have been described by milgram : stage 1 , when there is no secondary necrosis ; stage 2 , when there is partial necrosis ; stage 3 , when there is complete secondary necrosis and dystrophic calcification . since we did not find areas of necrosis or dystrophic calcification in the present case , we considered it was in stage 1 of milgram 's staging .
microscopically , the lesion must be distinguished from a well - differentiated liposarcoma , normal fibro - fatty bone marrow , and osteoporotic bone marrow defect .
diagnosis of normal fibro - fatty bone marrow was ruled out because of the lack of hematopoietic tissue and the expansile nature of the lesion .
furthermore , absence of any history of previous trauma to the site excluded the possibility of an osteoporotic bone marrow defect .
others have proposed that iol are reactive changes secondary to infarcts , infections , or trauma .
another hypothesis is that they are simple conglomerations of fatty marrow , formed as part of the normal ageing bone process .
however , in the present case , the young age of the patient and the absence of any trauma to the region , all point towards a benign neoplastic nature of the lesion . in symptomatic lesions and those prone to undergo pathologic fracture , curettage is the treatment of choice .
recurrence of the intraosseous lipoma is rare and it has been reported in only two cases . though intraosseous lipomas are considered benign , milgram reported four cases of malignant transformation ; however , no recurrence or malignant changes of mandibular intraosseous lipomas have been reported .
though , reported cases of osseous lipomas of the jaws are very few in number , this case highlights the importance of including it in the differential diagnosis of jaw lesions and properly documenting each new case . | lipomas are the most common form of benign mesenchymal tumors and are composed of mature adipocytes .
they can occur anywhere in the body where fat is found and thus , called as the universal tumor or the
ubiquitous tumor. intraosseous lipomas ( iol ) are among the rarest ( 0.1% ) of primary bone tumors and are very rarely seen in head and neck bones .
they have been subdivided based on the site of origin within bone , into intramedullary and intracortical .
of the two , few cases of intramedullary lipoma have been reported intraorally and none of the latter .
intraosseous lipomas are usually asymptomatic and are detected incidentally on radiographs taken for other complaints . here
, we report a case of intraosseous lipoma in the mandible presenting as a large swelling . |
the development of strategies to preserve
functional capacity of the geriatric population is therefore of paramount importance in
improving elderly quality of life and reducing health care costs2 .
there is growing evidence that the prevalence of
malnutrition in elderly people is increasing3 .
malnutrition in the elderly was often associated with negative
outcomes , for example , an increased risk of falling , anemia , immune dysregulation , impaired
cognitive status , reduced muscle function , and increased morbidity and mortality3,4,5,6 .
it
is also known that hospitalized elderly patients are at high risk for malnutrition;7 the prevalence of malnourished elderly is
high in rehabilitative settings8 and
malnutrition is associated with poor rehabilitative outcomes9 , 10 and worse functional
performance11 .
therefore , it is
important to assess nutritional status in elderly patients in these settings in order to
deliver them appropriate care .
the mini nutritional
assessment ( mna ) is a validated and commonly- used tool .
it has high sensitivity and
specificity and is effective for evaluating the nutritional status of elderly persons living
in different settings12 , 13 .
the mna consists of 18 items covering four dimensions : anthropometry , dietary assessment ,
global assessment , and self - evaluation .
the first 6 items of the full mna constitute the mna
short - form ( mna - sf ) .
mna - sf is a screening test used for identifying those who are
potentially at risk for malnutrition .
those who are identified as at risk for malnutrition
are further assessed with the full mna14 .
usually the mna - sf correctly predicts the result of the full mna12 , 15 .
several
studies have shown that the mna score is associated with functional status and is able to
predict the functional decline16 of older
adults living in a community17 as well as
in institutions18 and acute inpatient
wards19 .
there is little data on the relationship between nutritional status and functional
performance in elderly inpatients in rehabilitative hospitals .
the present study aims to
examine the relationship between nutritional status evaluated with mna - sf , and functional
status measured by the barthel index ( bi ) in a cohort of elderly inpatients in a
rehabilitative hospital .
our overarching goals were the following : 1 ) providing support for
the hypothesis that in elderly rehabilitative inpatients , nutritional status and functional
performance are positively related , and 2 ) showing that functional and nutritional status
decline with aging .
in the present cross - sectional study , we observed the relationship between barthel index
( bi ) and mini nutritional assessment short form ( mna - sf ) in a cohort of elderly inpatients
in a rehabilitative center .
informed consent was obtained from all participants and
procedures were conducted according to the declaration of helsinki .
the local ethical
committee of the rehabilitation center approved the protocol used in the study .
the initial sample consisted of 344 patients who were admitted at the general
rehabilitation center .
the inclusion criteria were as follows : 1 ) patients between 65 and
100 years of age admitted at the general rehabilitation center were included ; and 2 ) the
ability to perform functional tests was required . the exclusion criterion consisted of the
presence of any illness that could represent a risk to the subject s health during the
functional assessment .
thus , two groups of patients were excluded from the items of the bi
related to mobility ( ambulation or transfers ) : those medically prohibited from walking or
standing , and those reporting dizziness or altered vestibular function during the initial
physical examination or immediately prior to the functional assessment .
bedridden patients were also excluded . nutritional status : nutritional status was measured using the mini nutritional assessment
short form ( mna - sf ) , a valuable tool for providing rapid and reliable nutritional screening
in elderly patients , derived from the mna .
mna is a tool that evaluates the individual s
nutritional status with 18 questions in four areas ( basic anthropometrics , dietary intake ,
global indicators , and self - assessed health status ) .
the mna is a practical , noninvasive ,
and cost - effective instrument to assess nutritional status in older people14 .
the mna is considered a screening and
assessment tool with a reliable scale and clearly - defined thresholds , usable by health care
professionals12 .
the mna short form , a
subset of the full mna , may come to replace the full mna7 as it is sensitive , specific , and accurate in identifying patients
at risk for malnutrition14 .
it is adequate in assessing nutrition status in hospitalized elderly patients8 and it is also able to predict hospital
readmission and mortality10 .
functional status : the bi is one of the most widely used measures of self - care performance .
among all such instruments ,
the bi has been regarded as the best in terms of sensitivity ,
simplicity , communicability , scalability , and ease of scoring20 , 21 .
the bi was developed as a measure of disability in patients with neuromuscular and
musculoskeletal conditions receiving inpatient rehabilitation22 .
the bi has been associated with other scales , for example , the
frenchay activities index ( fai ) , the nottingham extended activities of daily living
( neadl)23 , and the functional
independence measure ( fim)24 .
the items can be
divided into a group that is related to self - care ( feeding , grooming , bathing , dressing ,
bowel and bladder care , and toilet use ) and a group related to mobility ( ambulation ,
transfers , and stair climbing ) .
the score ranges from zero , representing a totally dependent
bedridden state , to one hundred , which represents the complete independence23 .
prior to
participation in the study , all patients signed an informed consent form ( icf ) .
all
underwent a comprehensive assessment , which consisted of two visits . during the first visit ,
the patient s history was recorded , a physical examination was performed and the patient
signed the icf . during the second visit 24 hours later , a functional assessment was
performed using bi and mna - sf .
the
kolmogorov - smirnov ( k - s ) one - sample tests were initially used to assess the normality of the
distributions .
next , we categorized patients into three age groups : 6574 yrs , 7584 yrs ,
and > 85 yr .
we used kruskal - wallis h ( k - w ) tests and mann - whitney u ( m - w ) tests to assess
differences among the age groups on the mna - sf and bi .
mna - sf scores ranged from 0 to
14 , and values below a threshold of 12 were used to identify at - risk patients .
bi cutoff scores were defined as 45 out of 100 for
better functional status and < 45 out of 100 for worse25 .
kendall tau - b ( t ) tests were used to evaluate the relationship
between bi and mna - sf , and chi - squared ( 2 ) tests were
used to compare proportions between age groups .
finally , spearman s rho ( rs ) test was used
to analyze the association between bi and items within the mna - sf .
three hundred forty - four participants ( mean sd 77.7 6.8 years ; 59.3% female ) met all
the inclusion criteria and agreed to participate .
no significant differences in the mean age
were observed between men ( mean sd : 76.5 6.4 years ) and women ( mean sd : 78.5
7.0 years ) ( p = 0.51 ) .
analyses of kendall s correlation coefficients indicated significant associations between
age distribution and the scores obtained with the bi ( t = 0.326 , p < 0.001 ) and mna - sf
( t = 0.303 , p < 0.001 ) .
follow - up kruskal - wallis testing indicated significant lowest
scores with increased age for the bi ( k - w : = 139.8 , gl = 92 , p < 0.001 ) and
mna - sf ( k - w : = 52.6 , gl = 12 , p < 0.001 ) . in table 1table
1.age affects functional statusassessment age% of subjectstotalinstrumentsgroupper score rangebarthel index04445100657423 ( 19.7%)94 ( 80.3%)117 ( 34%)*758474 ( 44.8%)91 ( 55.2%)165 ( 48%)>8547 ( 75.8%)15 ( 24.2%)62 ( 18%)mna - sf0111214657459 ( 50.4%)58 ( 49.6%)117 ( 34%)*7584128 ( 77.6%)37 ( 22.4%)165 ( 48%)>8556 ( 90.3%)6 ( 9.7%)62 ( 18% ) * indicates significant difference at the level of p<0.001 . mna - sf : mini nutritional assessment short form , barthel index scores and mna - sf scores are reported according to age ; with
aging , the number of subjects with better functional status ( barthel index 45100 ) and
normal nutritional status ( mna - sf 1214 ) decreases .
mna - sf : mini nutritional assessment short form the analysis of the correlation coefficients obtained for the various assessment items used
in the functional evaluation of the elderly indicated that the bi scores presented
significant correlations with some items of the mna - sf ( table 2table 2.spearmans rank correlation coefficients between the mna - sf score and the barthel
index scoresbasal metabolic dataspearman s r barthel - item - a 0.262*barthel - item - b0.247*barthel - item - c0.439*barthel - item - d0.001barthel - item - e0.403*barthel - item - f0.142*mna - sf : mini nutritional assessment short form . *
finally , subjects with better functional status ( barthel index 45100 ) tend
to have normal nutritional status ( mna - sf 1214 ) , while subjects with worse functional
status ( barthel index 044 ) are more likely to be malnourished or at risk for malnutrition
( mna - sf 011 ) , ( =357.956 , p<0.001 ) .
the primary finding of the current study is that nutritional status is associated with
functional status in a cohort of elderly inpatients in a rehabilitative center .
indeed ,
mna - sf scores and bi scores are positively associated , and it is shown that both nutritional
and functional status worsen with age .
mna - sf is also a
tool capable of predicting functional disability in the elderly .
it has been demonstrated to
be a good predictor of functional status in institutionalized elderly at risk of
malnutrition27 .
a low mna - sf score has
also been related to incident disability in older adults28 .
moreover , malnutrition identified by the mna and mna - sf has been
associated with functional status in different settings , for example in elderly persons
living in communities , institutions27 ,
acute care hospitals19 , 29 , and in those undergoing ambulatory rehabilitation26 , 30 .
we verified the hypothesis that the association between malnutrition
and functional status also exists for patients hospitalized in a rehabilitative department .
there are several reasons for this : reduced appetite
and energy expenditure , fluid and electrolyte imbalance , altered levels of cytokines and
hormones , delayed gastric emptying , and an impaired sense of smell and taste .
moreover ,
pathologic changes of aging such as chronic disease , depression , cognitive impairment ,
multiple morbidities , and polypharmacy play an important role in the complex etiology of
malnutrition in the elderly3 .
inadequate diet and malnutrition32 results from the lack of functional
autonomy , impairing the ability to look after oneself , to prepare food , and to eat properly .
impaired functional status increases vulnerability and may affect food consumption as well
as the quality and the quantity of food consumed29 .
furthermore , nutritional status becomes particularly critical in
patients with cognitive impairments , given that these individuals become progressively more
dependent on others to perform the basic activities of daily life , such as feeding
oneself31 .
nutritional status assessment is crucial in rehabilitative setting , as there is an high
prevalence of malnutrition in rehabilitative department33 ; it has also been demonstrated that rehabilitative
outcomes are influenced by nutritional status26 . in their study ,
charlton et al.7
concluded that the majority of older patients in the rehabilitation setting are
nutritionally compromised and that this adversely influences health outcomes , for example
length of hospital stay .
it has been shown that malnutrition correlates with
pressure sores , infections , and non - infectious complications34 .
the elderly are often transferred to rehabilitation units from acute - care departments ,
where they may have developed malnutrition as a result of their illness , of certain
treatments , or of hospital - associated deconditioning .
therefore , upon admission , prompt and
careful evaluation of malnutrition risk is crucial in order to achieve positive
rehabilitative outcomes and to reduce the economic burden of malnutrition in this growing
segment of the population35 .
the impact of malnutrition on rehabilitative outcomes carries with it a biological
assumption : malnutrition has a direct effects on both muscular action and the activity of
the nervous system36 .
therefore ,
malnutrition may affect both gait and balance as a result of muscle weakness and the
inability of the brain to deal with the associated sensorimotor and cognitive demands31 .
routine functional evaluation and
nutritional screening is recommended for all older patients admitted to the hospital ,
especially in a rehabilitation setting , in order to identify patients with functional
disability and at risk of malnutrition35 .
this study reinforces the importance of the mna - sf as a tool to assess the nutritional
status of the elderly hospitalized in a rehabilitative department .
the association between nutrition and functional level prompts us to deeply evaluate
nutritional status in elderly patients with functional disability .
nutritional status can be
an important predictor of disability in hospitalized patients , and should be addressed by
interventional strategies .
the prompt identification of hospitalized elderly patients at risk for malnutrition makes
it possible to set up specific nutritional treatments .
more studies are needed to better understand the mechanism of
association between nutritional status and functional status in elderly . at this point , the
relationship between nutritional intervention and health status in the elderly is still not
clear . the main limitations of this study were the lack of data on indicators of physical
function , which limited the interpretation of the impact of nutritional status on physical
functional ability .
in addition , dietary intake was not assessed , and therefore reasons for
malnutrition could not be identified with certainty . in conclusion , this study reinforces the concept that nutritional status is associated with
functional status in elderly patients hospitalized in a rehabilitative department , and that
both nutritional and functional status deteriorate with age . in order to understand whether
early identification of malnutrition might help to reduce disability in the elderly , further
research into the effect of nutritional intervention on disability | [ purpose ] in this study , we sought to evaluate the relationship between the barthel index
and the mini nutritional assessment short form in a cohort of elderly patients
hospitalized in the general rehabilitation center .
[ subjects and methods ] three hundred
and forty - four patients underwent an extensive evaluation , which included the following
tests : 1 ) a mini nutritional assessment short form to evaluate nutritional status ; and 2 )
a barthel index assessment to evaluate functional status .
we categorized patients into
three age groups ( 6574 yrs , 7584 yrs , and > 85 yrs ) .
barthel index cutoff scores were
defined as 45 out of 100 for better functional status and < 45 for worse functional
status .
[ results ] significant associations between age distribution and the scores
obtained with the barthel index and mini nutritional assessment short form were found ;
nutritional status measured with mini nutritional assessment short form and functional
status measured with the barthel index were positively related .
[ conclusion ] this study
shows that the mini nutritional assessment short form value was associated with the
barthel index score , and that these scores varied with age . |
as part of a survey of fish diseases , lake whitefish ( coregonus clupeaformis ) were collected in fall 1995 from the st .
lawrence river 15 km upstream of quebec city , quebec , canada , to assess the prevalence of liver lesions .
a total of 141 fish were captured and necropsied , and three standard sections of liver were taken for histological examination .
prevalences of altered hepatocyte foci , hepatocellular carcinoma , cholangioma , and cholangiocarcinoma were 0.7% , 2.1% , 0.7% , and 2.1% , respectively .
thus , the overall prevalence of liver neoplasia was 4.9% ( 7/141 ) .
hepatic tumors were only observed in fish 7 years old or older .
fish age was significantly and positively correlated with the index assessing the number and size of macrophage aggregates ( p<0.001 ; rs = 0.16 ) .
hepatocyte vacuolation , anisokaryosis , lymphocytic infiltration , and bile duct hyperplasia were also observed but were not related to the age , length , sex , or condition factor of the fish .
these results represent the first report on a series of hepatic tumors in a wild salmonid species.imagesfigure 1figure 2figure 3figure 4figure 5 | |
gastric antral vascular ectasia ( gave ) is an uncommon cause of occult gastrointestinal ( gi ) bleeding and iron - deficiency anemia , accounting for 4% of nonvariceal upper gi hemorrhage.1 the clinical presentation of gave varies from a more indolent , chronic occult gi blood loss requiring serial transfusions to acute gi hemorrhage.1 first described by jabbari et al . in 1984,2
gave is also named watermelon stomach due to the gross endoscopic appearance of red stripes radiating from the antrum converging on the pylorus .
gave is commonly associated with cirrhosis of the liver and , in particular , portal hypertension;3 however , it has also been associated with such underlying chronic diseases as autoimmune connective tissue disorders , chronic renal failure , systemic lupus erythematosus , and diabetes mellitus ( table 1).47 in patients with cirrhosis , gave is found more commonly in patients with the most advanced liver disease.8 gave has two distinct endoscopic forms , diffused or punctate , which is more commonly associated with cirrhosis910 and striped ( linear ) , which typically occurs in the absence of cirrhosis.10 despite the difference in endoscopic appearance , the histopathologic parameters of gave in patients with cirrhosis are identical to patients without cirrhosis11 and is characterized by a combination of four findings : vascular ectasia of mucosal capillaries , focal thrombosis , spindle cell proliferation , and fibrohyalinosis.12 the diagnosis of gave can be difficult , as it can be confused with portal hypertensive gastropathy .
however , the pathophysiology of these two diseases is entirely different , as gave is more likely to be driven by the neurohumoral factors prostaglandin e and possibly gastrin , vasoactive intestinal peptide ( vip ) , and/or 5-hydroxytryptamine ( 5-ht),1316 leading to vasodilation and impaired motility.17 in general , there is a lack of epidemiologic evidence supporting differences in clinical outcomes in gave patients with and without cirrhosis .
one series of 30 patients by ito et al . observed lower hemoglobin levels at baseline and a greater need for transfusion when comparing patients with gave who did not have cirrhosis to those with cirrhosis.6 here , we examined whether there is a difference in clinically significant gi bleeding secondary to gave in a cohort of patients with and without cirrhosis .
we conducted a retrospective case - control study of patients who were found to have gave at the university of virginia between 1 january 2000 and 30 june 2014 using international classification of diseases , ninth revision , clinical modification ( icd-9 ) codes .
patients were queried from the clinical data repository , and a primary cohort of patients with gave was constructed .
if a disagreement on the diagnosis of gave occurred , a third independent gastroenterologist / hepatologist reviewed the report for final decision on inclusion .
the diagnosis of cirrhosis was based initially by screening icd-9 codes and confirmed by available histologic sampling by liver biopsy or by imaging studies ( both cross - sectional and ultrasound ) .
patients who had an additional gi bleeding source ( e.g. gastroesophageal varices , gastric ulcer , etc . ) were excluded .
baseline covariate characteristics were reviewed , including age , gender , race , etiology of liver disease , severity of liver disease based on model for end - stage liver disease ( meld ) , and child - pugh scores , laboratory values [ platelet counts , creatinine , blood urea nitrogen , sodium , albumin , international normalized ratio ( inr ) , and total bilirubin ] , gave risk factors [ hypothyroidism , bone marrow transplant , chronic kidney disease ( ckd ) , end - stage renal disease ( esrd ) , diabetes , proton pump inhibitor ( ppi ) use , connective tissue disease ] , and gi bleeding risk factors [ active smoking , active alcohol use , anticoagulant or antiplatelet agent use , and non - steroidal anti - inflammatory drug ( nsaid ) use ] .
primary outcomes were objective evidence of active gi bleeding , which was defined as symptoms of melena , hematochezia , hematemesis , change in hemodynamics , or gross bleeding on esophagogastroduodenoscopy ( egd ) .
secondary outcomes included number of transfusions , baseline hemoglobin , need for endoscopic intervention [ including argon plasma coagulation ( apc ) ] , rebleeding rates , need for surgery , and death .
patients with gave and cirrhosis were compared statistically to those without cirrhosis in multiple factors , including demographics , laboratory values , gave risk factors , and gi bleeding risk factors .
univariate comparisons were performed using the student - t test , wilcoxon sign rank test , chi - square test , and fisher exact test , as appropriate . when the normality of continuous variables was not assumed or when the equality of variances was not observed , logarithmic transformation was done .
multivariable models were constructed to assess statistical associations and risk factors for the development of bleeding from gave using logistic regression and analysis of the maximum likelihood estimates .
odds ratios ( or ) were calculated and were used as an estimation of risk under the rare disease assumption .
individual factors were included in the multivariable model if they were statistically significant ( p<0.20 ) in the univariate analysis , were clinically important , or have been shown in the literature to be important.18,19 multiple iterations of the model were performed .
variables included in the final iteration of the model included presence or absence of cirrhosis , platelet count , hypothyroidism , diabetes , ppi use , nsaid use , hemoglobin , type of gave , egd indication , and ckd .
all data set manipulation and statistical analyses were carried out using sas ( version 9.4 , cary , usa ) .
all statistical tests for significance were two sided and a significance level of p0.05 was considered statistically significant .
patients with gave and cirrhosis were compared statistically to those without cirrhosis in multiple factors , including demographics , laboratory values , gave risk factors , and gi bleeding risk factors .
univariate comparisons were performed using the student - t test , wilcoxon sign rank test , chi - square test , and fisher exact test , as appropriate . when the normality of continuous variables was not assumed or when the equality of variances was not observed , logarithmic transformation was done .
multivariable models were constructed to assess statistical associations and risk factors for the development of bleeding from gave using logistic regression and analysis of the maximum likelihood estimates .
odds ratios ( or ) were calculated and were used as an estimation of risk under the rare disease assumption .
individual factors were included in the multivariable model if they were statistically significant ( p<0.20 ) in the univariate analysis , were clinically important , or have been shown in the literature to be important.18,19 multiple iterations of the model were performed .
variables included in the final iteration of the model included presence or absence of cirrhosis , platelet count , hypothyroidism , diabetes , ppi use , nsaid use , hemoglobin , type of gave , egd indication , and ckd .
all data set manipulation and statistical analyses were carried out using sas ( version 9.4 , cary , usa ) . no data involving prisoners were included in this analysis .
all statistical tests for significance were two sided and a significance level of p0.05 was considered statistically significant .
a total of 478 patients underwent egd and were diagnosed with gave between january 2000 and june 2014 .
eighty - four patients were in the cirrhosis group ( 76.4% ) and 26 were in the noncirrhosis group ( 23.6% ) .
in the cirrhosis group , alcohol was the most common etiology of cirrhosis ( 34.5% ) , and the mean meld score was 13.3 . in the noncirrhosis group ,
the most common concomitant disorder was diabetes mellitus ( n=12 ) , followed by ckd ( n=8 ) .
the less commonly associated conditions were bone marrow transplantation recipients , hypothyroidism , and connective tissue disease .
baseline characteristics amongst patients with and without cirrhosis were in general similar with several exceptions ( table 2 ) .
of the covariates analyzed , the platelet count and albumin level in patients without cirrhosis were higher than patients with cirrhosis ( p<0.05 ) .
the indication for initial egd was also similar between the two groups when dichotomized to those performed for suspected gi blood loss versus other indications .
use of anticoagulant or antiplatelet agents was rare amongst our patients , and only one patient in the noncirrhosis group was on an anticoagulant .
aih , autoimmune hepatitis ; dili , drug induced liver injury ; hcv , hepatitis c virus ; nash , nonalcoholic steatohepatitis ; ns , not significant ; nsaid , nonsteroidal anti - inflammatory inhibitor ; pbc , primary biliary sclerosis ; ppi , proton pump inhibitor ; psc , primary sclerosis cholangitis . in univariate analysis ,
active gi bleeding was more common in the noncirrhosis group than the cirrhosis group ( 63.4% vs. 32.1% , p=0.003 ) ( table 3 ) .
there was no statistically significant difference in the baseline levels of hemoglobin or hematocrit between the two groups .
the proportion of patients with cirrhosis who had the striped ( linear ) form of vascular ectasia was 68.0% compared to 46.2% in the noncirrhosis group ( p=0.048 ) .
endoscopic treatment with apc was required more often in patients without cirrhosis than patients with cirrhosis and approached statistical significance ( 27.0% vs. 10.7% , p=0.056 )
. however , there was no difference between the two groups in bleeding severity , as evidenced by similar rates of rebleeding , need to escalate therapy to surgery , and death .
apc , argon plasma coagulation ; bicap , bipolar circumactive probe ; gave , gastric antral vascular ectasia ; ns , not significant .
after adjusting for the presence confounders , our multivariable regression analysis ( table 5 ) showed that absence of cirrhosis was the strongest predictor of active bleeding from gave with or : 5.151 ( 95% ci : 1.084 - 24.480 , p=0.039 ) .
no other variable included in our model , except for inr , was significantly associated with an increased risk of active gi bleeding from gave .
the model was deemed significant ( p=0.041 for likelihood ratio test statistic ) and had good predictability , as the c - statistic of the model was 0.85
. gave , gastric antral vascular ectasia ; inr , international normalized ratio ; ns , not significant ; nsaid , non - steroidal anti - inflammatory drug ; ppi , proton pump inhibitor .
gave is an uncommon cause of acute upper gi bleeding and is traditionally thought to be associated largely with cirrhosis . in our retrospective case - control study , we have found the opposite .
patients with gave in the absence of cirrhosis had a higher risk of developing active gi bleeding and required endoscopic treatment more frequently than those with cirrhosis .
in fact , after adjusting for the presence of comorbidities associated with gave , risk factors for gi bleeding and coagulopathy , the only significant predictor for clinically evident gi bleeding in the setting of gave was the absence of cirrhosis . however , there was no difference in the severity of gi bleeding measured by levels of hemoglobin and hematocrit or the total number of transfusions prior to presentation .
electrocoagulation was the mainstay therapy used in most cases , and the outcome of the therapy was similar between the two groups .
irrespective of cirrhosis , patients had a similar response to treatment in terms of rebleeding events , escalation of treatment to surgery , and mortality .
our findings confirmed those of ito et al6 and expanded them on a much larger scale . in a retrospective study of 30 patients ,
of which 25 had cirrhosis , they observed that patients with gave in the absence of cirrhosis had lower hemoglobin levels .
however , they failed to control for the presence of confounders in their analysis , and the indication for endoscopy in their patient population included variceal screening , which could introduce lead - time bias in the cirrhosis group in which endoscopies were routinely performed to screen for esophageal varices and could recognize gave in asymptomatic patients . in our study , we reviewed each patient s indication for initial egd , which included esophageal varices screening , chronic anemia , and overt gi bleeding .
after excluding esophageal varices screening in asymptomatic patients and only comparing symptomatic patients , we found that egd was done more frequently in the cirrhosis group in the setting of anemia or overt gi bleeding than that of the noncirrhosis group .
the small sample size of symptomatic patients is certainly a limitation ; however , it reflects the fact that gave associated nonvariceal gi bleeding is a rare event .
regardless , ito et al.6 argued that there was an inherent difference in either the pattern of gave ( punctate vs. linear ) or the presence or absence of cirrhosis , but they could not distinguish the independent contribution of these factors with their analysis .
our multivariable analysis argues against this , as the pattern of gave did not reach statistical significance in predicting a patient s likelihood of having active gi bleeding .
in addition , the two patterns of gave were indistinguishable histologically , suggesting that the two entities may be more similar than was previously realized . yet , there is a difference in circulating neurohumoral factors ( prostaglandin e , vip , and 5-ht ) between patients with cirrhosis and those without.1316 further study is needed to define the clinical importance of these biomarkers and to investigate any clinical utility .
our study had several limitations . while it contained a larger sample size than previous studies and was well powered , it was nonetheless retrospective .
missing data was also problematic as roughly of patients who underwent egd and were coded as having gave were excluded for this reason .
another limitation of our study was a lack of follow - up , as our rebleeding rates were calculated at 72 h due to limitations in our retrospective dataset .
this is problematic with gave , in particular , as these patients often required multiple treatment sessions ( 3060% ) , sometimes months after their first treatment.2022
based on our findings and the work of others , we argue that the dogma of gave as largely associated with cirrhosis and/or portal hypertension may in fact be incorrect and that gave should be considered on the differential diagnosis for any patient with objective evidence of acute upper gi bleeding . if gave is diagnosed endoscopically , a thorough investigation for not only cirrhosis of the liver but also other noncirrhosis disease processes associated with gave should ensue .
future studies should focus on the mechanistic differences between gave patients with and without cirrhosis in order to determine if neurohumoral factors differ as a potential explanation for the differences observed clinically . | background and aims : gastric antral vascular ectasia ( gave ) is commonly found in patients with cirrhosis , but it is also associated with other diseases in the absence of cirrhosis . whether gave confers a different severity of gastrointestinal ( gi ) bleeding between patients with and without cirrhosis remains unknown .
we aim to examine whether there is a difference in clinically significant gi bleeding due to gave in patients with or without cirrhosis .
methods : this is a retrospective case - control study of patients who were diagnosed with gave between january 2000 and june 2014 . patients were categorized into cirrhosis and noncirrhosis groups , and those with an additional gi bleeding source were excluded .
univariate comparisons and multivariable models were constructed using logistic regression .
results : in total , 110 patients diagnosed with gave on esophagogastroduodenoscopy ( egd ) were included in our analysis ; 84 patients had cirrhosis ( 76.4% ) and 26 ( 23.6% ) did not .
active gi bleeding was more prevalent in patients without cirrhosis ( 63.4% vs. 32.1% , p=0.003 ) despite similar indications for egd , and endoscopic treatment with argon plasma coagulation ( apc ) was required more often in this group , approaching statistical significance ( 27% vs. 10.7% , p=0.056 ) .
there was no difference in bleeding severity , as evidenced by similar re - bleeding rates , surgery , or death attributed to uncontrolled bleeding .
the strongest independent risk factor for gi bleeding was the absence of cirrhosis ( odds ratio ( or ) : 5.151 ( 95% confidence interval ( ci ) : 1.08 - 24.48 , p=0.039 ) .
conclusions : patients with gave in the absence of cirrhosis are at higher risk for active gi bleeding and require more frequent endoscopic treatment than similar patients with cirrhosis
. it may be worthwhile to treat gave in this population even in the absence of active bleeding . |
we performed this study in the soconusco region of chiapas , mexico across nine coffee plantations ranging from 600 to 1,300 m above sea level ( masl ) .
the soconusco region is largely dominated by coffee agriculture ( 94% ) with small forest fragments mixed throughout the coffee matrix ( 6% ) ( philpott et al .
we established 38 sites that differed in a number of local management and landscape level characteristics and established a minimum distance of at least 300 m between sites .
these sites fell within nine large coffee plantations , ranging in size from 1000 to 6 ha , but most farms were 300 ha .
we selected an intermediate distance ( 300 m ) between sites so as to avoid extreme differences within a given farm while still having varied landscape - level factors between nearby sites .
we used a gps ( garmin gpsmap76csx ) to map an approximate 1-ha circle around each site and documented the abundance and richness of tree species > 10 cm dbh within that area .
2012 ) was used to approximate the total number of tree species within a site .
a prior study in the same area of mexico revealed that leafhopper species respond to the density of inga spp .
trees ( burdine et al . 2014 ) , therefore we calculated the percentage of inga spp . per hectare in each plot .
to estimate the density of coffee bushes , we established a 15 15 m sub - plot at the center of the 1-ha plot and counted all the coffee plants within the plot .
we recorded shade cover using a convex spherical densiometer ( forestry suppliers , inc . ,
jackson , ms , usa ) at three spots in each site , 5 m away from the center of the site at 0 , 120 , and 240. in each of the three spots we measured the shade cover in all four cardinal directions .
we also measured groundcover extent and groundcover species richness in five 0.5-m quadrats set at 5 m from the plot center every 72. cover was estimated using the following ranges : 01 , 25 , 620 , 2140 , 4160 , 6180 , and 81100% . to obtain landscape level factors at each sampling site , we used geographic information systems ( gis ) and measured landscape heterogeneity by digitizing the borders of forests and plantations of varying shade tree management intensity using a basemap in arcgis 10 ( esri 2011 ) .
plantation boundaries were used to define rough categorizations of landscape shade management intensity based on the average percent shade cover of plantations : high- ( > 70% ) , medium- ( 3070% ) , and low- ( < 30% ) shade management .
some plantations had large areas of more than one category of shade intensity level ; therefore we delineated these areas and categorized each area into its appropriate level .
for each site , we calculated percent forest , low - shade , medium - shade , and high - shade coffee land - use types within a 100 , 250 , 500 , and 1,000 m radius surrounding each site .
we also calculated the shannon s diversity index ( ln(p)p ) of the habitat types at these scales .
the response of dependent variables to each landscape factor across these four scales were correlated ( supplementary material ) , therefore we proceeded with only the 1,000 m scale to maximize the differences between the local and landscape scales used in analysis .
we tested for spatial autocorrelation in our data using mantel test of geographic distance among sites and abundance , richness , and shannon s diversity of leafhoppers .
we found no significant correlations between geographic distance and abundance ( mantel r = 0.1010 , p = 0.963 ; r ade4 package ) , richness ( mantel r = 0.0897 , p = 0.884 ) , and shannon s diversity ( mantel r = 0.0640 , p = 0.892 ) of leafhoppers ( legendre and legendre 1998 ) .
table 1.mean and range values of local and landscape factorsmanagement factorsmeanrangelocal factors coffee density ( per 225 m)7421154 shade tree density ( per ha)17363337 shade tree richness ( estimated per ha)291073 inga spp . in plot ( % ) 601694 herbaceous height6.52.412.8 herbaceous cover ( % ) 56.54100 herbaceous spp.6211landscape factors high - shade coffee land - use ( % ) 28.4084 medium - shade coffee land - use ( % ) 37.2096 low - shade coffee land - use ( % ) 26.6086 forest ( % ) 7.7018 habitat diversity ( shannon s index)0.810.171.24other factors elevation ( masl)9425951273alandscape factors were measured at a 1000 m radius surrounding site centers .
mean and range values of local and landscape factors landscape factors were measured at a 1000 m radius surrounding site centers .
leafhoppers were collected by sweep netting 15 coffee bushes in transects on 2 occasions between may and july of 2012 between the hours of 7:00 and 14:00 .
each bush received four upward sweeps of the net starting at the base of the bush and moving upward .
in addition to sweep netting , we also sampled the leafhopper community by using pan - traps in the center of each site on one occasion per site .
we placed two white , yellow , and blue pans ( 350 ml volume bowls ) at the center of each plot .
three bowls , one of each color , were elevated 15 cm above the ground surface and another three bowls were elevated 45 cm above the ground , using pvc piping .
pan - traps were filled with a water solution of salt and liquid dawn dish soap . to make the solution , we mixed 2 kg of table salt and 80 ml of dawn dish soap dissolved in 20 liters of water ( as in burdine et al .
we opened pan - traps at 8:00 and collected pan - traps at 14:00 , maintaining the same order so that all traps were open for roughly the same amount of time .
we then combined the contents of all six pan - traps ( per site ) after sorting , rinsing , and storing contents in 70% alcohol .
each site was defined as a replicate , with individuals combined from the two sweep netting events and the pan trap sample .
we later identified all individuals of the family cicadellidae from both the sweep net and pan - trap sampling and assigned each individual to tribe within subfamilies and morphospecies based on morphological traits and the keys in wilson et al .
( 2009 ) , dmitriev ( 2003 ) , and nielson and godoy ( 1995 ) .
we constructed a species accumulation curve ( maotao estimation ) in the program estimate s ( colwell et al .
2012 ) to determine if we had sampled sufficiently to capture the leafhopper community . to determine the relative significance of local and landscape factors in explaining leafhopper diversity , we conducted multivariate analysis using generalized linear mixed models ( glmm ) .
due to a large number of independent variables and a large number of potential interactions between these independent variables , we first produced two smaller models , one with only local scale factors and another with only landscape scale factors before combining the most parsimonious model into a final local - landscape model ( as in stenchly et al .
we tested for co linearity between independent variables via comparison of variance inflation factor ( vif ; neter et al .
1996 ) and considered a vif above 3 as a factor that was strongly collinear .
we removed the landscape factor percent medium shade and the local factor percent herbaceous cover from models because these significantly increased vif and were thought to be ecologically insignificant or redundant with other factors . in all models , plantation was introduced as a random effect to account for unequal sample - size between plantations .
each landscape model considered percent forest , low shade coffee , high shade coffee , and habitat diversity ( shannon s diversity ) as independent variables ( at 1000 m radii ) .
each local model considered herbaceous height , herbaceous richness , coffee density , shade tree density , estimated shade tree richness , and percent inga tree spp . as independent variables .
we performed model selection via comparison of akaike information criteria corrected for sample size ( aicc ) and parameter number , keeping the most parsimonious models ( burnham and anderson 2002 ) . for the final local - landscape model , the most parsimonious model was selected using aicc and all models with aicc < 2 were examined and finally , we used model averaging to determine the importance of each independent variable ( supplementary material ) .
we constructed local , landscape , and local landscape models for the following leafhopper - dependent variables : total abundance , richness , and the abundance of the five most numerous leafhopper species ( macugonalia redundans [ fowler 1899 ] , sibovia sp . , isogonalia sp . , agallia sp . 1 , and agallia sp .
2 ) . to correct for effects of density on richness and shannon s diversity index ,
abundance was included as a covariate in models investigating variation in richness and in the shannon s index .
we tested for the normality of dependent variable distributions by comparing the residuals of the model with q
abundance and shannon s diversity index followed normal distributions we therefore analyzed these variables with linear mixed effects models with the function lme in the nlme package in the program r ( r 3.0.1 ) ( pinheiro et al .
we analyzed all other dependent variables with glmm assuming a poisson distributed error distributions and log - link functions , with the function
we calculated marginal r of the final models using the r.squaredglmm function from the mumin package ( barto 2013 , nakagawa and schielzeth 2013 ) .
we found a total of 299 individuals of 38 morphospecies . although the observed density was small , the estimated species accumulation curve approached asymptotic species richness for this coffee leafhopper community , suggesting our sampling had captured a significant portion of the leafhopper community ( fig .
2 ( nielson and godoy 1995)made up 37% ( 110 individuals ) of total leafhopper abundance .
fig .
solid line represents mean of estimated richness and dotted lines represent the upper and lower bounds of the 95% confidence interval .
solid line represents mean of estimated richness and dotted lines represent the upper and lower bounds of the 95% confidence interval .
results of the linear mixed effects models revealed that the proportion of low - shade coffee land - use was positively related to the abundance of leafhoppers ( r=0.15 ; table 2 ; fig .
3 ) and shannon s diversity index for leafhoppers increased with coffee density in the plot and was found to be statistically significant ( r=0.58 ; table 2 ; fig .
results for the breusch pagan test revealed no heteroscedasticity of residuals for both models ( p = 0.53 for richness and p = 0.22 for shannon s diversity index ) .
fig .
2.abundance of leafhoppers increased with proportion of low - shade management of coffee at the landscape level.fig .
3.leafhopper richness increased with increase in average height of the local herbaceous layer ( a ) as well as estimated local shade tree richness ( b ) and a decrease in local tree density ( c).fig .
4.shannons diversity index for leafhoppers increased with coffee density in the plot.table 2.results of mixed effects models for response of abundance , richness and shannon s diversity index to local and landscape factorsestimate semzpabundance low - shade coffee land - use9.3 3.62.570.0163*richness abundance0.069 0.0097.41.16e13 elevation0.001 0.00052.40.0154*shannon s diversity index abundance0.08 0.0136.30 * * * coffee density0.006 0.0031.990.0573 abundance of leafhoppers increased with proportion of low - shade management of coffee at the landscape level .
leafhopper richness increased with increase in average height of the local herbaceous layer ( a ) as well as estimated local shade tree richness ( b ) and a decrease in local tree density ( c ) .
results of mixed effects models for response of abundance , richness and shannon s diversity index to local and landscape factors local and landscape factors had differential effects on populations of individual leafhopper species . the abundance of agallia sp . 1 was significantly decreased with habitat diversity in the surrounding landscape as well as the proportion of inga spp .
sites dominated by low - shade management in the surrounding landscape and at higher elevations had more sibovia sp . individuals . the effect of herbaceous height on m. redundans depended on the low - shade management of coffee in the surrounding landscape ( r= 0.37 ) .
2 was most abundant in sites at higher elevations but decreased with diversity of habitats in the surrounding landscape .
2 was influenced by the proportion of forest in the surrounding landscape ( r= 0.54 ; table 3 ) .
table 3.results of mixed effects models for the response of individual leafhoppers to local and landscape factorsestimate semzpsibovia sp .
elevation0.004 0.0022.080.03769 * low - shade coffee land - use2.3 1.032.240.02511 * agallia sp . 1 inga spp . in plot5.66
redundans herbaceous height0.3 0.21.510.13133 low - shade coffee land - use4 . 2.41.810.07088 .
low - shade coffee land - use herbaceous height0.92 0.352.620.00883 * * isogonalia sp .
2 habitat diversity ( shannon s index)3.49 1.163.010.00257 * * elevation0.01 0.0022.660.00782 * * inga spp . in plot7.93
2.583.060.00220 * * forest19.11 12.061.580.1132 forest inga spp . in plot48.75 21.272.290.02193
* results of mixed effects models for the response of individual leafhoppers to local and landscape factors
the results of this study suggest that local and landscape level coffee management factors are primarily responsible for leafhopper diversity .
in particular , we found that low - shade coffee land use at large scales leads to greater leafhopper abundance , while elevation and coffee density explained leafhopper richness and shannon s diversity , respectively .
the increase in total leafhopper abundance with increased landscape intensification ( low - shade coffee land use ) supports the rch ( root 1973 ) .
the rch suggests that total abundance of the pest community should increase when host plant density increases . in this case
, high - shade management should dilute the density of coffee relative to low - shade management . at the same time , diversified agriculture at the local and landscape scales may support higher predator populations , which could limit pests from the top down in these habitats ( bianchiet al.2006 , chaplin - kramer et al .
indeed , within coffee agroecosystems , bird predation of pests is known to be greater in high shade management ( perfecto et al . 2004 ) and in plantations surrounded by a greater proportion of forest ( karp et al .
the observed increase in leafhopper abundance with landscape intensification in our study may be the result of both the concentration of resources and lower predation pressure in intensified areas .
nonetheless , syntheses of the literature have not always supported this hypothesis ( chaplin - kramer et al .
for example , in two reviews of the literature , neither chaplin - kramer et al .
our findings are therefore important in supporting the rch and/or the natural enemies hypothesis at the landscape scale .
our results reflect that leafhopper richness and shannon s diversity find variable support for the rch , which suggests that leafhopper diversity should increase with increased non - crop vegetative abundance or diversity .
leafhopper richness increased with elevation and shannon s diversity increased with coffee density , contrary to the rch .
an increase in pest diversity with increased focal crop density could be expected if most herbivores found are specialists of the focal crop , where each species would increase due to a concentration of resources .
for example , a review of the insect herbivore abundance in polyculture and monoculture crop fields found that 51.9% ( 130/220 ) of monophagous ( specialist ) herbivores increased in monocultures , while only 7.7% ( 17/220 ) increased in polycultures ( andow 1991 ) .
further , 28.4% ( 19/67 ) of polyphagous ( generalist ) herbivores increased in monocultures , while 40.3% ( 27/67 ) increased in polycultures ( andow 1991 ) .
however , while we are aware that at least a few of the species we studied consume coffee , none should be considered coffee specialists ( gonthier , personal observation ) .
given that low - shade coffee systems have increased fertilization and planting density , it is possible that this productivity may promote herbivore diversity .
greater productivity can lead to greater herbivore diversity through a number of mechanisms including : increased abundance of rare resources , greater persistence of rare species , or through density dependent effects ( siemann1998 ) .
leafhoppers , like other xylem - feeding insects , are nitrogen limited ( mattson 1980 ) , thus increased nitrogen fertilization may improve the persistence of species that feed on coffee .
yet another alternative explanation of increased richness with intensification could be that greater predation in high - shade coffee results in fewer species ( as in perfecto et al .
hence , it is plausible that the combined effects of more readily available resources and lower predation pressure could lead to greater richness .
however , we can only provide speculation and more research is needed to determine the mechanism behind this pattern .
montane tropical communities , in particular , are affected by climatic changes as the temperature at lower elevations shifts to higher elevations
. studies of bird and moth assemblages showed that these groups have shifted up slopes to cope with climate change ( chen et al .
if climate change continues in this study region , we might expect to see more leafhopper species at higher elevations .
it is also possible that more leafhopper species were found at higher elevations because they may be released from their natural enemies .
a recent study conducted across the same region as this study , reported a lower abundance and richness of spiders at higher elevations .
as spiders are generalist predators this finding may represent a reduction in the potential biocontrol services found at higher elevations ( hajian - forooshani et al .
a simultaneous shift of biological control to lower elevations and herbivore diversity to higher elevations may be problematic to coffee production because although leafhoppers are only considered minor pests in coffee , they can be important plant disease vectors .
some leafhopper species carry the bacterium ( x. fastidiosa ) , responsible for causing diseases in a number of important commercial plants like almonds , grapes , citrus , alfalfa , coffee , and ornamentals ( li et al .
most of the species found in the cicadellinae subfamily have been observed to transmit x. fastidiosa ( redak et al . 2004 ) .
recent reports of x. fastidiosa infecting coffee in costa rica and brazil ( beretta et al .
although incidence of coffee leaf scorch or crespera disease caused by x. fastidiosa is not reported in the soconusco region of mexico , it is possible that leafhoppers pose a future risk in vectoring these diseases .
our study highlights the need to understand the factors that affect the population distribution of leafhoppers in order to mitigate potential risks .
only two other studies , to our knowledge , have investigated the distribution of leafhoppers across an intensification gradient of coffee agroecosystems .
ramos ( 2008 ) found that species richness was positively correlated with fallows in the surrounding landscape .
in addition , forest cover in the landscape positively correlated with leafhopper abundance at 100 m scale , but not at the 500 m scale .
contrarily , pasture at both 100 and 500 m scales was negatively correlated with total abundance .
the differences between our findings and ramos ( 2008 ) could be due to the difference in the regional land use composition . while our study area was dominated by coffee land use ( 94% ) , with only small forest fragments consisting of
roughly 6% of the regional land area , ramos study area had a greater diversity of agricultural land uses ( 28% pasture , 14% coffee , 6% sugarcane , and 3% other crops ) , and a higher percentage ( 40% ) of forest cover as compared with our study area .
besides agallia sp.2 , forest fragments in the surrounding landscape had no effect on any of the other most abundant leafhoppers in our study .
perhaps the larger forest fragments in ramos ( 2008 ) study region resulted in strong effects of forests on leafhopper abundance .
burdine et al . ( 2014 ) found that the richness and abundance of leafhoppers increases with the number of inga spp .
( fabaceae ) form n - fixing associations with bacteria and often have higher nitrogen content than other plant species .
these plants could be important for leafhopper communities because leafhoppers are often nitrogen limited ( mattson 1980 ) .
trees locally , did not correlate with the overall leafhopper abundance , but did correlate with the abundance of agallia sp .
isogonalia sp . while our study contained a greater number of factors related to coffee agricultural intensification and covered a much larger regional extent across many more farms , burdine et al .
( 2014 ) studied leafhoppers during both the rainy and dry seasons , but did not investigate the effects of landscape characteristics .
furthermore , in our study larger - scale landscape effects of management may have influenced the strength of the effect of local shade tree composition .
interaction between local and landscape factors were also seen in the effects of herbaceous height on abundance of m. redundans , which depended on the proportion of low - shade coffee land use .
indeed , effects of local level management factors can change with surrounding landscape and this has been hypothesized in other systems ( tscharntke et al .
we found that abundance of only two out of the five most abundant species sibovia sp . and
m. redundans was influenced by low shade coffee use in the landscape despite our results for total leafhopper abundance . in the case of m. redundans ,
the effect of the height of herbaceous cover was influenced by the proportion of low - shade coffee land use .
however , we also found that habitat diversity in the landscape negatively affected the abundance of both the agallia spp . , suggesting a possible preference for a more homogeneous or simple landscape .
our results indicate that coffee management at both the local and landscape level are important drivers of leafhopper diversity .
although these insects in the family cicadellidae are not considered a major threat to coffee , some of them are vectors for diseases and pose potential risk to future coffee production .
our results are also important in explaining the factors that play a key role for the conservation of these insects in light of global land - use change .
this research was supported by a nsf doctoral dissertation improvement grant ( # 1309786 ) to d. gonthier and i. perfecto and an nsf biology postdoctoral fellowship ( # 1401874 ) to d. gonthier .
| the intensification of agriculture drives many ecological and environmental consequences including impacts on crop pest populations and communities .
these changes are manifested at multiple scales including small - scale management practices and changes to the composition of land - use types in the surrounding landscape . in this study
, we sought to examine the influence of local and landscape - scale agricultural factors on a leafhopper herbivore community in mexican coffee plantations .
we sampled leafhopper ( hemiptera : cicadellidae ) diversity in 38 sites from 9 coffee plantations of the soconusco region of chiapas , mexico . while local management factors such as coffee density , branches per coffee bush , tree species , and density were not important in explaining leafhopper abundance and richness , shade management at the landscape level and elevation significantly affected leafhoppers . specifically , the percentage of low - shade coffee in the landscape ( 1,000-m radius surrounding sites ) increased total leafhopper abundance .
in addition , shannon s diversity of leafhoppers was increased with coffee density .
our results show that abundance and diversity of leafhoppers are greater in simplified landscapes , thereby suggesting that these landscapes will have higher pest pressure and may be more at - risk for diseases vectored by these species in an economically important crop . |
acute myeloid leukemia ( aml ) is a malignant clonal disorder of immature myeloid hematopoietic cells that is the most common type of acute leukemia among adults .
reported mean age of aml patients is 60 . some factors such as viruses , radiation , chemotherapy , benzene exposure and smoking increase risk of aml ( 1 - 4 ) .
also , agents such as age , molecular and genetic changes such as flt3 , npm1 , mll disorders and hematologic indices are important prognostic factors in aml process ( 5 - 11 ) .
in addition to mentioned genes , brain and acute leukemia - cytoplasmic ( baalc ) gene is an important factor in treatment and prognosis determination ( 12 - 16 ) .
overexpression of baalc gene is reported in aml subjects with trisomy 8 abnormalities in gene expression studies ( 12 ) .
it is a new marker in hematopoietic progenitor cells , which its effects are indeterminate on cellular mechanism ( 12,13,17 - 19 ) .
different studies emphasis on gene expression level impression on different physiologic and pathologic circumstances prognosis . generally , baalc expression is a suitable prognostic marker which can be used to assign survival rate in normal cytogenetic aml ( cn - aml ) individuals ( 12,14 ) .
no study was found in aml patients in mentioned field in north east of iran , thus , the aim of this study was to define amount of gene expression and frequency of up and down regulation of baalc gene , and to determine the survival rate of affected patients .
in this case - control study , 94 individuals referred to molecular pathology research center of ghaem hospital in mashhad city during 2012 - 2015 were recruited in 2016 . from all , 47 cases were normal cytogenetic aml and others were healthy individuals that were studied as control group .
all patients were categorized based on french - american - british ( fab ) criteria .
extracted information included peripheral blood and bone marrow blast count , red and white blood cells and platelets , mean cell volume ( mcv ) , mean cell hemoglobin ( mch ) , mean cell hemoglobin concentration ( mchc ) and clinical signs including splenomegaly , hepatomegaly , and lymphadenopathy and bleeding protests .
it is necessary to mention that this research was approved by ethics committee of mashhad university of medical sciences , code 839 .
peripheral blood mononuclear cell were separated by ficoll gradient centrifugation method and sigma aldrich kit ( sigma aldrich company , saint louis , mo , usa ) . in the following rna extraction
was done by tri - pure solution ( tri - pure kit , cat no .
revertaid h minus first strand cdna synthesis kit ( fermentase company , thermo scientific 100 reactions ) was applied for cdna synthesis .
gpi gene control primers were evaluated for cdna qualification confirmation , by pcr method ; the results were checked on agarose gel .
qualification of synthesized cdna was evaluated by nano drop instrument ( thermo scientific 2000 , made in finland ) in 260 nm wavelength.
all samples concentration was > 450ng/ l . studied genes sequences were obtained from baldus .et al study ( 13 ) . to determine
primers specificity and evaluation of sequence homology blast software were applied which is available in ncbi site .
forward primers for gpi gene were 5'- tcttcgatgccaacaaggac-3 ' and 5'-cttttgcaggcattctcttagca-3 ' for baalc gene .
the reverse primers was 5-gcatcacgtcctccgtcac-3 ' for gpi and 5'-cttttgcaggcattct cttagca-3 ' for baalc gene .
the used probe sequences were 5-ttcagcttgac cctcaacaccaac-3 ' for gpi and 5'-ctcttttagcctctgtggtctgaaggccat-3 ' for baalc.
real time pcrs were applied in thermo cycler abi ( made in usa ) .
all samples were tested in duplicate . rt - pcr reaction master mix ( 20l for each reaction ) contained 10l taq man master mixes , 0.5l rox stain , 1l of each forward and reverse primers ( 10 pm ) , 1l taq man probe ( 5 pm ) , 2l cdna , 4.5l double distilled sterile water .finally the following temperature patterns were planned in termocycler ; 50c for 2 minutes , 95c for 15second , 95c for 15 seconds , and 60c for 1 minute . the second , third and fourth steps were repeated for 40 times .
melting curve analysis was done for products specificity confirmation.
chi - square , t - test , kruskal - wallis and mann- whitney tests were applied for analysis based on data distribution ( checked using kolmogorov - smirnov test ) , through spss v.21 .
there were 26 ( 55.3% ) male and 21 ( 44.7% ) female in patient group .
patients were categorized to child and adult ; the first one was 15 and consisted of 13 ( 27.5% ) and the second was > 15 which consisted of 34 ( 72.5% ) approached subjects .
most of patients were in m1 ( 29.78% ) and m2 ( 27.65% ) subtypes based on fab categorization .
mean of peripheral blood and bone marrow blasts were 39.0% and 66.2% in aml cases , respectively .
mean of hb , hct and rbc indices were significantly different between the two groups ( p=0.03 ) , which is justifiable with occurred pancytopenia . in comparison of baalc gene expression
there was no any significant relation between amount of gene expression and clinical and hematological parameters ( p=0.02 ) . among hematologic indices ,
rbc count had significant invert relation with amount of baalc gene expression ; patients ' age and sex were related to baalc expression too ( p>0.05 ) .
gene evaluation was associated with up regulation in 47% of cases and down regulation in 53% of patients .
survival rate was assessed during 36 months in 47 aml patients and it was 26 months in down regulation subjects and 33 months in up regulation individuals .
there was significant relation between up regulation of baalc and survivability reduction ( p=0.04 ) .
in the recent decades , genomic profile has been used to identify prognostic factors of leukemia .
erg , baalc , mn1 and wt1 genes are considered as categorization indices based on gene expression patterns ; they can be used to distinguish therapeutic intervention and disease process in aml patients ( 13,20 ) .
although baalc overexpression mechanism has not been recognized yet , but it is reported in myeloid leukomogenesis . this up regulation prevents myeloid blast differentiation and is associated with poor prognosis in aml and acute lymphoblastic leukemia ( all ) individuals .
different research revealed that baalc up regulation has been observed in glioblastoma , melanoma and gastrointestinal cancers ; they suggested an oncogenic role for baalc ( 21 ) .
this gene expression is known as a prognostic and survival rate marker in normal cytogenetic aml cases ( 12 ) .
baalc up regulation was seen in 47.00% of aml group members of this study ; damiani .et al research on 175 aml patients by rt - pcr method revealed up regulation in 50% of studied cases , and recommended it as a suitable marker for minimal residual disease ( mrd ) assignment ( 15 ) .
high baalc expression was reported in 48.51% of 125 cn - aml individuals of yoon 's study in korea too ( 22 ) .
baldus study on baalc gene expression and flt3 mutation in normal cytogenetic aml patients under 60 years found no significant differences between age , sex and bone marrow blast count and amount of expressed baalc gene , before treatment ; he mentioned poor prognosis of baalc up regulation too ; while cited gene overexpression and peripheral blood blasts increment were seen in m1 and m0 subtypes . in current study ,
parts of our findings were supported by baldus ( 13 ) and weber 's ( 12 ) studies which expressed that there were no any significant relationships between baalc expression , gender and hematological parameters ( wbc , hb , bone marrow and peripheral blood blasts count ) .
yahya rs et al in their study about prognostic role of baalc gene in aml patients in egypt in 2013 represented up regulation in 48.9% of studied cases ; while they did not report any relations between baalc up regulation and clinical parameters .
they reported baalc up regulation in association with patient 's survival rate reduction and indicated to its prognostic role ( 16 ) .
we calculated amount of cited gene expression in each morphologic group ( based on fab ) , the most up regulated cases were m1 and m2 ; as mentioned before it is reasonable according to blast count in these groups .
these findings are similar to baldus .et al reported result about normal cytogenetic aml patients under 60 ; as it mentioned before they observed overexpression of baalc along with peripheral blood blast increment in m1 and m0 and down regulation in m5b subtypes .
their findings support our results about poor prognosis of baalc over expression too ( 13 ) .
liu et al stated that baalc overexpression is seen in undifferentiated cells ; they found the most up regulation in m0 , m1 and m2 categories too , same as our findings ( 23 ) .
current study emphasized that baalc up regulation is increased in aml patients , and it is more common in m1 and m2 subtypes . | background : acute myeloid leukemia ( aml ) is known as one of the most common leukemia among adults .
environmental and different genetic factors affect disease process , prognosis and treatment . among different
genetic factors
npm1 , flt3 , mll and baalc genes are the most effective on patient 's survival rate .
the aim
of this study was to assess amount of baalc gene expression in aml patients , and its relation to survival rate .
methods : in this case - control study , from all 94 individuals referred to ghaem medical center during 2012 - 2015 , 47 cases were normal cytogenetic aml and others were healthy individuals that were studied as control
group .
real - time pcr method was applied for gene expression evaluation .
other information of patients was
extracted from medical documents .
spss v.21 was used for data processing .
results : mean age of studied cases was 31.50 years . the most of baalc gene expression was seen in m1
and m2 subtypes , and the less was in m5 .
a significant relation was found between amount of gene expression
and patient 's survival rate .
conclusion : baalc gene expression was increased significantly in aml cases .
baalc expression had reverse
relation with patients ' survival rate in north - east of iran . |
a reactive leucocytosis , with a significant increase in early neutrophil precursors , exceeding 50,000/mm is referred as leukaemoid reaction .
these reactions are generally benign , but may imitate more serious conditions such as chronic myelogenous leukaemia .
leukemoid reaction may be a paraneoplastic manifestation of several cancers such as lung , gastrointestinal , genitourinary , ovarian , head and neck cancers and hepatocellular carcinoma .
non - cancerous conditions that can precipitate a leukemoid like state include those of situations of stress , infection and inflammation .
the cytokines implicated in this process include granulocyte - macrophage colony - stimulating factor ( csf ) , granulocyte - csf ( g - csf ) , interleukin-3 ( il-3 ) and il-6 .
a 54-year - old female with angina ( nyha - class ii ) , double vessel disease ( left main coronary artery equivalent , distal left anterior descending artery - 95% , distal circumflex artery - 80% ) with good left ventricular ( lv ) function was referred for coronary revascularisation .
pre - operative investigations revealed a white blood cell ( wbc ) count of 12,900/cmm ( neutrophils 61% , monocytes 6% lymphocytes 29% eosinophils 4% ) and urinalysis was within the normal limits . the raised total leukocyte count ( tlc )
was possibly attributed to her type 2 diabetic status , the radial artery angiogram procedure that was done 2 days prior to the pre - anaesthetic check - up and possibly to the recent angina that the patient had at the time of admission ( 1 week ) .
although she remained asymptomatic an early coronary artery bypass grafting ( cabg ) was planned .
cabg was performed using the left internal mammary artery and left great saphenous vein graft . cardiopulmonary bypass ( cpb )
time was 110 min with aortic cross - clamp for 58 min , using antegrade cold crystalloid cardioplegia .
post - operatively ( on the day of surgery ) routine blood tests demonstrated a mild leucocytosis ( 20,900/cmm ) with marked neutrophilia ( 71% ) , lymphocytosis ( 20% ) , and monocytosis ( 9% ) .
on the first post - operative day the tlc increased to 25,000/cmm and to 29,500/cmm on second post operative day .
this increasing trend in the wbc prompted us to change the antibiotic from first line antibiotics ( cefuroxime + ofloxacin ) to second line antibiotics ( cefoperazone + sulfactum + ofloxacin ) assuming a subclinical infection due to her diabetic status .
blood , urine and sputum culture showed no growth of organisms after 48 h. laboratory work - up for infective ( bacterial and parasitic ) aetiology revealed negative results .
the usual cardiac supports were tapered off on the third post - operative day and the patient shifted to intermediate care unit on the fifth day in spite of her rising total wbc count .
the wbc count reached 33,000/cmm on the third day and 41,000/cmm on the 4 post - operative day without any signs of infection .
on the fifth post - operative day the tlc increased to 49,400/cmm ( neutrophils 54% , lymphocytes 30% , and monocytes 16% ) .
inflammatory parameters were mildly elevated ( c - reactive protein [ crp ] 38 mg / l ) .
a peripheral smear showed marked leucocytosis , predominately neutrophils and mild shift to left was noted with presence of myelocytes and band forms .
red blood cell ( rbc ) were chiefly normocytic , with increased polychromasia and occasionally normoblast .
serum procalcitonin to rule out sepsis was not assessed as the patient was clinically stable with mildly elevated crp levels and also keeping in mind the limited resources , the cost factor and delay in reporting .
chest x - ray and ultrasound abdomen did not reveal obvious causes of possible solid viscera tumours that can cause a leukemoid reaction .
computed tomography ( ct ) of chest , abdomen and pelvis were postponed , to be obtained at review . on 10 post - operative day
thus a provisional diagnosis of leukemoid like reaction - post cabg surgery was made and patient discharged on the 10 post - operative day with an advice for follow - up 1 week later .
the tlc was 15,000/cmm with neutrophils 68% , eosinophil 2% , monocytes 6% , and lymphocytes 24% on her next visit which confirmed the diagnosis of leukaemoid like reaction .
leukocytosis exceeding 50,000 wbc / cmm with a significant increase in early neutrophil precursors is referred to as a leukaemoid reaction . in most cases
certain types of chronic infection such as mononucleosis , malaria , and tuberculosis , certain types of cancers , hodgkin 's disease and autoimmune anaemia may cause leukemoid reactions [ table 1 ] . thus it is a clinical syndrome in which changes are found in the peripheral blood similar to what occurs in people with leukaemia , but not the result of leukaemic disease .
leukaemoid reaction has also been reported to occur in allergic reaction of phenobarbital , an aromatic anticonvulsant drug [ table 1 ] .
common causes of leukaemoid reaction in our patient , the possible cause of the raised tlc with monocytosis during the pre - operative time would have been due to the angina pectoris , which she had at the time of admission .
the study by maekawa et al . showed that the peak monocyte count had a significant positive correlation with lv end - diastolic volume obtained from pre - discharge left ventriculography .
patients with pump failure or lv aneurysm had higher peak monocyte counts than those without these complications .
multivariate analyses showed that a peak monocyte count 900/cmm was an independent determinant of pump failure , lv aneurysm and long - term adverse cardiac events .
post cabg our patient presented with a rise in tlc which was due to increased neutrophil count along with relative lymphocytosis and monocytosis .
the possible causes resulting in the scenario could be malignancies ( carcinoma , plasma cell myeloma , or lymphoma ) , chronic infections or autoimmune disorders .
all laboratory work - up for acute and chronic infective ( bacterial and parasitic ) aetiology revealed negative results .
blood peripheral smear showed marked leucocytosis , with predominately neutrophils with presence of myelocytes and band forms .
the other possible reason for this abnormal behaviour may be due to inflammatory response to the extra - corporeal circulation .
studies have proved that after revascularisation the total blood monocyte counts tend to rise temporarily , peaking between day 2 and 3 after intervention .
the resident macrophages in the myocardium are activated and synthesize and secrete humoral factors that cause monocytopoiesis .
cardiac surgery produces an altered activation of the inflammatory response due to the combination of surgical trauma , cpb and ischemia - reperfusion injury .
in the case of our patient , it appears that the inflammatory storm generated by cardiac surgery with cpb precipitated a dramatic myelomonocytic leukemoid reaction , with no clinical picture of sirs and multiple organ dysfunction syndromes .
the question that arose was why our patient responded to the inflammatory stress of surgery in such an unpredictable fashion .
our prime suspect was that of undiagnosed underlying chronic myelomonocytic leukaemia ( cmml ) or any other malignancies , which may progress to the acute phase of leukemoid reaction , a complex and poorly understood response by the bone marrow to stress . in this patient
, it appears that the inflammatory storm generated by cardiac surgery with cpb precipitated a dramatic myelomonocytic leukemoid like reaction .
a leukemoid reaction may rarely prove rapidly fatal although the cause of disease acceleration and potential remitting factors remain obscure .
our patient was planned for a later bone marrow biopsy to exclude leukaemia and finally demonstrated a lack of myeloid clonality .
thus with the provisional diagnosis of leukaemoid reaction , investigations such as bone marrow biopsy , lap score and ct thorax abdomen pelvis were deferred in consultation with the physicians and surgeons .
investigation of the bone marrow including immunophenotyping could have helped us to differentiate between leukaemia and a leukaemoid reaction .
lap scoring could confirm our provisional diagnosis , while ct thoracoabdominopelvis can help to rule out solid tumours that commonly produce leukaemoid reaction .
a paraneoplastic leukaemoid reaction can be caused by increased serum levels of g - csf or other growth factors , which are considered to be produced by the malignant cells , mostly from an endothelial tumour .
the only report of off - pump cab in a patient with cmml , performed via a median sternotomy , highlighted concerns over leukaemoid transformation and the assumed benefits of a reduction in cytokine release .
when wbc counts increase grossly , a cause outside the bone marrow has to be searched for .
the leucocyte count was showing increasing trend throughout the post operative period after cabg in the present case and no clear source could be found , including sepsis and hence , we termed it as leukaemoid ( wbc count > 50,000/cmm ) like reaction . | the presentation of leukemoid reaction in patients post - cardiac surgery is rare with limited prior reports in the english language literature .
we report a case of raised leukocyte count with no evidence of infection in a patient post coronary artery bypass graft surgery . the exaggerated inflammatory response by the patient to extra - corporeal circulation
was drastically elevated , but fell short of the leukaemoid reaction definition
so we have defined it as a leukaemoid like reaction .
a clear correlation between the extra - corporeal circulation and inflammatory response is documented . |
in recent years , the field of radiology has advanced from reviewing films on a view box to a digital era with viewing on a computer monitor .
the digital environment has opened the doors to new image processing techniques that have capitalized on the three - dimensional ( 3d ) datasets of computed tomography , magnetic resonance imaging ( mri ) , and positron emission tomography .
for example , maximum intensity projection and volume rendering provide a two - dimensional representation of the 3d volume and can better define some complex anatomy ; however , these techniques are limited by overlapping structures1,2 ( figure 1 ) . in this paper
, we will discuss several features of the depth 3-dimensional ( d3d ) augmented reality imaging system .
the d3d user wears a headset , similar to that used in 3d gaming , which provides a separate image to each eye , yielding binocular disparity and depth perception.3
d3d also provides convergence of the eyes to a particular focal point of interest , tracks the individual s natural head movements , and provides corresponding images to each eye s display ; thus , an immerse environment is established .
the d3d algorithm provides an enhanced discrimination of separated points in space that might otherwise appear to overlap when viewed by conventional methods .
s enhanced visualization is accompanied by a joystick control system , similar to that of a pilot such that the radiologist can fly through the image while selecting objects of interest with a 3d cursor .
while d3d technology can be applied to multiple types of radiological imaging , the specific focus in the article will be related to viewing of microcalcifications in mammography .
breast cancer is one of the leading causes of death in women.4,5 mammography can detect suspicious microcalcifications associated with preinvasive or invasive cancers.6 while breast calcifications are extremely common , and can be present in up to 86% of mammograms,7 a minority of calcifications biopsied tend to be preinvasive or invasive cancers in asymptomatic patients .
a linear and branching distribution of microcalcifications is suspicious for ductal carcinoma in situ ( dcis).8,9 standard mammographic views may not reveal the true linear and branching distribution due to a suboptimal viewpoint . from the wrong viewpoint
, a linear distribution of calcifications can appear as an amorphous aggregate , leading to false negatives .
on the other hand , depending on the angle of view , a cluster of calcifications can be wrongly interpreted to follow a linear pattern , causing false positives .
consequently , full 3d viewing of a group of calcifications has the potential advantage of reducing both false negatives and false positives . to test this hypothesis , in the present study
, we generated image sets from a simulated dataset of microcalcifications and evaluated the discrimination of a branched or linear pattern when viewed with d3d .
a simulated 3d dataset of microcalcifications in a linear , branching pattern was created using a total of 34 voxels in a 646464 matrix by an in - house script , with each microcalcification being of the same size . using a cartesian coordinate system ,
the simulated 3d dataset was uploaded such that each microcalcification was assigned to a unique ( x , y , z ) voxel within the volume of interest and a total of 262,144 ( 643 ) voxels were created of which 34 were assigned the value of 1 to represent calcification displayed as a white pixel .
all other voxels within the volume of interest were assigned the value of 0 to represent noncalcified tissue .
an initial left eye viewing perspective was assigned an ( x , y , z ) position outside the volume of interest . from the left eye viewing perspective
, a series of cones , each being defined by the cone angle and the trajectory angles and , were sent into the volume of interest to record the highest voxel in its path , either a 0 or a 1 ( figure 2 ) .
finally , the values recorded for each cone were displayed in the head display unit with a unique image sent to each eye such that binocular disparity and depth perception are achieved ( figure 3 ) .
the number of cones for each eye viewing perspective is determined by factors including : size of the pixel array in the head display unit , acceptable latency time for the user , and the computer processor speed .
after the initial left and right eye images are established ( figure 4a ) , a new image is provided to each eye display each time any of the following occurs : changing the interocular distance to provide greater binocular disparity ( figure 4b ) ; changing the angular field of view ( figure 4c ) ; volume of interest is translated or rotated ( figure 4d ) ; user rotates their head with roll , pitch , or yaw maneuvers , by translation or combination thereof ( figure 4e ) .
depth perception is the ability to distinguish the relative distance of an object in one s visual field .
depth perception requires binocular disparity , which is the difference in image location seen by the left and right eyes .
the simulated data were viewed with the d3d system by the author ( ew ) , who is a board - certified radiologist with 11 years of experience in evaluating the distribution of the microcalcifications .
using simulated data allows one to visualize differing orientations and gain an appreciation of the importance of 3d visualization from multiple viewing points in obtaining an accurate diagnosis .
a simulated 3d dataset of microcalcifications in a linear , branching pattern was created using a total of 34 voxels in a 646464 matrix by an in - house script , with each microcalcification being of the same size . using a cartesian coordinate system ,
the simulated 3d dataset was uploaded such that each microcalcification was assigned to a unique ( x , y , z ) voxel within the volume of interest and a total of 262,144 ( 643 ) voxels were created of which 34 were assigned the value of 1 to represent calcification displayed as a white pixel .
all other voxels within the volume of interest were assigned the value of 0 to represent noncalcified tissue .
an initial left eye viewing perspective was assigned an ( x , y , z ) position outside the volume of interest . from the left eye viewing perspective
, a series of cones , each being defined by the cone angle and the trajectory angles and , were sent into the volume of interest to record the highest voxel in its path , either a 0 or a 1 ( figure 2 ) .
finally , the values recorded for each cone were displayed in the head display unit with a unique image sent to each eye such that binocular disparity and depth perception are achieved ( figure 3 ) .
the number of cones for each eye viewing perspective is determined by factors including : size of the pixel array in the head display unit , acceptable latency time for the user , and the computer processor speed .
after the initial left and right eye images are established ( figure 4a ) , a new image is provided to each eye display each time any of the following occurs : changing the interocular distance to provide greater binocular disparity ( figure 4b ) ; changing the angular field of view ( figure 4c ) ; volume of interest is translated or rotated ( figure 4d ) ; user rotates their head with roll , pitch , or yaw maneuvers , by translation or combination thereof ( figure 4e ) .
depth perception is the ability to distinguish the relative distance of an object in one s visual field .
depth perception requires binocular disparity , which is the difference in image location seen by the left and right eyes .
the simulated data were viewed with the d3d system by the author ( ew ) , who is a board - certified radiologist with 11 years of experience in evaluating the distribution of the microcalcifications .
using simulated data allows one to visualize differing orientations and gain an appreciation of the importance of 3d visualization from multiple viewing points in obtaining an accurate diagnosis .
when the author visualized the microcalcifications with a single viewing perspective , the microcalcifications were classified as a cluster , which is indeterminate for cancer ( figure 5a ) .
when the author visualized the microcalcifications with d3d , the system was rotated and the microcalcifications were classified as a linear pattern ( figure 5b ) , which is suspicious for breast cancer . finally , the image was rotated further and the microcalcifications were classified as a branching pattern , which is highly suspicious for dcis breast cancer ( figure 5c ) .
the d3d s augmented reality imaging technique is an extremely computationally demanding process that is operated only through the recent advances in computer processing and head display unit technology . using a simulated image , d3d achieves a higher level of convergence and improved human machine interface ( hmi ) to discriminate linear or branching calcifications .
while conventional two - view mammography can not assign microcalcifications to a specific 3d coordinate system , digital breast tomosynthesis and dedicated breast computed tomography are able to provide 3d spatial localization ; thus , d3d can only be used for these types of breast imaging.1015 d3d suggests that the preferred method of looking at calcifications is a fly - through such that the radiologist can better appreciate the 3d pattern .
dcis can be multifocal , with regional variation in the histologic grade that predicts the risk for progression to invasive cancer .
therefore , it is crucial to identify and treat all areas of dcis for the best possible outcome . whether viewing with contiguous slices or volume rendering , characterizing the 3d pattern of microcalcifications can be extremely difficult because there is no depth perception and no convergence of the viewing perspectives to a focal point .
d3d provides an immersive fly - through perspective such that a 3d map of the suspicious microcalcifications can be drawn and highlighted . beyond improving the display of microcalcifications
, d3d holds promise for enhanced visualization and discrimination of malignant breast masses within a dense breast or behind areas of benign pathology or previous surgery . in addition
, d3d can give the viewer the ability to fly - around a breast mass and highlight the change in the shape or volume of the mass before and after therapy , by comparing the current mass to a 3d ghost of the mass recalled from a pretreatment baseline .
there is strong rationale to support the value of dynamic 3d view of a tumor mass throughout the course of therapy .
while the efficacy of neoadjuvant chemotherapy ( nact ) in advanced breast cancer is known to correlate to tumor shrinkage,16 data from the recent investigation of serial studies to predict your therapeutic response with imaging and molecular analysis ( i - spy ) trial demonstrated the importance in breast tumor morphology . in this study ,
the particular phenotype of the breast tumor , such as well - defined margins or multilobulated appearance , correlated with the pathologic response to nact and contributed to the clinical recommendation for breast conservation therapy instead of mastectomy.17 in fact , the mri findings were a better predictor of pathological response to nact compared with clinical assessment.18 in addition to enhanced visualization of the changing 3d morphology of the tumor , d3d can also help make an accurate volume and shape comparison of the tumor at two or more time points as a quantitative measure of effectiveness of nact . in the current practice , shifts in patient position from scan to scan can change the position and orientation of a breast tumor , which obscures subtle differences between treatment cycles .
d3d can register the image to a 3d grid that is tied to an anatomic reference point .
proper position and orientation of the ghost images representing prior states of the mass can permit color - coding of the regions of the tumor that are expanding or shrinking with an automatic calculation of the volume of change .
in addition to mammography , the d3d s augmented reality technique can be applied to many of the radiological subspecialties . in neuroradiology ,
characterization of a cerebral aneurysm s morphology , orientation , neck , and relationship to other vessels is extremely important in both follow - up and in surgical planning .
d3d can facilitate understanding the interrelationship of comminuted fracture fragments to guide surgery and to accurately orient the placement of prosthetic joints . in pulmonary imaging , the depth perception by d3d may help to better identify , characterize , and follow - up pulmonary nodules . in virtual colonoscopy , the improved hmi may speed up the examination and offer improved lesion detection . for all of these applications , and others to be conceived , the fact that d3d technology can input conventional digital radiologic images will permit thorough and rapid side - by - side comparison with the current standards .
new radiological advances such as additional mri sequences , new contrast agents , and thinner imaging planes are expanding the information presented to the radiologist .
it is becoming impractical for the radiologist to scroll through hundreds of slices to evaluate each item on the checklist .
there is a growing need to optimize presentation of radiological images in a practical manner , whereby the radiologist can view the whole volume of data at once .
not only does d3d of the future offer such an improved visual display , but also it will provide nonvisual sensory information to the user , including auditory and tactile feedback .
for example , mr elastography provides information on the stiffness of tissue and d3d s augmented reality presents this as a tactile response to the stiffness by a glove .
d3d s augmented reality provides new opportunity for enhanced visualization and improved hmi , creating the potential for improved diagnosis and response to therapy .
d3d is an augmented reality medical imaging system that provides stereoscopic 3d imaging with binocular disparity .
furthermore , d3d provides hmi in the form of a gaming joystick to fly inside the image and view the 3d lesion from any angle .
we have illustrated , using a simulated dataset of breast microcalcifications , how the d3d system can reveal a linear branching morphology that would have otherwise been invisible under conventional imaging .
future testing of the d3d should be performed to determine the utility of the system in mammography and other radiological subspecialties . | purposethe purpose of this article is to present images from simulated breast microcalcifications and assess the pattern of the microcalcifications with a technical development called
depth 3-dimensional ( d3d ) augmented reality.materials and methodsa computer , head display unit , joystick , d3d augmented reality software , and an in - house script of simulated data of breast microcalcifications in a ductal distribution were used . no patient data was used and no statistical analysis was performed.resultsthe d3d augmented reality system demonstrated stereoscopic depth perception by presenting a unique image to each eye , focal point convergence , head position tracking , 3d cursor , and joystick fly-through.conclusionthe d3d augmented reality imaging system offers image viewing with depth perception and focal point convergence . the d3d augmented reality system should be tested to determine its utility in clinical practice . |
benign primary intramural tumors of the esophagus are rare and account for approximately 2% of all esophageal tumors .
most of them are esophageal leiomyomas ; less frequent are schwannomas or malignant masses such as gastrointestinal stromal tumors ( gist ) .
endoscopic ultrasound biopsy or frozen section biopsy during the operation does not differentiate the tumors .
we report a case of benign esophageal schwannoma causing progressive dysphagia in a 37-year - old woman .
a 37-year - old woman presented due to symptoms of chest pain during swallowing of solid food for 3 months .
the physical examination was noncontributory . computed tomography of the chest showed a soft tissue mass in the lumen of the upper esophagus ( fig .
endoscopic ultrasound identified the mass at 19 cm from the incisors , but the biopsy was nondiagnostic .
computed tomography scan showing concentric soft tissue lesion surrounding the esophagus bronchoscopy and pulmonary function test of the patient were normal .
the right chest was opened through a right anterolateral thoracotomy at the fifth intercostal space .
the intramural tumor 3.5 3 3 cm was enucleated without erosion of the mucosa ( fig .
the esophageal wall allowed primary closure of the muscular layers , and the native esophagus was preserved .
enucleated esophageal schwannoma ( 3.5 3 3 cm ) histopathological examination of the tumor revealed spindle - shaped cells in a fasciculated and disarrayed architecture and nuclei in a palisading pattern ( fig .
immunohistochemically the tumor was positive for s100p and gfap , and negative for cd34 , cd117 , sma , desmin and h - caldesmon ( fig .
spindle - shaped cells in a fasciculated and disarrayed architecture and nuclei in a palisading pattern .
a ) 40 , b ) 200 immunohistochemical studies revealed s100 protein positivity postoperative recovery was uneventful , and there has been no evidence of recurrence to date .
esophageal schwannoma is uncommon and difficult to diagnose in preoperative investigations due to its similarity to other intramural esophageal tumors ( leiomyoma , gist , oesophageal cysts or cancer ) .
these tumors more frequently develop in women and are often located in the upper and mid esophagus .
malignant schwannoma has also been reported [ 1 , 2 ] . symptoms of this tumor include dysphagia , dyspnea [ 1 , 3 ] , chest pain , and hematemesis , and appear when the tumor increases in size .
imaging studies ( positron emission tomography , computed tomography , and magnetic resonance imaging ) are useful for the confirmation of esophageal tumor [ 1 , 4 , 5 ] .
endoscopic ultrasonography - guided fine needle aspiration biopsy is not always useful for diagnosis , as in our cases .
the value of frozen section is limited , and it can lead to diagnostic pitfalls .
final diagnosis is possible upon positive immunohistochemical studies for s-100 protein and negative staining for smooth muscle markers such as sma , cd34 , and cd117
. surgical excision of the tumor is the method of choice , because chemotherapy and radiotherapy are ineffective .
the option of enucleation is recommended when : the tumor is well encapsulated , a clear margin is achievable , and there is sufficient redundant mucosa to close the defect . in our case
enucleation with video - assisted thoracoscopic surgery ( vats ) is possible for small tumors ( 2 cm ) . for large tumors ( 8 cm ) with broad adjacent to the muscular layer and an extensive mucosal defect , esophagectomy with gastric pull - up is recommended [ 3 , 7 ] .
| schwannomas of the esophagus are rare peripheral nerve tumors . a 37-year - old woman with dysphagia
was found to have an intramural tumor of the upper esophagus .
the lesion was revealed on computed tomography .
endoscopic ultrasound biopsy was nondiagnostic .
through right thoracotomy , the mass was enucleated from the wall of the esophagus .
benign schwannoma was diagnosed only after immunological staining examination . |
generally , vesicle trafficking occurs via three important steps : vesicle budding from the donor organelle , movement towards a target organelle , and fusion .
several proteins are involved in docking and fusion of vesicles for target recognition and driving membrane fusion ( malsam et al . , 2008 ) . among the proteins for vesicle trafficking ,
snares ( soluble n - ethylmaleimide - sensitive fusion protein attachment protein receptors ) were described a critical role in membrane fusion ( sollner et al . , 1993 ) .
snares can be categorized into two types : v - snares , as snares on the vesicle , and t - snares , as snares on the target membrane .
when a vesicle moves close to its target , a v - snare on the vesicle interacts with three t - snares on the target membrane , forming a hetero - tetrameric trans - snare complex that drives membrane fusion ( mcnew et al . , 2000 ) . according to the core amino acid in the hydrophobic heptad repeats in the snare motif
, snares can be divided into four groups : qa- , qb- , qc- , and r - snares ( fasshauer et al . , 1998 ) . in mammals , a typical snare complex consists of qa- ( syntaxin 1 like ) , qb- ( n - terminal half of snap25 like ) , and qc- ( c - terminal half of snap25 like ) snares on the target membrane , and r - snares on the vesicle ( bock et al . , 2001 ) .
it has been reported that there may be as many as 65 snares in arabidopsis ( table 1 ; sanderfoot , 2007 ; saito and ueda , 2009 ) .
a large number of snares in arabidopsis may help facilitate trafficking in a complex endomembrane system that include distinct secretory and vacuolar trafficking steps mediated by two times more snares than unicellular or mammalian systems ( sanderfoot , 2007 ) .
studies to date on the same family of plant snares have often revealed redundancy but also functional specificity . here , we summarize the roles of snares in arabidopsis and present additional thoughts for future snare studies .
localization of snares are either experimentally investigated or predicted ( uemura et al . , 2004 ;
sanderfoot , 2007 ; saito and ueda , 2009 ) . snares with multiple localization are specified in the table with references .
in the endoplasmic reticulum ( er ) and golgi apparatus , 21 snares ( three qa - snares : atsyp81 and two atsyp3 ; seven qb - snares : atsec20 , two atmemb1 , two atgos1 , and two atuse1 ; seven qc - snares : three atsyp7 , two atbs14 , and two atsft1 ; four r - snares : two atsec22 , atvamp714 , and atvamp723 ) have been localized at a subcellular level by microscopy with fluorescent protein fusions along with in silico analyses ( uemura et al . , 2004 ; chatre et al . , 2005 ; sanderfoot , 2007 ; bubeck et al . , 2008 ) .
in general , a qa - snare is the core snare that regulates a snare complex formation at the target membrane .
interestingly , several qa - snare null mutants have been reported to be lethal , supporting that qa - snares are essential ( sanderfoot et al . , 2001b ) .
two qa - snares ( atsyp81 and atsyp31 ) have been shown to be involved in er
when secretory and er retention markers were used to investigate alteration of er golgi traffic , it was found that atsyp81 overexpression inhibited both anterograde and retrograde membrane traffic , while atsyp31 overexpression only inhibited anterograde traffic ( bubeck et al . , 2008 ) .
in yeast , the ortholog of atsyp31 has been reported to interact with the yeast counterparts of atsec22 and atmemb11 ( tsui et al . , 2001 ) .
interestingly , in tobacco leaf epidermis a strong redistribution of golgi proteins into the er was also observed in conditions of overexpression of atsec22 or atmemb11 ( chatre et al . , 2005 ) , implicating also these proteins in traffic at the er / golgi interface .
for example , yeast sec22p was found to have a role in both anterograde and retrograde traffic between er and golgi ( spang and schekman , 1998 ) .
furthermore , components of the copii machinery in mammalian cells , sec24a and sec24b , were found to interact directly with sec22p ( mancias and goldberg , 2008 ) . in plants
, we do not know whether atsec22 interacts with either copi or copii ; however , an essential role in er / golgi membrane traffic has been further supported by analyses of loss - of - function mutants .
specifically , the atsec22 mutation was reported to affect golgi morphology and cause er retention of a plasma membrane ( pm ) snare in pollen ( el - kasmi et al . , 2011 ) .
similarly to mammalian cells , atsec22 might interact with copii coat components ; however , in general , an interaction between coat proteins and snares has not been reported in plants yet .
the arabidopsis genome encodes a large number of genes for coat proteins , often outnumbering other eukaryotes isoforms ( robinson et al . , 2007 ) .
copii protein interactions may at least partially explain the diversification of the er and golgi snares and coat proteins in plants .
although in conditions of overexpression atsyp71 was found in the er , atsyp71 expressed using its endogenous promoter was found to localize at pm in the arabidopsis root as well as er and pm localization in the dividing root cells ( suwastika et al . , 2008 ) .
this suggests that syp71 might form at least two different snare complexes on the er and pm , or that atsyp71 is a snare for the er pm traffic during cell division .
no atsyp71 homolog has been identified in mammals , suggesting that atsyp71 may be involved in plant - specific membrane traffic events .
thus , identification of other snares in the atsyp71 snare complex may clarify the nature of the plant - specific role of atsyp71 , perhaps by providing important insights on understudied traffic route(s ) that may occur between the er and pm . two types of electron - dense vesicles originating from the er have been suggested for the transport of storage proteins or proteases to storage or lytic vacuoles in pumpkin seeds and castor bean ( hara - nishimura et al .
however , trafficking components required for budding and cargo selection in such traffic routes have not been identified yet .
proteomics analyses of these dense vesicles may reveal important clues on the machinery and snares that allows membrane transport to plant - specific organelles such as the protein storage vacuole .
at the tgn , endosome , and vacuoles , 21 snares ( six qa - snares : three atsyp4 and three atsyp2 ; four qb - snares : atvti1 ; five qc - snares : atsyp61 , three atsyp5 , and two atsyp5-like proteins ; six r - snares : two atykt6 , three atvamp71 , and atvamp727 ) have been identified ( uemura et al . , 2004 ;
the tgn is a tubular and vesicular organelle facing the trans side of the golgi apparatus .
mounting evidence supports that the tgn represents a crossroad for post - golgi protein traffic because it appears that vesicles containing vacuolar or secretory cargo can bud from the tgn , but also that endocytic markers such as the dye fm4 - 64 can be internalized to the tgn ( nakamura et al .
, 1993 ; ahmed et al . , 1997 ; dettmer et al . , 2006 ;
thus , complex vesicle budding and fusion events take place at the tgn , and it is likely that membrane transport is highly regulated at this organelle . in this view , specific snares should facilitate anterograde or retrograde traffic between the tgn and endosomes , vacuoles , and pm . in particular , two qa - snares , atsyp41 and atsyp42
were found to form separate complexes in distinct tgn domains ( bassham et al . , 2000 ) .
furthermore , the corresponding knock - out mutants were found to be gametophytic lethal , suggesting that these snares have specific and essential functions ( sanderfoot et al . , 2001b ) .
recently , another snare , atsyp61 , has been implicated in traffic at the tgn .
atsyp61 is a component of the atsyp4 snare family complexes and it is involved in salt and drought stress tolerance ( sanderfoot et al . , 2001a ; zhu et al . , 2002 ) .
proteomic analysis of vesicles containing atsyp61 has identified trafficking components at the tgn and pm along with enzyme cargos ( drakakaki et al . , 2011 ) .
these results suggest that atsyp61 has possibly a dual role as a t - snare at the tgn but also as a snare for tgn pm traffic . previously , atsyp42 was co - immunoprecipitated with atsyp61 ( sanderfoot et al . , 2001a ) ,
it would be interesting however to assay the proteome of the vesicles containing either atsyp41 or atsyp42 because we may learn about the function of atsyp41 and atsyp42 , and , perhaps , determine whether the tgn exists as a uniform compartment or whether there may be tgns with different composition due to specific functions or maturation stages .
for example , three atsyp2 family members , atsyp21 , atsyp22 , and atsyp23 , have been shown to localize on the prevacuolar compartment ( pvc ) , vacuole , and cytoplasm , respectively .
individual single mutants are viable , with only a gravitropic defect reported for the atsyp22 mutant ( yano et al . , 2003 ) .
however , the atsyp21/atsyp22 double mutant is known to be lethal ( shirakawa et al . , 2010 ) . a complete complementation of atsyp22 with atsyp21 under atsyp22 promoter suggests that atsyp21 and atsyp22 may have redundant functions ( uemura et al . , 2010 )
overexpression of syp21 resulted in homotypic fusion of pvc as well as the accumulation of vacuolar cargo in the pvc and partial secretion of vacuolar proteins ( foresti et al . ,
2006 ) , suggesting that overexpression of syp21 inhibits anterograde trafficking in the pvc vacuole route .
these observations open the question on the molecular basis for the role of atsyp21 in two different biological events . in other words
, it would be interesting to test whether vacuolar trafficking defects and homotypic fusion of pvc observed in conditions of atsyp21 overexpression are due to the collapse of one or multiple fusogenic machineries .
atsyp23 has a similar protein sequence with atsyp21 , but it does not have transmembrane domain .
atsyp23 can make a snare complex with atvti11 and atsyp5 , and high expression of atsyp23 can complement an atsyp22 mutant ( shirakawa et al . , 2010 ) .
thus , atsyp23 may function in vacuolar trafficking like atsyp21 and atsyp22 . considering the lack of transmembrane domain in atsyp23
therefore , either other regulatory proteins may recruit atsyp23 at the active site for the snare complex formation or that atsyp23 may interact with a preformed snare complex . in vitro liposome fusion assays may allow distinguishing the two models .
similar to atsyp61 , members of the atvamp71 family ( atvamp711 , atvamp712 , atvamp713 , and atvamp714 ) have been found to be involved in drought stress response by regulating stoma closure ( leshem et al . , 2010 ) .
stoma aperture is directly regulated by the volume of the vacuole ( blatt , 2000 ; sirichandra et al . , 2009 ) .
therefore , the atvamp71 family may be involved in membrane fusion to tonoplast or vesicle budding from vacuole so as to control the size of vacuole through modulating the tonoplast size .
so far , the composition of the atvamp71 family complexes has not been determined , but the presence of three atvamp71 snares ( atvamp711 , atvamp712 , atvamp713 ) on the tonoplast suggests that these snares could have either overlapping functions or that they may form distinct snare complexes on the tonoplast for tissue- and developmental - specific roles .
another r - snare , atvamp727 is localized on the endosome / pvc and pm ( uemura et al . , 2004 ;
it forms a complex with atsyp22 , atvti11 , and atsyp51 on the pvc / vacuole and forms a complex with atsyp121 at the pm ( ebine et al . , 2008 , 2011 ) .
roles in vacuolar trafficking was shown that in the atsyp22/atvamp727 double mutant , storage vacuolar cargo such as 2 s albumin was secreted ( ebine et al .
the double mutant contained multiple small vacuoles instead of large vacuoles found in wild - type and in atvamp727 and atsyp22 single mutants ( ebine et al . , 2008 ) , supporting that atsyp22 and atvamp727 may form a snare complex for membrane fusion between pvc and vacuole .
in addition , rab - gtpase , ara6 has been identified to regulate the formation of atvamp727 snare complex with atsyp121 at pm under salt stress ( ebine et al . , 2011 ) , suggesting atvamp727 has another role in pm , presumably triggered by salt stress .
analyses of the diverse snare complexes at the tonoplast will provide further valuable information about vacuole biogenesis and vacuolar trafficking pathways possibly in relation to cell type specificity , along with plant development and growth , and plant may have developed specific traffic route to overcome environmental stress .
a total of 23 snares ( nine qa - snares : two atsyp11 , five atsyp12 , and two atsyp13 ; three qb - snares : atnpsn1 ; three qb + qc - snares : atsnap29 , atsnap30 , and atsnap33 ; eight r - snare : six atvamp72 and two tomosyn ) have been localized at the pm ( uemura et al . , 2004 ; sanderfoot , 2007 ) .
a large number of qa - snares at the pm may allow the delivery of different types of vesicles to the pm in response to specific development and biotic / abiotic cues .
for example , it has been shown that atsyp121 is involved in the regulation of k channels and resistance against barley powdery mildew ( collins et al .
, 2003 ; sutter et al . , 2006b ; honsbein et al . , 2009 ) .
however , an atsyp122 mutant did not show any defects on non - host resistance , contrary to the atsyp121 mutant , although both atsyp121 and atsyp122 are involved in the regulation of salicylic acid and jasmonic acid ( assaad et al . , 2004 ;
growth defects were only seen in the atsyp121/atsyp122 double mutant ( assaad et al . , 2004 ) . therefore , atsyp121 and atsyp122 might have overlapping functions in plant growth and development , but distinct roles in disease resistance , presumably by mediating fusion of different types of vesicles containing cargo to resist pathogens .
two atsyp121 snare complexes have been identified with atsnap33 and atvamp721 or atvamp722 as components in the complexes ( kwon et al . , 2008 ) .
atsnap33 also forms a snare complex with atsyp111 and individual mutants showed incomplete cytokinesis , a process that requires massive vesicle traffic at the cell plate ( lukowitz et al . , 1996 ;
the existence of multiple atsnap33 complexes suggests that this protein may have multiple roles in plant development , and transport machinery of atsnap33 to either cell plate during cell division or pm may rely on specific traffic machinery .
for example , investigation on the trafficking of atsyp111 during cytokinesis has established that atsyp111 is delivered to the cell plate by secretory pathways . in mitotic cells ,
atsyp111 was found to localize at the tgn and cell plate , and inhibition of er golgi trafficking accumulated atsyp111 in the er ( reichardt et al . , 2007 ; chow et al . , 2008 ) .
these results suggest that atsyp111 traffics through the secretory pathway , and that distribution of atsyp111 at the cell plate depends on a clathrin and dynamin - related protein a dependent endocytosis ( boutte et al . , 2010 ) . however , there is no evidence that atsyp111 is directly delivered from tgn to cell plate .
considering endocytosis dependent polar distribution of auxin transporter ( pin1 ; dhonukshe et al . , 2008 ) , atsyp111 may be secreted to pm first by the default pathway and then relocalized to the cell plate by endocytosis .
recent discovery for a role of qa - snares ( atsyp11 , atsyp12 , and atsyp13 families ) at the pm supports a diversified but redundant function of qa - snares at this compartment .
it was found that atsyp132 could complement an atsyp111 mutant ( reichardt et al . , 2011 ) .
also , replacement of the snare domain of atsyp111 with that of atsyp132 rescued the atsyp111 mutant . however , neither atsyp121 nor the atsyp111 chimera containing the snare domain of atsyp121 was able to complement the atsyp111 mutant ( reichardt et al . , 2011 ) .
these results support that while atsyp132 may have an overlapping function with other snares at the pm , atsyp111 , and atsyp121 may have distinct roles , perhaps by forming highly specialized complexes .
, 2001 ; kwon et al . , 2008 ) , atsyp111 may form a snare complex with different r - snares .
identification of the r - snares in the atsyp111 and atsyp121 complexes along with structural analyses of the snare complexes by x - ray crystallography may provide fundamental information on the difference between atsyp111 and atsyp121 snare complexes .
the large number of snares in arabidopsis implicates diversified roles of snare complexes in membrane traffic events during growth and development as well as responses to biotic and abiotic stresses .
it is also important to consider that other proteins may contribute to the function of snares , thus increasing the level of complexity as well as fidelity and regulation of the biological processes mediated by snares .
for example , the sec1/munc18 ( sm ) protein family was reported to regulate a snare complex formation by controlling a qa - snare in mammalian and yeast cells ( malsam et al . , 2008 ) .
similarly in plants , atsyp41 and atsyp42 form distinct snare complexes at different subdomains of tgn with atsyp61 , atvti12 , and a sm protein , atvps45 ( bassham et al .
atvps45 rnai plants showed decreased amount of atsyp41 compared to wild - type , suggesting that atvps45 may be important for the stability of atsyp41 ( zouhar et al . , 2009 ) .
these findings support that sm proteins regulate snare - mediated membrane fusion , as well as stability or turnover of snares . in arabidopsis , six putative sec1-like proteins in the sm protein family have been predicted to exist ( schwacke et al . , 2003 ) .
thus , investigation of the uncharacterized sm proteins will be necessary to define one of the possible regulatory mechanisms of snare complexes .
related to this , tethering factors and gtpases can play important roles in membrane fusion ( malsam et al . ,
tethering factors are involved in the snare complex assembly and docking for membrane fusion by interacting with gtpases ( price et al . , 2000 ; seals et al . ,
2002 ) , supporting that snare complex assembly is a multifaceted process based on the interplay of multiple components .
therefore , a study focused on snare specificity in vesicle trafficking should take into account an analysis of several components for membrane fusion to better define an overall picture of the function of the snares .
so far , valuable approaches to characterize the role of snares have been developed and have provided important data in plants . however , there are still several unsolved questions on the function of snares and the nature of the complexes that they can form in vivo in plant cells .
arabidopsis has a large number of snares and several snare complexes that partially share the same components .
we need to understand the physiological relevance of the number of snare and of the snare complexes . with no doubt
, we are living in an exciting moment for research in plants thanks to a wealth of genetics , genomics , and molecular resources . in vitro fusion assays using snares and other regulatory proteins
the combination of in vitro and in vivo studies on cargo trafficking and membrane fusion will also provide important insights into the mechanisms and regulatory pathways of vesicle transport along with a better understanding of their role in plant growth , development , and response to stress .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | secretory organelles are engaged in a continuous flux of membranes , which is believed to occur mostly via transport vesicles .
being critical in maintaining several cellular functions , transport vesicles are membrane - enclosed sacs that temporarily store and then deliver membrane lipids , protein , and polysaccharides .
snares have a crucial role in vesicle traffic by driving membrane fusion and conferring fidelity through the formation of specific snare complexes .
additionally , specific roles of snares in growth and development implicate that they are versatile components for the life of a plant . here , we summarize the recent progress on the understanding of the role of snares and highlight some of the questions that are still unsolved . |
this study was approved by the university of south australia and the australian government department of veterans affairs ( dva ) human research ethics committees .
a retrospective cohort study was undertaken from 1 july 2001 to 30 june 2008 using data from the dva health administrative claims database .
the dva database contains details of all prescription medicines and medical and allied health services and hospitalizations subsidized by dva for a treatment population of 290,000 veterans , war widows , and widowers . over 70% of the population
are 70 years of age or older ; 54% are male and 9.8% live in residential aged care , and they are dispensed an average of 11 unique medicines annually ( 12 ) . within the dataset , medicines are coded according to the world health organization ( who ) anatomical and therapeutic chemical ( atc ) classification ( 13 ) and the schedule of pharmaceutical benefits item codes ( 14 ) .
hospitalizations are coded according to the who australian modification of the icd-10 ( icd-10-am ) classification ( 15 ) .
subjects were included if they were eligible for all health services subsidized by dva in the 12 months prior to the date of their first ( index ) dispensing of an oral antidiabetic medicine .
the study included new users of either metformin or sulfonylurea medicines , defined by those who did not have a dispensing of these medicines in the 12 months prior .
users of other classes of diabetes medicines ( insulin , other oral medications , and combination of metformin ) were excluded , as prescription of these medicines at the initiation of diabetes treatment is not recommended as first - line pharmacotherapy and may relate to unusual clinical circumstances ( 16 ) .
newly initiated medicine users were selected for study as these patients are less likely to have existing complications than those who are on second- and third - line agents ( insulin or other oral medications ) ( 16 ) .
comorbid copd was identified from medicine use , defined as two dispensings of tiotropium or ipratropium ( atc codes r03bb04 and r03bb01 , respectively ) in the 6 months preceding the first dispensing of the oral antidiabetic medicine .
the total cumulative corticosteroid dose ( both inhaled and systemic [ oral ] , atc codes r01ad - r03ba and h02ab01-ab10 , respectively ) was calculated in the 12 months from study entry and converted to a defined daily dose ( ddd ) per day ( 17 ) . in order to account for the systemic bioavailability of inhaled corticosteroids by comparison with that of oral corticosteroids ,
the who - defined dose of inhaled corticosteroids was reduced by a factor of 10 ( 18 ) .
users of corticosteroids were stratified into three groups : > 0 to < 0.25 , 0.25 to < 0.83 , and 0.83 ddd / day .
the study end point was time until first hospitalization for a diabetes - related complication , defined by the icd-10-am codes e11 and e13e14 ( 15 ) .
these include diabetic hyperosmolarity with or without coma , diabetic ketoacidosis with or without coma , diabetic nephropathy , diabetic retinopathy , diabetic neuropathy , diabetic angiopathy , and diabetic arthropathy .
we used an intention - to - treat approach to compare risk of diabetes complications between users and nonusers of corticosteroids in our diabetes and copd cohort .
these analyses assessed the effect of treatment in the first 12 months from incident antidiabetes medicine use . characteristics of nonusers and users of corticosteroids were compared using a mann - whitney u test for continuous variables and test for categorical variables . as death is a competing risk event in this elderly study population that may preclude the onset of the outcome of interest ( time to hospitalization for a diabetes - related complication ) , we calculated the cumulative incidence of outcome of interest in the presence of competing risk events .
competing risk regression analyses were conducted using the fine and gray approach , which extends the cox model to competing risks data by considering the subdistribution hazard ( 19 ) .
the strength of the association between each variable and the outcome was assessed using the subhazard ratio ( shr ) , which is the ratio of hazards associated with the cumulative incidence function under different values of the covariates .
covariates included age , sex , socioeconomic status ( 20 ) , residential status ( community dwelling or aged - care resident ) , number of comorbid conditions ( identified using the pharmaceutical - based comorbidity index , rx - risk - v , excluding those conditions used to identify patients ) , number of prescribers , number of unique medicines ( excluding those medicines used to identify patients ) , number of visits to an endocrinologist , number of visits to a pulmonary specialist , number of hospitalizations for conditions other than diabetes , exposure to corticosteroids in the 12 months prior to study entry , and glycated hemoglobin ( hba1c ) testing in the 12 months prior to study entry .
all covariates were time varying , adjusted at study entry and then annually , apart from age , socioeconomic status , prior corticosteroid exposure , and hba1c testing , which were adjusted at baseline only .
the subproportional hazards assumption was confirmed by examining log[log(survival ) ] curves , and no violation was detected .
we also examined cause - specific cox proportional hazard models , with death censored and adjusting for the same covariates as included in the competing risks regression analyses , to provide a more complete understanding of the association between corticosteroid use and time until diabetes complication ( 22 ) .
this study was approved by the university of south australia and the australian government department of veterans affairs ( dva ) human research ethics committees .
a retrospective cohort study was undertaken from 1 july 2001 to 30 june 2008 using data from the dva health administrative claims database .
the dva database contains details of all prescription medicines and medical and allied health services and hospitalizations subsidized by dva for a treatment population of 290,000 veterans , war widows , and widowers . over 70% of the population
are 70 years of age or older ; 54% are male and 9.8% live in residential aged care , and they are dispensed an average of 11 unique medicines annually ( 12 ) . within the dataset , medicines are coded according to the world health organization ( who ) anatomical and therapeutic chemical ( atc ) classification ( 13 ) and the schedule of pharmaceutical benefits item codes ( 14 ) .
hospitalizations are coded according to the who australian modification of the icd-10 ( icd-10-am ) classification ( 15 ) .
subjects were included if they were eligible for all health services subsidized by dva in the 12 months prior to the date of their first ( index ) dispensing of an oral antidiabetic medicine .
the study included new users of either metformin or sulfonylurea medicines , defined by those who did not have a dispensing of these medicines in the 12 months prior .
users of other classes of diabetes medicines ( insulin , other oral medications , and combination of metformin ) were excluded , as prescription of these medicines at the initiation of diabetes treatment is not recommended as first - line pharmacotherapy and may relate to unusual clinical circumstances ( 16 ) .
newly initiated medicine users were selected for study as these patients are less likely to have existing complications than those who are on second- and third - line agents ( insulin or other oral medications ) ( 16 ) .
comorbid copd was identified from medicine use , defined as two dispensings of tiotropium or ipratropium ( atc codes r03bb04 and r03bb01 , respectively ) in the 6 months preceding the first dispensing of the oral antidiabetic medicine .
the total cumulative corticosteroid dose ( both inhaled and systemic [ oral ] , atc codes r01ad - r03ba and h02ab01-ab10 , respectively ) was calculated in the 12 months from study entry and converted to a defined daily dose ( ddd ) per day ( 17 ) . in order to account for the systemic bioavailability of inhaled corticosteroids by comparison with that of oral corticosteroids ,
the who - defined dose of inhaled corticosteroids was reduced by a factor of 10 ( 18 ) .
users of corticosteroids were stratified into three groups : > 0 to < 0.25 , 0.25 to < 0.83 , and 0.83 ddd / day .
the study end point was time until first hospitalization for a diabetes - related complication , defined by the icd-10-am codes e11 and e13e14 ( 15 ) .
these include diabetic hyperosmolarity with or without coma , diabetic ketoacidosis with or without coma , diabetic nephropathy , diabetic retinopathy , diabetic neuropathy , diabetic angiopathy , and diabetic arthropathy .
we used an intention - to - treat approach to compare risk of diabetes complications between users and nonusers of corticosteroids in our diabetes and copd cohort .
these analyses assessed the effect of treatment in the first 12 months from incident antidiabetes medicine use
. characteristics of nonusers and users of corticosteroids were compared using a mann - whitney u test for continuous variables and test for categorical variables . as death is a competing risk event in this elderly study population that may preclude the onset of the outcome of interest ( time to hospitalization for a diabetes - related complication ) , we calculated the cumulative incidence of outcome of interest in the presence of competing risk events .
competing risk regression analyses were conducted using the fine and gray approach , which extends the cox model to competing risks data by considering the subdistribution hazard ( 19 ) .
the strength of the association between each variable and the outcome was assessed using the subhazard ratio ( shr ) , which is the ratio of hazards associated with the cumulative incidence function under different values of the covariates .
covariates included age , sex , socioeconomic status ( 20 ) , residential status ( community dwelling or aged - care resident ) , number of comorbid conditions ( identified using the pharmaceutical - based comorbidity index , rx - risk - v , excluding those conditions used to identify patients ) , number of prescribers , number of unique medicines ( excluding those medicines used to identify patients ) , number of visits to an endocrinologist , number of visits to a pulmonary specialist , number of hospitalizations for conditions other than diabetes , exposure to corticosteroids in the 12 months prior to study entry , and glycated hemoglobin ( hba1c ) testing in the 12 months prior to study entry .
all covariates were time varying , adjusted at study entry and then annually , apart from age , socioeconomic status , prior corticosteroid exposure , and hba1c testing , which were adjusted at baseline only .
the subproportional hazards assumption was confirmed by examining log[log(survival ) ] curves , and no violation was detected .
we also examined cause - specific cox proportional hazard models , with death censored and adjusting for the same covariates as included in the competing risks regression analyses , to provide a more complete understanding of the association between corticosteroid use and time until diabetes complication ( 22 ) .
a total of 1,077 ( 5.9% ) of the 18,226 patients with diabetes had comorbid copd . in the 12 months after study entry , corticosteroids were used by 724 ( 67.2% ) of those with diabetes and copd . for those who received a corticosteroid , the median corticosteroid dose over the 12-month period was 0.34 ddd / day ( iqr 0.110.83 ) .
those who were users of corticosteroids had a statistically significant greater number of comorbid conditions and hospitalizations and were more likely to visit an endocrinologist or pulmonary specialist .
demographic and clinical characteristics of study cohort ( n = 1,077 ) after 1 year , there was little difference between the proportion of those who had a hospitalization for a diabetes complication between nonusers ( 6.3% [ 95% ci 4.019.63 ] ) and users ( 7.1% [ 5.299.16 ] ) of corticosteroids ( p = 0.37 ; data not shown ) . after 5 years of follow - up , 19.8% ( 18.7223.45 ) of corticosteroid users had a diabetes - related hospitalization , by comparison with 16.2% ( 16.9122.84 ) for nonusers of corticosteroids ( p = 0.18 ) . shown in fig .
2 is the dose - dependent analysis of corticosteroid use and time to hospitalization for a diabetes complication .
stratification of corticosteroid dose demonstrated that a total dose of 0.83 ddd / day , over the 12 months from study entry , resulted in a 94% increased risk of diabetes complication ( shr 1.94 [ 95% ci 1.143.28 ] , p = 0.014 ) by comparison with those who did not receive a corticosteroid ( table 2 ) . despite observing a dose - dependent increase in risk ,
this was not statistically significant for the lower two corticosteroid dose treatment groups ( table 2 ) .
hazard ratios ( hrs ) for both the lower two doses of corticosteroids using competing risk or cox proportional hazards regression analyses were similar .
the hr for the higher - dose grouping of corticosteroid 0.83 using cox proportional hazards regression analysis demonstrated an increased risk ( hr 2.46 [ 95% ci 1.454.18 ] , p = 0.001 ) by comparison with competing risk ( shr 1.94 [ 1.143.28 ] , p = 0.014 ) , highlighting the increasing influence of the competing risk of death , for this dose . cumulative incidence of time to hospitalization for a diabetes complication stratified by total cumulative dose ( ddd ) of corticosteroids in the 12 months from study entry .
competing risk and cox proportional hazards regression analyses for time to hospitalization for diabetes complication , stratified by total dose ( ddd ) of corticosteroid / day in the 12 months from study entry we examined the risk of diabetes hospitalizations in those who received inhaled only , oral only , and both .
inhaled corticosteroid only was received by 24.8% , with a median total dose of 0.08 ddd / day ( iqr 0.030.13 ) ; 46.5% received an oral corticosteroid only , with a median total dose of 0.49 ddd /day ( iqr 0.210.99 ) ; and 28.7% received both an inhaled and oral corticosteroid with a median total dose of 0.64 ddd / day ( iqr 0.073.04 ) ( table 3 ) . competing risk analysis stratified by corticosteroid use ( inhaled ,
oral , and both ) showed no significant association between corticosteroid use and risk of a diabetes - related hospitalization ( table 3 ) .
competing risk regression analyses for time to hospitalization for diabetes complication , stratified by inhaled corticosteroids , oral corticosteroids , or both
in this large population - based study of older people with copd and diabetes , a dose of corticosteroids of 0.83 ddd / day in the 12 months after starting diabetes medication was associated with a 94% increased risk of having a hospitalization for a diabetes complication . this increased risk was not evident for doses < 0.83 ddd / day . to our knowledge , this is the first study to have examined the effect of corticosteroid dose on longer - term diabetes complications for patients with diabetes and comorbid copd .
older patients and those with a comorbidity are generally excluded from randomized clinical trials used to generate the evidence for clinical guidelines , decreasing the applicability of trial results to real - world populations ( 23 ) .
the results of this study suggest that , when they are necessary to use , corticosteroids should be used in their minimally effective dose for older patients with diabetes and copd .
the use of inhaled corticosteroids for patients with copd has been the subject of much debate ( 5 ) .
inhaled corticosteroids have been shown to reduce the frequency of exacerbations and decline in quality of life in people with copd but have no effect on the rate of decline in lung function or mortality ( 5 ) .
although they are recommended for use in those with moderate to severe copd , inhaled corticosteroids are used widely , with reports of use in > 70% of patients with copd ( 24 ) . whereas systemic corticosteroids are known to increase diabetes risk ( 25 ) , the effects of inhaled corticosteroids are less well known . in a recent large population based study of 388,584 patients with respiratory disease , treatment with inhaled corticosteroids
was associated with a 34% ( rr 1.34 [ 95% ci 1.291.39 ] ) increased risk of diabetes onset ( defined by commencement of an oral hypoglycemic agent ) , with the highest dose associated with the greatest risk ( 11 ) .
furthermore , in a subset of patients with respiratory disease and newly diagnosed diabetes treated with oral hypoglycemic agents , inhaled corticosteroid use was associated with a 34% ( rr 1.34 [ 1.171.53 ] ) increased likelihood of progression to insulin use , with higher doses associated with a 54% increased risk ( 11 ) .
this demonstrates the short - term systemic effects of inhaled corticosteroid use , resulting in hyperglycemia and the subsequent need for the use of oral antidiabetic agents or intensification of treatment .
we stratified our analyses by type of corticosteroid dispensed ( inhaled , oral , or those who received both ) and found no significant association with risk of diabetes - related hospitalization .
however , the median doses of each of these groups were lower than 0.83 ddd / day , consistent with our results , where an association of increased risk of diabetes - related hospitalization was only observed at these higher doses .
previous studies have shown that corticosteroids are associated with insulin resistance , hyperglycemia , and loss of diabetic control in a dose - dependent manner in patients with diabetes ( 10,25 ) .
corticosteroids have been shown to inhibit a number of steps in the insulin signaling network , leading to insulin resistance .
these include increased proteolysis , lipolysis , and free fatty acid production , which can contribute to insulin resistance .
the results of the current study provide an insight into potential safety concerns of corticosteroid use in a real - world setting for the treatment of copd in patients with comorbid diabetes , where both oral and systemic corticosteroids are used .
this highlights the potential for longer - term adverse effects of higher doses of both oral and systemic corticosteroids on diabetes complications in older patients with diabetes and copd .
diabetes and copd are common comorbid disorders , and both the prevalence and incidence of type 2 diabetes are higher in patients with copd , by comparison with those without copd ( 27,28 ) .
the presence of comorbidity adds considerable complexity to the therapeutic management , with > 60% of those with diabetes having a comorbid condition that may cause a treatment conflict , affecting both the benefits and risks associated with treatment choices ( 2 ) .
the presence of both diabetes and copd has been reported to be associated with greater care complexity ( 6 ) .
greater numbers of comorbid conditions or the presence of comorbid cardiovascular disease , hypertension , anxiety , or depression are associated with increased respiratory impairment and risk of hospitalization and mortality ( 5,28 ) .
our results highlight the importance of the need for shared decision making in this population , where treatment prioritization of the more symptomatic conditions , such as copd , needs to be balanced with both the short - term and long - term harms associated with use of corticosteroids for patients with comorbid diabetes .
although we have shown that longer - term adverse effects on diabetes complications were only evident at higher doses of corticosteroids , use of corticosteroids ( both inhaled and oral ) in this patient population should be associated with close monitoring of blood glucose levels and a review of efficacy within 48 weeks of commencing inhaled therapy .
regular use of high - dose corticosteroids should be avoided where possible ( 0.83 ddd / day ) for those individuals with comorbid diabetes .
the identification of patients with copd in our study was based on dispensings of either- the short or long - acting anticholinergic agent ipratropium or tiotropium , respectively .
we have not included patients who are only taking -2 agonists as these drugs may also be used for asthma or in those with mild or newly diagnosed copd , and as such , we may have excluded patients with less severe copd in our study .
a recent australian study of patients with copd showed that the majority ( 89% ) used tiotropium or ipratropium as their regular copd therapy ( 29 ) .
the use of an intention - to - treat analysis may have resulted in misclassification of corticosteroid exposure during follow - up ; however , the median dose of corticosteroids within the second year after study entry was 0.27 ddd / day ( iqr 0.090.66 ) , similar to that of the first 12 months , suggesting that this was minimized .
although we included a number of covariates in our analyses to adjust for several potential confounders , residual confounding from unmeasured covariates may still exist .
the issue of confounding by indication also deserves consideration ; those with more severe copd are more likely to receive a corticosteroid , which may be associated with the outcome of a diabetes - related hospitalization . despite a lack of evidence to support that severity of copd
is on the causal pathway to an increased likelihood of a diabetes - related hospitalization , there is , however , the potential that a nonbiological causal bias exists ; that is , competing priorities , whereby those who have more severe copd may have less capacity to focus on management of their comorbid diabetes and therefore have poorer diabetic control .
a number of covariates were included in our analyses as a proxy for copd disease severity , including number of unique medicines ( which included -agonists and antibiotics ) , number of visits to a pulmonary specialist , and number of hospitalizations ( including those that are copd related ) .
we also adjusted for potential differences in diabetes severity at study entry due to prior corticosteroid exposure in our analyses .
our study end point mainly included microvascular diabetes complications and did not include macro / cardiovascular complications , as almost all of the study population ( > 92% ) had pre - existing cardiovascular comorbidity at study entry .
the results of the current study are likely to be applicable to the older australian population ; age - specific comparisons of dva gold card holders with no service - related disability with the wider australian population have shown similar rates of general practitioner visits , use of prescriptions , and hospitalizations ( 30 ) . in conclusion , use of a higher dose of corticosteroids is associated with an increased risk of diabetes complications in diabetes patients with comorbid copd .
use of corticosteroids in patients with diabetes and copd should be accompanied by regular review to ensure that minimally effective doses are used , efficacy of treatment , and close monitoring of blood glucose control .
treatment with higher doses of corticosteroids in patients with diabetes and copd should be limited to optimize the balance between risk of benefits and longer - term adverse effects . | objectiveto identify if there is a dose - dependent risk of diabetes complications in patients treated with corticosteroids who have both diabetes and chronic obstructive pulmonary disorder ( copd).research design and methodsa retrospective study of administrative claims data from the australian government department of veterans affairs , from 1 july 2001 to 30 june 2008 , of diabetes patients newly initiated on metformin or sulfonylurea .
copd was identified by dispensings of tiotropium or ipratropium in the 6 months preceding study entry .
total corticosteroid use ( inhaled and systemic ) in the 12 months after study entry was determined .
the outcome was time to hospitalization for a diabetes - related complication .
competing risks and cox proportional hazard regression analyses were conducted with adjustment for a number of covariates.resultsa total of 18,226 subjects with diabetes were identified , of which 5.9% had copd . of those with copd ,
67.2% were dispensed corticosteroids in the 12 months from study entry .
stratification by dose of corticosteroids demonstrated a 94% increased likelihood of hospitalization for a diabetes complication for those who received a total defined daily dose ( ddd ) of corticosteroids 0.83/day ( subhazard ratio 1.94 [ 95% ci 1.143.28 ] , p = 0.014 ) , by comparison with those who did not receive a corticosteroid .
lower doses of corticosteroid ( < 0.83 ddd / day ) were not associated with an increased risk of diabetes - related hospitalization.conclusionsin patients with diabetes and copd , an increased risk of diabetes - related hospitalizations was only evident with use of high doses of corticosteroids .
this highlights the need for constant revision of corticosteroid dose in those with diabetes and copd , to ensure that the minimally effective dose is used , together with review of appropriate response to therapy . |
the most common malignancies associated with malignant pleural effusions are carcinomas of the breast , lung , gastrointestinal tract , ovary and lymphomas .
a 72-year old female patient presented to us with shortness of breath for the last 2 months .
a contrast - enhanced computed tomography ( cect ) scan of her - thorax revealed only bilateral pleural effusion with absence of any mass lesion or any mediastinal lymphadenopathy . a cytologic examination of pleural fluid revealed adenocarcinoma cells .
a cect of her abdomen and pelvis revealed heterogenous thickening of omentum with nodular appearances and small amount of ascites .
her ovaries were normal and no other mass lesion was detected . a histological examination of a peritoneal lesion was suggestive of adenocarcinoma .
the patient was diagnosed with a rare case of primary peritoneal adenocarcinoma with bilateral pleural effusion .
in male patients about 50% of malignant effusions are caused by lung cancer , 20% by lymphomas or leukemia , 7% from gastrointestinal primaries , 6% from genitourinary primaries , and 11% from tumors of unknown primary site . in female patients ,
about 40% of malignant effusions are caused by breast cancer , 20% from tumors arising in the female genital tract , 15% from lung cancer , 8% from lymphomas or leukemia , 4% from gastrointestinal tract primaries , 3% from melanoma , and 9% from tumors of unknown primary site .
here we present a rare case of bilateral malignant pleural effusion which was due to primary peritoneal adenocarcinoma .
a 72-year old female patient presented to us with shortness of breath for the last 2 months . there was no history of fever , cough , chest pain , haemoptysis , abdominal pain , vomiting or bleeding per vaginum .
there was no lymphadenopathy , clubbing , or pedal edema , but pallor was present .
the complete blood cell count ( cbc ) , blood sugar , renal function test and liver function test were all found to be within normal ranges .
1 ) was done which showed homogenous opacity in the bilateral lower lung field along with blunting of both costophrenic angles ( cpa ) suggestive of bilateral pleural effusion .
a posterior to anterior view chest x - ray ( cxr - pa ) showed homogenous opacity in the bilateral lower lung field along with blunting of both costophrenic angles ( cpa ) suggestive of bilateral pleural effusion . a cytologic examination of pleural fluid revealed adenocarcinoma cells ( fig 2a ) .
a contrast - enhanced computed tomography ( cect ) scan of the thorax was done which revealed only bilateral pleural effusion with absence of any mass lesion or any mediastinal lymphadenopathy ( fig .
an ultrasound of the abdomen and pelvis showed only ascites ; no other abnormality or mass lesion was detected . a cytologic examination of ascitic fluid also revealed adenocarcinoma cells ( fig .
nuclear crowding , overlapping and acini formation ( arrow ) is seen suggestive of papillary adenocarcinoma ( h&e x400 ) .
2(b ) ascitic fluid cytology showing pleomorphic malignant cells , nuclear overlapping , acini formation ( arrow ) and cytoplasmic vacuoles in few cells suggestive of adenocarcinoma ( h&e x400 ) .
a contrast - enhanced computed tomography ( cect ) scan of the thorax showed bilateral pleural effusion .
a cect scan of the abdomen and pelvis revealed heterogeneous thickening of omentum with nodular appearance and fatty streakiness into the adjacent soft tissues extending up to the anterior abdominal wall and adjacent bowel loops , including transverse and descending colon , suggestive of omental caking with small amount of ascites .
a laparoscopic examination of the abdomen showed omental thickening along with multiple nodules on the peritoneum , but the ovaries and other organs were found to be normal .
a biopsy was taken from a peritoneal lesion . a histologic examination of the biopsy was suggestive of adenocarcinoma ( fig .
a test for cancer antigen 125 ( ca 125 ) showed that the level of ca 125 was elevated ( 3950 u / ml ) , but a test for carcinoembryonic antigen ( cea ) was negative .
the patient was treated with cisplatin chemotherapy , which resulted in a decrease in tumor size , a reduction of ca 125 level , and a reduction of ascites and pleural effusion .
histopathology of the peritoneal nodular lesion showing complex branching papillary pattern ( h&e x100 ) .
primary peritoneal adenocarcinoma is a rare malignancy , which was first reported by swerdlow in 1959 .
it arises from the peritoneum lining the pelvis and abdomen and is characterized by disseminated intraperitoneal carcinomatosis involving the peritoneum and omentum , with normal or minimally involved ovaries , and no identifiable primary tumor .
it occurs almost exclusively in menopausal and post - menopausal women and may be associated with bilateral oophorectomy for benign disease or prophylaxis[46 ] .
however , a case of primary peritoneal adenocarcinoma in men was reported by shmeuli et al .
primary peritoneal adenocarcinoma is also known as serous surface papillary carcinoma , multiple focal extraovarian serous carcinoma , primary peritoneal papillary serous adenocarcinoma , serous surface carcinoma of the peritoneum , extraovarian peritoneal serous papillary carcinoma , normal - sized ovary carcinoma syndrome , papillary serous carcinoma of the peritoneum , and peritoneal papillary carcinoma .
primary peritoneal adenocarcinoma is identical to serous ovarian carcinoma in its clinical presentation , histologic features , and treatment pattern , but differs from it by the presence of ovaries of normal size or ovaries that are enlarged due to a benign process , extraovarian involvement greater than ovarian involvement , and ovarian surface penetration of less than 5 mm .
according to rothacker et al , primary peritoneal adenocarcinoma may account for 10% of the cases with a presumed diagnosis of ovarian cancer .
both stain positive for estrogen receptor ( er ) , cytokeratin 7 ( ck7 ) , wilm 's tumor suppressor gene ( wt1 ) , and cancer antigen 125 ( ca 125 ) , but negative for cytokeratin 20 ( ck 20 ) , progesterone receptor ( pr ) , calretinin , carcinoembryonic antigen ( cea ) , gross cystic disease fluid protein ( brst-2 ) , and thyroid transcription factor 1 ( ttf1 ) .
primary peritoneal adenocarcinoma shows widespread intraperitoneal carcinomatosis , usually involving the omentum and upper abdomen , and can be misdiagnosed as peritoneal carcinomatosis from metastatic gastrointestinal cancers or ovarian cancer .
however , it can be differentiated by no or minimal involvement of the ovaries and by no primary lesion in the gastrointestinal tract or other organs .
extraovarian primary peritoneal carcinoma spreads mainly transperitoneally ; but lymphatic and blood - borne metastases have also been reported .
metastases to the liver , brain , and lymph node have been reported . in primary peritoneal adenocarcinoma , the level of ca 125 is found to be elevated and correlates with the clinical status of the disease and response to therapy .
the most common presenting symptoms include abdominal distension , pain , nausea , vomiting , dyspepsia , or change in bowel habits .
interestingly , our patient presented with massive bilateral pleural effusion , which is unusual in presence of small amount of ascitis as pleural involvement is a rare event.the histological picture is characterized by a predominantly serous pattern , but non serous types such as mucinous , clear cell , endometrioid , and mixed mllerian tumors have also been reported .
patients with primary peritoneal carcinoma are treated by cytoreductive surgery followed by cisplatin - based chemotherapy .
this treatment is similar to the treatment of ovarian carcinoma and the response achieved by this treatment is also similar to that achieved in ovarian carcinoma .
a cisplatin - based chemotherapy regimen is the most common first - line chemotherapeutic regimen used in primary peritoneal carcinoma .
patients treated by cisplatin - based regimens have a longer survival compared to other chemotherapy regimens . at the same time , patients treated with combination chemotherapy regimens show longer survival compared to single agent regimens .
patients who undergo optimal cytoreductive surgery show significantly higher response rates to chemotherapy as well as increased survival rates compared to patients for whom optimal cytoreductive surgery was not achievable.the grade of tumor does not appear to affect the response to chemotherapy .
the median survival time varies between 7.0 and 27.8 months , while 5-year survival rates range from 0% to 26.5% .
the survival rate of patients with primary peritoneal carcinoma is poorer than the survival rate of patients with serous ovarian cancer .
the diagnosis of primary peritoneal adenocarcinoma should be considered in elderly female patients with malignant pleural effusion containing adenocarcinoma cells if intraperitoneal carcinomatosis is found , but no primary tumor is identified . | context : the most common malignancies associated with malignant pleural effusions are carcinomas of the breast , lung , gastrointestinal tract , ovary and lymphomas .
primary peritoneal adenocarcinoma is a very rare cause of malignant pleural effusion.case report : a 72-year old female patient presented to us with shortness of breath for the last 2 months .
a contrast - enhanced computed tomography ( cect ) scan of her - thorax revealed only bilateral pleural effusion with absence of any mass lesion or any mediastinal lymphadenopathy . a cytologic examination of pleural fluid revealed adenocarcinoma cells
. a cect of her abdomen and pelvis revealed heterogenous thickening of omentum with nodular appearances and small amount of ascites .
her ovaries were normal and no other mass lesion was detected .
a histological examination of a peritoneal lesion was suggestive of adenocarcinoma.conclusions:the patient was diagnosed with a rare case of primary peritoneal adenocarcinoma with bilateral pleural effusion . |
while blood transfusions are commonly used for prophylaxis and treatment for acute chest syndromes and strokes in sickle cell patients , accumulation of excess iron resulting in secondary hemochromatosis remains a rare disease .
chelation is the mainstay for preventing and treating iron overload to deter potential end - organ damages ; it is rare when therapy fails .
a 52-year - old african american woman with chronic anemia secondary to sickle cell anemia and history of multiple blood transfusions presented with elevated serum ferritin ( 8000 ng / ml ) and bilirubin ( 16.8 mg / dl ) .
she had no previous personal or family history of liver disease . a magnetic resonance cholangiopancreatography ( mrcp ) and a liver biopsy confirmed the secondary hemochromatosis with marked fibrosis and 4 + iron deposits , but since she was therapeutically on deferasirox , her treatment regimen involved only closer monitoring .
her hemochromatosis led to readmission within a year for rapid progression of cardiac and hepatic failure .
since chronically transfused sickle cell patients are at a significantly higher risk of mortality due to the secondary hemochromatosis and end - stage organ damage , knowledge of prophylactic iron chelation is important . minimizing unnecessary transfusions should be strongly emphasized to reduce the sequelae as iron burden remains a threat .
the effectiveness of iron - chelating therapy is best monitored via periodic magnetic resonance imaging , liver transaminases , bilirubin , creatinine , ferritin , and cardiac function tests . despite the prophylactic treatment and quarterly blood work , in this case
the initial presentation did not correlate with the severity of end - stage liver failure .
the damage was not discovered until proven by liver biopsy and mrcp , too late to deter the sequelae and the mortality exactly 1 year after diagnosis of the secondary hemochromatosis .
secondary hemochromatosis remains a rare , underdiagnosed disorder when presented atypically with bronze diabetes and cirrhosis in sickle cell patients .
blood transfusions serve as both prophylaxis and treatment for strokes and acute chest syndromes.1,2 however , excess transfusional iron accumulates in tissues and forms reactive oxygen species resulting in end - organ damages .
chelation is often initiated prophylactically in patients who have received greater than 10 units of packed red blood cells.3 in our case , despite prophylactic chelation and phlebotomy , our asymptomatic patient developed significant hepatocellular iron deposition with fibrosis leading to mortality within a year .
a 52-year - old african american woman with chronic anemia due to sickle cell disease , and history of multiple transfusions , presented for routine outpatient blood work and found to have elevated serum ferritin ( 8000 ng / ml ) and bilirubin ( 16.8 mg / dl ) .
her past medical history was significant only for cholecystectomy , with no previous personal or family history of liver disease .
an magnetic resonance cholangiopancreatography ( mrcp ) ( figure 1 ) was done to rule out an obstruction . to everyone
the biopsy later confirmed the presumptive diagnosis of the secondary hemochromatosis with marked fibrosis and 4 + iron deposits ( figure 2 ) . since the patient was therapeutically receiving phlebotomies every 45 months in addition to her iron chelator deferasirox for the last 10 years , her treatment regimen post discharge involved further monitoring every 3 months with imaging and laboratory tests , including ferritin , transferrin , and hepatic panel .
laboratory investigations represented significant cirrhosis secondary to hemochromatosis along with evidence of renal , pulmonary , and cardiac failures , severe anemia , and supratherapeutic international normalized ratio resulting in her death within 7 days of admission .
long - term transfusion therapy is now a routine , life saving treatment for patients with intractable anemia resulting from sickle cell disease , thalassemia , bone marrow failure , or aggressive treatment of cancer .
repeated transfusion leads to rapid iron loading : each unit of blood contains 200250 mg of iron.4 the american association for the study of liver diseases recommends evaluating for hemochromatosis in patients with abnormal iron studies , even in the absence of symptoms , and in all patients with evidence of liver disease.5 they suggest diagnosing hemochromatosis if transferrin saturation is > 45% in combination with elevated ferritin levels .
however a previous clinical trial suggested that serum ferritin ( range : 15003000 ng / ml ) is not always accurate to interpret the hepatic iron store concentrations because it does not directly correlate with the degree of cirrhosis.5 the effectiveness of iron - chelating therapy is best monitored via periodic magnetic resonance imaging ( mri ) , liver transaminases , bilirubin , creatinine , ferritin , and cardiac function tests .
while this patient presented with an atypical , asymptomatic presentation , her routine labs conveyed only increased ferritin and bilirubin so hepatic failure was not considered . after the liver biopsy , a concern was raised for hemochromatosis even though she was prophylactically on iron chelator .
this case raises important questions to consider : does initiating therapy early really help to increase the life expectancy of a patient with the secondary hemochromatosis ?
despite chelation therapy , iron burden in chronically transfused patients remains a threat . since her chronic conditions were well - known , would a liver biopsy earlier in the course have indicated the degree of her serious hepatic and cardiac failures ?
should routine quarterly mri and echocardiogram be used in asymptomatic patients to prevent some sequelae of end - organ damages ?
since chronically transfused sickle cell patients are at significantly higher risk of mortality due to the secondary hemochromatosis and end - organ damages , knowledge of prophylactic iron chelation is of paramount importance.2,3 minimizing unnecessary transfusions should be strongly emphasized early .
the effectiveness of iron - chelating therapy may be best monitored via periodic mri , liver transaminases , bilirubin , creatinine , ferritin , and cardiac function tests . in this atypical case ,
the initial presentation did not correlate with the severity of end - stage liver failure until proven by liver biopsy and mri , which still could not deter the sequelae and the mortality exactly 1 year after the diagnosis of secondary hemochromatosis . | backgroundwhile blood transfusions are commonly used for prophylaxis and treatment for acute chest syndromes and strokes in sickle cell patients , accumulation of excess iron resulting in secondary hemochromatosis remains a rare disease .
chelation is the mainstay for preventing and treating iron overload to deter potential end - organ damages ; it is rare when therapy fails.case reporta 52-year - old african american woman with chronic anemia secondary to sickle cell anemia and history of multiple blood transfusions presented with elevated serum ferritin ( 8000 ng / ml ) and bilirubin ( 16.8 mg / dl ) .
she had no previous personal or family history of liver disease .
a magnetic resonance cholangiopancreatography ( mrcp ) and a liver biopsy confirmed the secondary hemochromatosis with marked fibrosis and 4 + iron deposits , but since she was therapeutically on deferasirox , her treatment regimen involved only closer monitoring .
her hemochromatosis led to readmission within a year for rapid progression of cardiac and hepatic failure.conclusionsince chronically transfused sickle cell patients are at a significantly higher risk of mortality due to the secondary hemochromatosis and end - stage organ damage , knowledge of prophylactic iron chelation is important . minimizing unnecessary transfusions
should be strongly emphasized to reduce the sequelae as iron burden remains a threat .
the effectiveness of iron - chelating therapy is best monitored via periodic magnetic resonance imaging , liver transaminases , bilirubin , creatinine , ferritin , and cardiac function tests . despite the prophylactic treatment and quarterly blood work , in this case
the initial presentation did not correlate with the severity of end - stage liver failure .
the damage was not discovered until proven by liver biopsy and mrcp , too late to deter the sequelae and the mortality exactly 1 year after diagnosis of the secondary hemochromatosis . |
the juvenile hemangioma ( jh ) is a form of capillary hemangioma which occurs during infancy at a rate of about 1 in every 200 live births . during the early stage
it may resemble a common birthmark in that it is a flat , red lesion that intensifies in color when the infant strains or cries . with time it acquires an elevated , protruding appearance that distinguishes it from birthmarks and
deeply situated lesions impart little color to the overlying skin and consequently may be misdiagnosed preoperatively .
these tumors may be located on any body surface but are most common in the region of head and neck , particularly parotid , where they seemingly follow the distribution of cutaneous nerves and arteries .
although described as congenital they actually appear within few weeks after birth and rapidly enlarge over a period of several months , achieving the largest size in about 6 - 12 months , after which they regress over a period of a few years .
regression is usually accompanied by fading of the lesion from scarlet to dull red - gray and by concomitant wrinkling of the once - taut skin .
it has been estimated that by age 7 years , 75 - 90% have involuted , leaving a small pigmented scar . in the lesions that have ulcerated
early lesions are characterized by plump endothelial cells that line vascular spaces with small inconspicuous lumens .
mast cells and factor - viii positive interstitial cells are a consistent feature of these tumors .
the former may be important in the production of angiogenic factors that regulate the growth of these tumors . at this early stage of development
the vascular nature of the tumor may not be readily apparent unless a reticulin preparation is done that demonstrates connective tissue fibers encircling myriad tiny vessels .
as the lesions mature and blood flow through the lesion commences , the endothelium becomes flattened and resembles that seen in adult forms of capillary hemangioma .
regression of the jh is accompanied by a progressive , diffused interstitial fibrosis and is believed to be mediated by way of apoptosis . in unusual cases ,
a 3 year old male child was referred to the department of oral and maxillofacial surgery at nims dental college , jaipur , rajasthan , due to a painless , dome - shaped lesion of the upper lip , extending upto the cheek on the right side , light red in color , measuring 2 4 cm , present since 20 days after birth , when it first appeared .
parents revealed that the lesion would change color to a deep red when the child cried . on intraoral examination
the lesion , firm on palpation , was excised under local anesthesia , and the specimen was sent to the department of oral pathology , nims dental college , jaipur , rajasthan , for histopathological evaluation .
five micrometer sections were made for staining with h and e and 3-m sections were made for immunohistochemical analysis for the traditional markers cd 34 and cd 31 .
the immunohistochemical analysis was performed in the department of histopathology at santokba durlabhji memorial hospital , jaipur , rajasthan . the tissue received after excisional biopsy had a wrinkled surface [ figure 1 ] and revealed a cream colored , gelatinous cut surface [ figure 2 ] .
the gross tissue appears small due to decrease in size after compression of the lesion to drain out the blood during and after performing excisional biopsy procedure .
wrinkled surface of excised lesion cream - colored gelatinous cut surface sections stained with h and e were observed under 4 , 10 and 40 to reveal normal epithelium , under which was a band of normal connective tissue .
the lobules contained plump endothelial cells lining vascular spaces with inconspicuous lumens [ figure 4 ] .
some areas were more solid and organized containing a proliferation of endothelial cells [ figure 5 ] .
on the basis of clinical and histopathological features , a diagnosis of jh was made .
lesional area showing lobules below normal epithelium and connective tissue ( h and e , 4 ) plump endothelial cells lining vascular spaces with inconspicuous lumens ( h and e , 10 ) solid areas containing proliferation of endothelial cells ( h and e , 40 ) the sections were positive for cd 34 [ figure 6 ] and cd 31 [ figure 7 ] , which confirmed the diagnosis of our lesion which was in its proliferating phase in most regions of the neoplasm and involuting in a few areas .
cd 34 expression in lesional areas ( 10 ) cd 31 expression in lesional areas ( 40 )
the tissue received after excisional biopsy had a wrinkled surface [ figure 1 ] and revealed a cream colored , gelatinous cut surface [ figure 2 ] .
the gross tissue appears small due to decrease in size after compression of the lesion to drain out the blood during and after performing excisional biopsy procedure .
sections stained with h and e were observed under 4 , 10 and 40 to reveal normal epithelium , under which was a band of normal connective tissue .
the lobules contained plump endothelial cells lining vascular spaces with inconspicuous lumens [ figure 4 ] .
some areas were more solid and organized containing a proliferation of endothelial cells [ figure 5 ] .
on the basis of clinical and histopathological features , a diagnosis of jh was made .
lesional area showing lobules below normal epithelium and connective tissue ( h and e , 4 ) plump endothelial cells lining vascular spaces with inconspicuous lumens ( h and e , 10 ) solid areas containing proliferation of endothelial cells ( h and e , 40 )
the sections were positive for cd 34 [ figure 6 ] and cd 31 [ figure 7 ] , which confirmed the diagnosis of our lesion which was in its proliferating phase in most regions of the neoplasm and involuting in a few areas .
cd 34 expression in lesional areas ( 10 ) cd 31 expression in lesional areas ( 40 )
in our case the clinical and histopathological features suggest that the lesion was in the proliferating and involuting phase .
the solid areas gave a clear indication that the lesion was a long - standing one .
the clinical phases of jh have distinctive underlying physiological differences , which have been correlated with equally distinctive immunophenotypic profiles by takahashi et al . during the early proliferative phase ( 0 - 12 months ) the tumors can be shown immunohistochemically to express proliferating cell nuclear antigen ( pcna ) , vascular endothelial growth factor ( vegf ) , and type iv collagenase , the former two localized to both endothelium and pericytes , and the last to endothelium .
all of these substances are associated with proliferation and growth of vessels . during the involuting phase ( 1 - 5 years ) these
are dramatically reduced , whereas the tissue inhibitors of metalloproteinases ( timp ) , anti - angiogenic factors , are markedly elevated .
the traditional vascular markers cd 31 , von willebrand factor ( vwf ) , and smooth muscle actin ( pericyte marker ) are present during the proliferating and involuting phases , but are lost after the lesion is fully involuted .
jh also expresses glut1 , a glucose transport receptor , the expression of which is independent of proliferative activity , and is not found in other forms of hemangiomas although it is present rarely in angiosarcomas .
this protein is also expressed by human placenta and has led to the novel hypothesis that the proliferative component of jh may be derived from embolized placental vascular cells .
neonatal and infantile hemangiomas grow rapidly initially , but the majority spontaneously involute by age 7 years .
surgical excision during the proliferative phase should be avoided due to risk of major blood loss and injury to facial nerve .
diagnosis may be made by aspiration cytology and conservative nonoperative management includes steroids to reduce growth , compression therapy , or embolization .
no treatment may be required , however , a small percentage of hemangiomas present themselves with indications for prompt therapy ( including surgical excision ) , such as severe disfigurement , airway compromise , excessive bleeding , and ophthalmological complication . the patient was examined after 2 months . | hemangiomas occupy a grey zone between hamartomatous malformations and true neoplasms . they are frequently designated and regarded as neoplasms because of their usually localized nature and mass effect .
although clearly benign , they can become very large and unsightly , and can even be fatal if they affect vital structures .
they almost never become malignant , although a few documented examples of this complication are on record .
a high percentage occur in children , manifesting within the first month of life .
one half of these cases are in the head and neck area .
hemangiomas have been classified according to their clinical appearance and the caliber of vessel involved , namely , capillary , cavernous and venous .
capillary hemangiomas are made up of small vessels of capillary caliber .
one such capillary hemangioma , the juvenile hemangioma ( jh ) , is usually present at birth or appears during the first month and enlarges rapidly during the first few months of life ( infancy ) , only to stop growing when the child is approximately 6 years old .
we present one such jh , seen in a 3 year old male child , which appeared when the child was 2 months old .
routine histopathological ( h and e ) and immunohistochemical analysis ( cd 34 , cd 31 ) was done on biopsy received . |
it is important to limit the block level to minimise haemodynamic changes during spinal anaesthesia .
low - dose local anaesthetics can limit the block level and induce rapid recovery from anaesthesia with minimal haemodynamic changes . local anaesthetics - when used alone have a relatively short duration of action .
the addition of opioids to local anaesthetic produces good post - operative analgesia but has disadvantages , such as pruritus , post - operative nausea vomiting and respiratory depression .
dexmedetomidine ( dmt ) , a highly selective 2-agonist , is under evaluation as a neuraxial adjuvant .
it provides stable haemodynamic conditions , good quality of intra - operative analgesia and prolonged post - operative analgesia with minimal side effects .
however , there may be dose - related prolongation of the duration of motor blockade thus delaying discharge from post - anaesthesia care unit in day case surgeries .
this trial was conducted to investigate if reduction in dose of intrathecal dmt added to a low dose hyperbaric bupivacaine would produce clinically relevant analgesia without prolonging motor blockade in patients undergoing ambulatory perianal surgeries .
time to ambulation and duration of analgesia were the primary outcomes studied and sensory regression , haemodynamic changes and effect on sedation were secondary outcomes observed .
after obtaining approval from the institutional ethics committee , 50 adult patients of american society of anesthesiologists physical status i and ii undergoing elective perianal surgeries were enrolled in this prospective randomised double - blinded study .
patients were randomly allocated to two groups of 25 each using a sealed envelope method .
group c received dmt 3 g intrathecally and group d dmt 5 g along with the local anaesthetic agent . patients with past history of spine surgery , coagulation disorders and infection at site of lumbar puncture , neurological and psychiatric disturbances , diabetes and other endocrine disorders , hypertension and other cardiac disorders , and those on beta blockers , calcium channel blockers , 2 agonists and patients with history of drug allergy were excluded from the study . written informed consent
was obtained from all the patients who fulfilled the inclusion criteria and were willing to participate in the study .
premedication with oral ranitidine hydrochloride 150 mg and alprazolam 0.25 mg were given the night before the surgery and they were kept nil per oral for 8 h before induction of anaesthesia . during the pre - operative evaluation , patients were given instructions to use a 10-point visual analogue scale ( vas ) , with 0 indicating no pain and 10 indicating the worst imaginable pain .
ranitidine 50 mg intravenous ( iv ) . on shifting to operation theatre , electrocardiogram , peripheral oxygen saturation ( spo2 ) , non - invasive blood pressure monitors were connected and baseline values were recorded .
following infusion of 500 ml ringers lactate solution , lumbar puncture was performed at l3-l4 interspace or l4-l5 interspace with the patient in the sitting position using 27 gauge quincke needle .
group c received hyperbaric bupivacaine 0.5% ( 0.8 ml ) ( 4 mg ) + dmt 3 g intrathecally and group d received hyperbaric bupivacaine 0.5% ( 0.8 ml ) ( 4 mg ) + dmt 5 g intrathecally .
the required dose of dexmedetomidine was drawn using a tuberculine syringe ( 0.03 ml for 3 g and 0.05 ml for 5 g ) .
the total volume of solution to be injected intrathecally was made to one ml using normal saline .
the drug solutions were prepared under sterile precautions by an anaesthesiologist as per instructions in the envelope , who was not involved in administration and further monitoring of the patient .
the anaesthesiologist performing the spinal block and monitoring the patient was unaware of the drug administered .
after the spinal block , the patients were made to sit for 5 min and then made supine .
the level of sensory block , defined as the dermatomal segment with total loss of sensation to cold swab was assessed every 2 min after intrathecal injection till 10 min and every 5 min thereafter until maximum level of sensory block level stabilised .
the maximum level of sensory block was considered when the loss of sensation was recorded at the same dermatomal level for 2 consecutive time intervals .
( appendix ) onset of sensory block , defined as the time required for a sensory block to reach s1 was noted .
blood pressure and heart rate , was recorded every 2 min for initial 10 min and then every 5 min until the end of surgery and then at 30 min intervals in post - operative period until regression of block .
sedation was monitored using ramsay sedation scale and time for sensory and motor regression to s1 and b6 , respectively , were noted and were considered as the duration of sensory and motor blockade , respectively . if patient complained of pain during surgery , it was considered as failure of subarachnoid block and inj fentanyl 2 g / kg was given as bolus and if necessary , supplementation with general anaesthesia was done .
post - operatively , pain intensity was assessed using vas scale and inj diclofenac 75 mg administered intramuscularly as rescue analgesic , once the vas score was more than three .
duration of post - operative analgesia was noted from the time of sab to the first demand for rescue analgesic .
patient was made ambulant when the following criteria were achieved ( 1 ) able to perform partial knee bend ( bromage scale 6 ) ( 2 ) recovery of proprioception of great toe ( 3 ) return of perianal sensation ( 4 ) no postural hypotension on making the patient stand .
time for ambulation was recorded from the time of sab to the time when patient was made ambulant .
time for urination , sedation and side effects if any were also observed in the post - operative period .
side effects such as hypotension ( defined as a decrease in mean arterial pressure > 25% of the baseline value ) was treated with iv boluses of 6 mg ephedrine .
bradycardia defined as a pulse rate of < 50 beat / min was treated with bolus of 0.6 mg atropine iv , respiratory depression ( rr < 8 or spo2 < 95% ) was treated with oxygen supplementation and respiratory support if required , vomiting and others if any were noted .
we hypothesised that reduction in dose of dmt would reduce the time required for ambulation . keeping ( 1 ) error at 80% and two sided significance level ( error ) at 0.05 , to detect at least a 10% difference in time for ambulation between the two groups , the minimum sample size required was 21 in each group .
independent sample t - test was used to compare parametric and continuous data , and mann whitney u - test applied if variables were not uniformly distributed .
one patient in each group was excluded from analysis due to the failure of the subarachnoid block .
demographic characteristics such as age , weight , height , sex and duration of surgery were comparable .
demographic characteristics duration of post - operative analgesia was 337.86 105.11 min in group c whereas 340.78 101.81 min in group d ( p = 0.9 ) and time to ambulation in group c was 252.46 93.72 min and 253.64 88.04 min in group d ( p = 0.97 ) .
the onset of sensory block to s1 was 9.61 5.53 min in group c compared to 7.69 4.80 min in group d ( p = 0.35 ) .
median level of cephalad spread of sensory block was l1 ( t11 s1 interquartile range ) in group c and t11 ( t10 s1 interquartile range ) in group d. duration of sensory ( 145.28 83.17 min
c , 167.85 93.75 min d , p = 0.5 ) and motor block ( 170.53 73.44 min
intraoperative and post - operative haemodynamic parameters were comparable between the two groups [ figures 1 and 2 ] .
the mean sedation score ( average of mean sedation score of each patient ) was 2.008 0.03 in group d compared to 2 0.05 in group c ( p = 0.5 ) , and average post - operative sedation scores were two in both groups .
none of the patients had sedation scores more than four in either group at any point of time during the study period .
none of the patients in either group developed hypotension or bradycardia either in intraoperative period or post - operative period .
the present study showed that dmt 3 g administered as intrathecal adjuvant to a low dose hyperbaric bupivacaine provided similar duration of analgesia compared to 5 g of dmt as intrathecal adjuvant , but did not shorten the time required for ambulation . though the onset of sensory block was slightly shortened in group d , this was not statistically significant .
doses varying from 3 to 15 g of dmt have been added as an adjuvant to intrathecal bupivacaine .
the present study tried to assess the effects of lower doses of dmt in ambulatory surgeries , hence doses of 3 and 5 g were compared .
low - dose local anaesthetic for the subarachnoid block is commonly administered to limit the block level to minimise the haemodynamic changes .
a low dose of hyperbaric bupivacaine has been successfully used for ambulatory arthroscopic knee surgeries and also in perianal surgeries .
the addition of an adjuvant should further potentiate analgesia without prolonging motor blockade or causing other side effects .
the prolongation of sensory effect may result from synergism between local anaesthetic and 2-adrenoceptor agonist , while the prolongation of the motor block of spinal anaesthetics may result from the binding of 2-adrenoceptor agonists to motor neurons in the dorsal horn .
perineural administration of dmt also is reported to prolong the duration of the motor blockade in rats .
observed faster onset of sensory and motor block with addition of dmt , but the present study did not reveal any statistically significant difference in onset of sensory and motor block with 3 and 5 g of dmt . the shorter duration of motor block in the present study compared to kanazi et al .
may be attributed to the lower dose of local anaesthetic used . making the patient sit for 5 min may also limit the spread of drug further limiting the motor blockade .
administration of an 2-agonist like dmt via an intrathecal route provides an analgesic effect in the post - operative period without severe sedation .
this effect is due to the sparing of supraspinal central nervous system sites from excessive drug exposure , resulting in robust analgesia without heavy sedation . at spinal cord level , activation of both 2-c and 2-a receptors , in the neurons of superficial dorsal horn , especially lamina ii ,
directly reduces pain transmission , by suppressing the release of pro - nociceptive transmitter , substance p and glutamate from primary afferent terminals , and by hyperpolarising spinal interneurons via g - protein - mediated activation of potassium channels .
none of the patients in either group had excessive sedation and is comparable with other studies .
dmt 5 g when used as adjuvant to bupivacaine 6 mg prolonged duration of motor block and delayed ambulation in patients undergoing perianal surgeries . in this study
, an attempt was made to assess the effect of reduction in doses of both dmt and bupivacaine on ambulation and analgesia .
haemodynamic parameters were comparable in both the groups and there were no incidences of hypotension and bradycardia in the intraoperative or post - operative period .
the present study did not observe the effect of these doses of dmt on analgesic consumption in the 24 h period , which may be a limitation .
future studies may be targeted to assess the effect of these doses on analgesic efficacy in ambulatory surgeries .
intrathecal bupivacaine with dexmedetomidine 3 g does not produce faster ambulation compared to intrathecal bupivacaine with dexmedetomidine 5 g , though it produces comparable duration of analgesia for perianal surgeries .
| background and aims : dexmedetomidine ( dmt ) , as intrathecal adjuvant has been shown to successfully prolong duration of analgesia but delay the motor recovery .
hence , this study was designed to find out the dose of dmt which can provide satisfactory analgesia without prolonging motor block.methods:a total of 50 patients scheduled for elective perianal surgeries were randomly allocated to groups c or d ( n = 25 ) .
group d received hyperbaric bupivacaine 0.5% 4 mg + dmt 5 g and group c received hyperbaric bupivacaine 0.5% 4 mg + dmt 3 g intrathecally .
onset and duration of sensory and motor blockade , duration of analgesia , time for ambulation and first urination were recorded .
adverse effects if any were noted.results:demographic characters , duration of surgery were comparable .
the onset of sensory block to s1 was 9.61 5.53 min in group c compared to 7.69 4.80 min in group d ( p = 0.35 ) .
duration of sensory ( 145.28 83.17 min
c , 167.85 93.75 min d , p = 0.5 ) and motor block ( 170.53 73.44 min
c , 196.14 84.28 min , p = 0.39 ) were comparable .
duration of analgesia ( 337.86 105.11 min
c , 340.78 101.81 min
d , p = 0.9 ) and time for ambulation ( 252.46 93.72 min
c , 253.64 88.04 min
d , p = 0.97 ) were also comparable .
one patient in each group had urinary retention requiring catheterization .
no other side effects were observed.conclusion:intrathecal dmt 3 g dose does not produce faster ambulation compared to intrathecal dmt 5 g though it produces comparable duration of analgesia for perianal surgeries . |
it typically occurs within fourteen days of aneurysmal rupture , and it is associated with significant morbidity and mortality [ 1 , 2 ] . while pathophysiology remains incompletely understood , the interplay between inflammation and the innate immune response is strongly implicated .
following sah , increased blood - brain barrier ( bbb ) permeability engenders a proinflammatory milieu in the cerebral cisterns and extravascular space .
subarachnoid blood initiates leukocyte transmigration via cellular margination , adhesion , rolling , and diapedesis .
the process necessitates violation of the tight junctions between endothelial cells of the bbb and typically occurs in response to stimulatory chemoattractants or chemokines .
regulation of the extracellular matrix and basal lamina by matrix metalloproteinase ( mmp ) enzymes may play a critical role in vasospasm .
mmps have been studied extensively in the pathogenesis of ischemic stroke and the development of aortic and cerebral aneurysms .
recently , investigations have examined the role of mmps in the setting of sah . in this paper , we specifically review the function of mmps in cerebral vasospasm .
understanding the complex interactions between inflammation and degradation of the extracellular matrix may ultimately allow for better development of diagnostic markers and targeted therapies relevant to the management of delayed cerebral vasospasm .
mmps are a family of extracellular and membrane - bound proteases capable of degrading or proteolytically modifying the extracellular matrix ( ecm ) through interactions with collagenases , laminins , and proteoglycans .
they utilize zinc - dependent endopeptidases to regulate physiologic processes relevant to development , homeostasis , and tissue modeling .
mmps are secreted by macrophages , leukocytes , smooth muscle cells , endothelial cells , astroglia , and microglia in response to growth factors and inflammatory cytokines [ 712 ] .
activation through the zymogen in a cysteine switch was first demonstrated by van wart and birkedal - hansen .
demonstrated that mmp-3 is capable of activating pro mmp-1 , mmp-7 , mmp-8 , mmp-9 , and mmp-13 , while mmp-2 can activate pro mmp-1 , mmp-9 , and mmp-13 .
activated mmps have important roles in neuronal development , function , and differentiation [ 1418 ] .
inhibition is mediated by a family of proteins known as tissue inhibitors of metalloproteinases ( timps ) .
activated leukocytes are a major source of mmp production following brain injury [ 21 , 22 ] . as mmps degrade the basal lamina , cellular transmigration is facilitated .
degradation of the blood - brain barrier allows leakage of inflammatory mediators into the extracellular matrix .
the proinflammatory environment , in turn , may promote arterial dysregulation , stimulate leukocyte migration , enhance cytokine / chemokine production , and render the affected vessels susceptible to narrowing .
a review of the specific mmps follows ; only those that have been studied in the setting of delayed cerebral vasospasm ( mmp-1 , mmp-2 , mmp-8 , mmp-9 , and mmp-13 ) are included .
the effect is durably negated by mmp inhibitors [ 2325 ] . to date , a single investigation has examined the role of mmp-1 in cerebral vasospasm . in an experimental rodent model , satoh et al .
utilized immunohistochemical and western blot analysis of basilar arteries to quantify mmp-1 deposition . when compared to controls ,
this elevation peaked at 30 minutes post - sah and was temporally concordant with vessel narrowing .
only one group has examined the role of mmp-2 in the pathogenesis of cerebral vasospasm .
other investigators , however , have documented elevated mmp-2 levels in vessel walls of ruptured and unruptured cerebral aneurysms [ 2628 ] and in patients who suffered ischemic stroke . due to a paucity of large studies and conflicting data , the precise role of mmp-2 in the development of cerebral aneurysms and pathogenesis of vasospasm
the putative role of mmp-9 in cerebral vasospasm was first explored by mcgirt et al . .
they compared levels with individuals admitted for elective aneurysm clipping and patients who suffered from non - sah stroke .
further , they assessed mmp-9 levels in sah patients who developed cerebral vasospasm and those who did not .
onset of vasospasm was defined as transcranial doppler flowmetry velocities greater than 180 cm / s and/or new focal neurological deficits with angiographically confirmed vasospasm .
mean peak serum mmp-9 levels were increased in the sah cohort that developed vasospasm when compared to the group that did not .
further , peak mmp-9 levels for the sah cohort that developed vasospasm were significantly elevated when compared to the focal ischemia and control cohorts .
mmp levels of greater than 700 ng / ml independently increased the odds of subsequent vasospasm by 25-fold .
these findings first advocated mmp-9 as a potential diagnostic biomarker for cerebral vasospasm in the setting of sah and incited significant interest in related research . in a rat sah model ,
sehba et al . examined the role of mmp-9 on basal lamina integrity in the cerebral microvasculature .
collagen iv is a major structural component of the basal lamina , comprising approximately 90% of the total protein [ 32 , 33 ] .
the authors noted activation of four key signal transduction molecules ( p38 , erk1/2 , elk-1 , and activating transcription factor-2 ) and resultant transcription of ecm - related ( mmp8 , mmp9 , and mmp13 ) and inflammatory ( il6 , tnfa , il1b , cxcl1 , cxcl2 , ccl20 , and inos ) genes .
the findings suggested one signal transduction pathway by which mmps are activated in response to sah and elucidated potential targets for therapy .
examined serum mmp-9 concentrations in a relatively small cohort of sah patients and compared values to normal controls .
the authors found mmp-9 levels to be increased in the sah group , but they failed to demonstrate correlation with clinical hunt and hess scores .
feiler et al . examined the influence of mmp-9 on cerebral edema and functional outcomes following sah in mmp-9 knockout ( mmp-9/)mice .
they found no significant differences in acute intracranial pressure ( icp ) , cerebral blood flow , or mean arterial blood pressure between mmp-9/ and wild - type mice .
however , day 7 mortality was significantly lower in mmp-9/ mice ( p = 0.03 ; 20% versus 60% ) .
the mmp-9 knockout mice also demonstrated less brain edema , lower chronic icp , and improved neurological recovery when compared to the wild - type mice .
findings suggest a role for mmp-9 in the genesis of cerebral edema and increased intracranial pressure following sah . early brain injury ( ebi ) is believed to cause significant morbidity and mortality following sah .
the study examined ecm protein lamina integrity and anoikis / apoptosis in the hippocampal region .
injury increased from 12 to 72 hours , with peak activity evident at 24 hours .
similarly , laminin density decreased at 12 hours , reached a nadir at 24 hours , and began to recover at 48 hours .
brain water content , a surrogate measure for cerebral edema , increased at time points relevant to neuronal apoptosis .
the authors concluded that mmp-9 plays an important role in the pathophysiology of early brain injury following sah via degradation of laminin and resultant anoikis of hippocampal neurons .
a single long - term study has associated mmp-9 levels and inflammation with poor clinical outcomes following sah .
chou et al . examined mmp-9 and neutrophil levels in blood and csf of fifty - five sah patients .
the investigators examined associations between the following parameters : ( 1 ) serum and csf leukocyte and neutrophil and mmp-9 levels , ( 2 ) development of cerebral vasospasm , and ( 3 ) poor clinical outcome ( three - month modified rankin score ) .
development of cerebral vasospasm was independently associated with persistent elevation of blood leukocytes and neutrophils on post - sah days 014 but not with csf or serum mmp-9 levels .
poor three - month modified rankin scores correlated with elevated blood neutrophil counts on post - sah days 2 - 3 , elevated serum mmp-9 levels on post - sah days 4 - 5 , and elevated csf mmp-9 values on post - sah days 014 .
further , blood neutrophil levels correlated with mmp-9 levels on post - sah days 68 and 1014 . together , these findings suggest that neutrophils may represent a major source of mmp-9 production in the early post - sah time period .
as mmp-9 levels were associated with poor three - month outcomes , but not delayed cerebral vasospasm , the authors postulate a role for mmp-9 in alternate mechanisms of brain injury ( aside from vasospasm ) following sah .
wang et al . recently described the time course of basilar artery mmp-9 expression in an experimental murine model of sah .
further , the investigators explored the effect of sb-3ct , a selective mmp-9 inhibitor , on the development of cerebral vasospasm .
they discovered that expression of mmp-9 peaked on postbleed day 3 and returned to normal by day 14 which is consistent with the time course of delayed cerebral vasospasm .
the authors noted a significant reduction in vasospasm ( measured by cross - sectional area of the basilar artery ) on postbleed day 3 following the administration of sb-3ct .
these findings highlight an mmp-9 inhibitor which may hold therapeutic promise in the setting of cerebral vasospasm .
recent studies have assessed the utility of mmp inhibitors in translational models of stroke and cerebral ischemia .
however , few inhibitory strategies have been investigated in the setting of vasospasm . in a study by dittmar
et al . , various classes of mmp inhibitors including monoclonal antibodies , tissue mmp inhibitors , and broad - spectrum mmp inhibitors ( bb-94 , bb-1101 , and kb - r7785 ) proved to be effective in reducing tissue damage in an animal model of cerebral ischemia . in a rodent model ,
reijerkerk et al . demonstrated that bb-3103 , a broad - spectrum matrix metalloproteinase ( mmp ) inhibitor , decreased endothelial gap formation , occludin loss , and the ability of monocytes to traverse the endothelium .
future studies may seek to translate these positive findings from closely related systems into relevant cerebral vasospasm models .
mmps are a family of proteases that preserve the structure of blood vessels . under normal conditions ,
they regulate transfer of molecules , aid in adaptation of the ecm , and maintain the bbb . in state of disease
, they can mediate breakdown of the bbb and thus modify the local inflammatory response .
together , these factors may incite or propagate delayed cerebral vasospasm . in this capacity , mmps likely contribute to progressive injury following ischemia and aneurysmal rupture .
mmps could ultimately provide a potential biomarker for early diagnosis or a target for therapeutic intervention in the setting of delayed cerebral vasospasm following sah . | delayed cerebral vasospasm is a significant cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage ( sah ) .
while the cellular mechanisms underlying vasospasm remain unclear , it is believed that inflammation may play a critical role in vasospasm .
matrix metalloproteinasees ( mmps ) are a family of extracellular and membrane - bound proteases capable of degrading the blood - rain barrier ( bbb ) . as such
, mmp upregulation following sah may result in a proinflammatory extravascular environment capable of inciting delayed cerebral vasospasm .
this paper presents an overview of mmps and describes existing data pertinent to delayed cerebral vasospasm . |
a well - functioning permanent vascular access is crucial for the survival of patients on maintenance hemodialysis as it provides sufficient blood flow for dialysis . because adequate blood flow is provided by the arteriovenous fistula ( avf ) with the fewest complications , avf
the national kidney foundation 's kidney disease outcomes quality initiative ( nkf - kdoqi ) is keeping in attempts to improve the utilization of the avf in patients who receive maintenance hemodialysis .
it was reported by the united states renal data system ( usrds ) that hemodialysis access failure remains to be the most frequent cause of hospitalization .
all kinds of complications , including stenosis , thrombosis , abnormal establishment of collateral branches circulation , and so on , may lead to a dysfunction of the avf or inadequate dialysis , which eventually has an impact on the quality of life of the patients or even makes the vascular access unusable .
the buttonhole cannulation , also called the constant site cannulation , has been successfully used in home hemodialysis and dialysis centers to reduce bruising , prolonged bleeding , and aneurysm formation .
we report the case of a 47-year - old male ( chinese ) , who was diagnosed with end - stage renal disease ( esrd ) .
a mature avf on the left wrist was operated 5 years ago , and the wrist recovered its function 2 months after the operation . at the end of the fourth year of hemodialysis ,
the ultrasound dilution technique was applied for surveillance of the function of the fistula , showing a recirculation of zero ( transonic hd02 monitor ; ithaca , ny , usa ) and an access flow rate ( qa ) of 1,360 ml / min .
the spkt / v was 1.28 , which indicated that the avf was functionally effective .
two months later , the buttonhole cannulation technique was applied due to fistula punctuation difficulty with previous aneurysmal dilatation .
after 10 times of buttonhole cannulation , the patient experienced gradual edema on the left forearm as well as insufficient blood flow of the fistula [ fig . 1 ] .
the recirculation of the avf was 87% with a blood flow ( qb ) of 220 ml / min and a spkt / v of 0.75 , which indicated a dysfunction of the avf .
this was further confirmed by a vascular ultrasound of the avf , revealing abnormal fistula between the mature cephalic vein and the radial vein [ fig .
2 ] where blood shunt existed , together with thrombus in the proximal cephalic vein and aneurysmal dilatation in the distal segment [ fig .
3 ] that developed 3 years ago . the site for punctuation was changed , and a reduction of recirculation to 25% with qb 240 ml / min could be confirmed 2 weeks later .
the utilization of the new avf began 4 months after the operation and has been working well until now .
a well - functioning avf is the key to long - term maintenance hemodialysis in patients with esrd .
one of the major causes of dialysis inadequacy is the dysfunction of vascular access secondary to vascular access complications . despite the fact that the avf has the least complications and is the preferred method of dialysis access
, every permanent access may lead to complications associated with low blood flow and recirculation , such as failure of maturation , venous stenosis , thrombosis , and so on . in our case , thrombus was found in the proximal cephalic vein with aneurysmal dilatation in the distal segment .
emergency hospitalization for 15 days was necessary because of cardiopalmus , dizziness , and hyperkalemia .
in fact , the most common causes of hospitalization associated with access complications in dialysis patients include insufficient blood flow , vascular access - related infections , limb ischemia , and aneurysms . in the case reported herein , the complication of abnormal fistula on the avf occurred during the shift from traditional cannulation ( rope - ladder cannulation ) to buttonhole cannulation .
rope - ladder cannulation , which is characterized by the variation of the needling site , is considered the most frequently used technique in comparison with other kinds of cannulation .
the purpose of buttonhole cannulation is to form a scar tissue tunnel track between skin and vascular access to allow a dull - tipped needle to access the avf following the same path during every dialysis session by means of repeated needle puncture at exactly the same site and angle .
this technique may reduce the incidence of vascular access complications with the clinical manifestation of bruising , prolonged bleeding , and aneurysm formation .
several studies have shown that buttonhole cannulation is a valuable technique with less hematoma , aneurysm formation , and cannulation pain than the rope - ladder cannulation technique and that is easy to self - cannulate , which is particularly relevant for home hemodialysis [ 5 , 6 , 7 ] .
al - though growing evidence has shown the advantages of buttonhole cannulation , a generally accepted method of cannulating for the avf has not been defined yet . in theory , patients with buttonhole cannulation show fewer complications in the view of access , since buttonhole cannulation only needs one puncture site on the blood vessel wall and there is no regional injury of the elastic fiber on the blood vessel , thus reducing the incidence of aneurysm formation and vascular stenosis .
on the one hand , the angle , direction and depth of cannulation must be identified before the formation of a buttonhole .
however , even if the most skilled expert cannulators perform the procedure , slight or even invisible cannulation differences still exist between different operators or between several procedures performed by the same cannulator . on the other hand , in buttonhole cannulation ,
however , dry weight changes and variations in the patient 's fluid status , both intra- and extravascular , may lead to changes that render the cannulation more uncertain , illustrating the difficulty of punctuation . to make some progress in reducing the incidence of vascular access complications caused by buttonhole cannulation ,
ball strongly recommended that the same expert should create the tunnels in order to maintain the same angle and depth to the greatest extent .
one of them is to leave approximately 1/16 inch of the needles exposed when inserting them into the buttonhole tunnels to prevent the hub of the needles from touching the entrance sites , thus making it easy to punctuate , remove the scabs , clean the entrance site , and reduce the risk of injury and infection of the tunnel .
the complications induced by buttonhole cannulation itself are sometimes difficult to be separated . unfortunately , in this case , recirculation monitoring was only applied 2 months before cannulation , so the exact time of abnormal fistula formation in this patient could not be known . in this rare case ,
edema on the left forearm and insufficient blood flow of the fistula revealed recirculation , abnormal fistula , and thrombus in the previous aneurysmal dilatation , which was confirmed by the transonic hd02 monitor and ultrasound .
appropriate monitoring and surveillance for the detection of abnormalities concerning vascular access in dialysis patients is essential even under the best protection measures and the most skilled experts .
the nkf - kdoqi work group recommends an organized monitoring or surveillance approach with regular assessment of clinical parameters of the vascular access .
the detection of the access dysfunction contains three aspects : monitoring , surveillance , and diagnostic testing .
monitoring is defined by the nkf - kdoqi as the evaluation of the vascular access by means of physical examination to detect physical signs that suggest the presence of dysfunction , which can be used as a monitoring tool to exclude low blood flow associated with impending vascular access failures . inspection ( look ) , palpation ( touch ) , and auscultation ( listen ) are three important components of monitoring , which can help clinical doctors detect initial events of vascular access .
surveillance is an effective method to detect the presence of dysfunction by means of tests or even with special instruments .
surveillance methods include device - based methods such as sequential access flow , recirculation measurements ( recommended every month ) , sequential dynamic or static pressures , and so on . in the case
described , ultrasound dilution , which is a practical , noninvasive and reliable indicator of vascular access , was used for function surveillance of the avf , which is reflected by access flow ( qa ) and recirculation .
recirculation surveillance by ultrasound dilution thus not only indicates the existence of vascular stenosis but also other complex vascular complications .
every 3 months , transonic hd02 monitor was scheduled for patients at our center , since it is not covered by insurance .
for patients with a high risk of vascular access dysfunction as our case , such monitor sessions should take place every month .
in the view of the dialysis adequacy and kt / v , on line urr based on uv absorbance may be a more sensitive method to find the inadequate dialysis in real time .
however , no significant evidence has been found to prove prolonged access survival . although surveillance did not increase the likelihood of vascular access survival , it is critical to establish a team consisting of the patient , dialysis staff , vascular access coordinator , nephrologists , interventionalists , and vascular surgeon to take care of and improve the quality and outcomes of vascular access using monitor and surveillance .
a well - functioning avf is the key to long - term maintenance hemodialysis in patient with esrd . however , one of the major causes of dialysis inadequacy is the dysfunction of vascular access resulting from vascular access complications .
buttonhole cannulation compared to other cannulation methods is a technique that may lead to a reduction in many complications , but it can not entirely avoid all incidences of vascular access problems . therefore , before the formation of a buttonhole , the punctuation should be operated by a defined expert cannulator team to ensure that the identified angle , direction , and depth are maintained as well as possible and to pay attention to other details to minimize the incidence of complications as described above .
a successful surveillance system should be established , including the participation of patients and medical staff from various departments based on the adequate monitoring , online kt / v monitor , and the surveillance of the vascular access with access blood flow and recirculation to identify a possible dysfunction of the avf .
regular surveillance with the ultrasound dilation method before and during the process of construction of buttonhole cannulation is suggested to find a possible dysfunction of the avf so that intervention measures can be taken at the earliest convenience . | buttonhole cannulation has been suggested as a technique that may lead to a reduction in many complications when compared with other techniques such as rope - ladder cannulation . despite all above
, some complications still continue , which may lead to a dysfunction of the arteriovenous fistula ( avf ) and inadequate dialysis , having an impact on the quality of life of the patients or eventually making the vascular access unusable .
we report a 47-year - old chinese male with end - stage renal disease who had maintenance hemodialysis three times a week for 5 years .
the avf on the left wrist was operated 5 years ago and proved to be functionally effective with some monitoring and surveillance measures in the fourth year of hemodialysis .
two months later , the buttonhole cannulation technique was applied due to fistula punctuation difficulty by previous aneurysmal dilatation .
after 10 times of buttonhole cannulation , the edema on the left forearm of the patient indicated a dysfunction of the avf .
this was further confirmed by the hd02 hemodialysis monitor and a vascular ultrasound , revealing a recirculation of 87% , an abnormal fistula between the avf and the vein together with thrombus , and aneurysmal dilatation near the anastomosis .
the site of cannulation was changed to avoid heavy recirculation ( reduced to 25% ) , and a new avf on the left wrist was successfully operated .
abnormal fistula on the avf and vein during buttonhole cannulation is rarely reported , which reminds us that vascular access surveillance should be done regularly before and during the process of construction to find a possible dysfunction of the avf in patients undergoing buttonhole cannulation . |
our case is a 37-year - old male who hails from sivagangai district , tamil nadu .
the patient had minimal bleeding of 20 ml / day within the past 2 days which was not associated with pain , postural change , trauma or nose picking .
he had a history of headache , that was dull aching in type and confined to the frontal region .
the patient gives a classical history of residing in an endemic region with cattle rearing as his occupation and also gives a history of routine bathing in the ponds along the animals . on examination , a small single fleshy bluish pink pedunculated polypoidal mass
its surface was smooth , and it was not probed due to anticipation of bleeding .
all routine investigations were done , and contrast computed tomography paranasal sinuses was done which showed a single nonenhancing mass seen to be attached to the inferior turbinate with no signs of bony erosion and any other abnormalities . the patient was later planned for elective surgery , and an endoscopic excision was done and the base was cauterized to prevent recurrence .
the specimen was sent for histopathological examination where it showed that the polyp was lined by stratified squamous epithelium and it was infiltrated by numerous lymphocytes and plasma cells .
later follow - up of the case was done at 6 months and after 1-year where no signs of recurrence seen .
rhinosporidiosis is seen to be endemic is seen in about 70 countries but its incidence is very high in india and sri lanka , in india certain places tamil nadu like , madurai , sivagangai , ramanathapuram , chenglepet ( singaperumalkoil ) districts are highly endemic for the disease .
the organism causing the disease is described in textbooks as the fungi rhinosporidium seebri but latest research conclude that the actual organism belongs to clusters with novel group of fish parasites referred to as the dermocystidium , rossete agensts , icthyophorus and psorospermium clade .
the presentation of the disease classical as the person belongs to an endemic area , bathes in ponds and is also a cattle raiser all of which have documental evidence of being associated with rhinosporidiosis .
it is described that the polyp usually presents at multiple sites and is attached to the septum and lateral wall .
the presentation in our case is solitary , and the site is also very unique as it is attached to the inferior turbinate .
this also promotes us to the fact that we might have diagnosed the patient in the very early course of the disease and if not he would have progressed to the advanced stages and presented with the more textbook features .
the treatment which we have offered is simple endoscopic excision and cauterization of the base which is approved by standard textbooks another add - on therapy , which could have been added is dapsone to prevent recurrence .
the histopathological findings also are very standard when compared with some findings like pseudoepithelomatous squamous cell metaplasia [ figure 1 ] . at present ,
the latest methods of treatment include the use of ktp-532 potassium titanyl phosphate laser for treatment and has been showing promising results .
the classical findings like the fungal spores were also visualized which were similar to the images in figure 1 . | rhinosporidiosis is a unique disease , which is seen to be endemic in certain places in india such as tamil nadu and sri lanka .
the disease is caused by rhinosporidium seebri and it is transmitted by bathing in ponds contaminated by cattle feces containing spores of the organism .
the disease usually presents as multiple granulomatous bleeding polyps .
the case described here is a unique presentation where it occurs only in a single site and that too in an uncommon location where the suspicion of rhinosporidiosis is a last possibility . |
since ancient times , prospective parents have tried to influence the sex of their future children .
aristotle , for example , advised those wanting a boy to have intercourse when the wind is in the north .
other people have assumed that special diets , the timing of intercourse in relation to ovulation , binding up one of the testicles during intercourse , or the position during intercourse , might facilitate sex selection . apparently , every folk culture has its own strategies .
this is widely ( though not universally ) seen as a welcome development insofar as sex selection for medical reasons is concerned .
however , sex selection for non - medical reasons has been the subject of recurrent ethical and public policy debate in many countries .
the latest round in this debate was fuelled by the public consultation held in the united kingdom on the initiative of the human fertilisation and embryology authority ( hfea ) in 2002 , and the hfea s subsequent decision to continue to limit the use of sex selection techniques in licensed centres to cases in which there is a clear and overriding medical benefit ( hfea , 2002 ; hfea , 2003 ) .
this is now also given a further legal underpinning in the amended british human fertilisation and embryology ( hfe ) act of 2008 . a recent overview ( darnovsky , 2009 ) shows that a similar restrictive stance is adopted in all 36 countries with laws and policies on sex selection , including china , india , turkey , australia , canada , and 25 european countries ( among which are also belgium and the netherlands ) .
in the same vein , article 14 of the european convention on human rights and biomedicine ( oviedo convention ) of 1997 states that techniques of medically assisted reproduction shall not be allowed for the purpose of choosing a future child s sex , except where serious hereditary sex - related disease is to be avoided. some countries ( in europe : austria and switzerland ) go even further and prohibit sex selection for any reason .
although it may seem that the sheer weight of this prohibitive consensus between jurisdictions , at least in europe , has brought the debate about allowing sex selection for non - medical reasons to a closure , and although it is clear ( as again confirmed in the consultation held in the uk ) that among the public there are strong moral feelings against more liberal policies , we will argue that there are still good reasons for questioning the arguments behind this international consensus .
the focus of this paper is on medical methods for sex selection - not on the do it yourself methods .
sex selection can be performed at three stages : before conception / fertilization , where one can make a selection at the level of the sperm using sperm sorting .
this strategy is called preconception sex selection , primary sex selection or sex pre - selection ; just after fertilization , by means of in vitro fertilization ( ivf ) combined with preimplantation genetic diagnosis ( pgd ) , aiming at a selective transfer of an embryo of the desired sex .
this procedure is called preimplantation - or secondary sex selection . during pregnancy , where people might use prenatal diagnosis - chorionic villus sampling ( cvs ) , amniocentesis or even non - invasive prenatal diagnosis ( nipd ) ( newson , 2008 ; de jong , 2009 ; wright , 2009 ) - to decide about continuation or selective termination of the pregnancy in view of the sex of the foetus .
medical reasons are often defined in terms of preventing the conception or birth of a child suffering from a sex - linked disorder , like duchenne muscular dystrophy or haemophilia .
non - medical reasons are rather diverse ; people may just prefer a child of one sex over the other , either for personal reasons or for socio - cultural reasons , or they may want to
balance their family if they have one or more children of one sex and would like to have a further child of the other sex . but obviously , a third category of possible reasons should be added , which can be regarded as intermediate between medical and non - medical reasons ( de wert , 1993 ; de wert , 2005 ) .
think of males affected with an x - linked recessive disorder , like haemophilia . some of these patients would prefer to conceive boys only , because sons ( inheriting the non - affected y chromosome from their father ) will not carry the mutation , whereas all daughters would be ( in principle healthy , but ) obligate carriers of the mutation .
what , then , if one of these male patients ( and their partners ) were to request sex selection in order to avoid the conception or birth of a carrier - daughter ?
in such cases , the reason for choosing sex selection is not to avoid the birth of a child with a serious disease , but to avoid the birth of a child that later in her life may have to face difficult reproductive decisions herself , given her 1 in 4 risk of having children affected by her father s disease .
more in particular , this paper focuses on the ethics of preconception sex selection for non - medical and intermediate reasons , as preconception sex selection for medical reasons is widely considered to be acceptable from a moral point of view . in the next paragraph , we will first summarize the normative views of some relevant committees and some ( inter- ) national legal regulations ( section 2 ) .
next , we will provide an ethical evaluation of the main arguments in the debate ( section 3 ) .
the technology presently available for preconception sex selection will be sketched thereafter , with particular attention to its efficacy and safety ( section 4 ) .
finally , we will draw some conclusions and present some recommendations ( section 5 ) .
to give an impression of the reception of preconception sex selection for non - medical reasons this section contains a brief , chronological overview of relevant recommendations of ethics committees , advisory bodies and of some ( inter-)national regulations .
clearly , the overview is not exhaustive - but it is , we think , representative .
this american commission argued that sex selection for non - medical reasons reflects a morally objectionable attitude to the future child , an attitude which , taken to an extreme , treats the child as an artifact and the reproductive process as a chance to design children according to parental standards of excellence ( president s commission , 1983 ) . the warnock report ( 1984 ) .
warnock committee mentioned possible negative social effects of sex selection for non - medical reasons , like effects on the ratio of males to females .
these considerations made the committee dubious about the use of sex selection techniques on a wide scale , but because of the difficulty of predicting the outcome of any such trend the committee has not found it possible to make any positive recommendations on this issue .
nevertheless , the committee considered that the question of the acceptability of sex selection should be kept under review ( warnock report , 1984 ) . the glover report ( 1989 ) . according to this report , submitted to the european commission
, it could be argued that there is nothing intrinsically wrong with sex selection for social reasons .
but the committee is impressed by the dangers of an unbalanced sex ratio , and inclined to the view that the motives behind the choice of sex will often be ones society would do better to discourage than to satisfy .
it would be desirable for clinics to be banned from providing pre - conceptual techniques as a service. ( glover report , 1989 ) .
this piece of legislation contains a special section on inappropriate sex selection ( mibrauchliche geschlechtswahl ) : any person who fertilizes a human egg with a sperm cell which has been specifically chosen because of its sex chromosome is guilty of an offence ... this is not the case if the sperm has been specified by a physician in order to protect the child from possible severe , sex linked diseases ... ( embryonenschutzgesetz , 1991 ) . the law reform commission ( 1992 ) .
this canadian commission states : to eliminate the possibility of eugenic practices , the selection of gametes and embryos with specific qualities should be prohibited , except where the objective is to prevent the transmission of serious genetic diseases. selection of sex for non - medical reasons is considered to be unacceptable , as such practices ... disrupt the demographic and social balance between the sexes for future generations , and could have a tremendous impact on these made - to - measure children .
it therefore seems appropriate to limit individual freedoms in the name of respect for human dignity. ( law reform commission , 1992 ) .
health council of the netherlands , standing committee medical ethics and health law ( 1995 ) . according to this committee
governments ought , in principle , to respect the reproductive freedom of parents - any exception to this basic principle can only be justified if the interests of others are harmed . after a review of the scientific evidence ,
the committee concluded that there is no reason to suppose that this is the case. however , the committee did emphasize the provisional nature of this conclusion concerning social implications ; it can not be excluded that the availability of opportunities for sex selection as part of family planning might lead to parents finding the gender of their children to be more important than now appears the case .
developments in this area should , therefore , be monitored very critically ( health council , 1995 ) .
this committee concluded that if trials show that the techniques of preconception gender selection are safe and effective , the most prudent approach at present for the non - medical use of these techniques would be to use them only with the aim of establishing gender variety in a family .
if the social , psychological , and demographic effects of those uses have been found acceptable , then other nonmedical uses of preconception sex selection might be considered ( ethics committee of the asrm , 2001 ) dutch and belgian embryos acts ( 2002 , 2003 ) .
the dutch embryos act prohibits performing procedures with gametes or embryos with a view to selecting the sex of a potential child. an exception to this prohibition is made for cases in which there is a risk of a serious sex - linked hereditary disease in the child and the procedures are intended to prevent that disease(embryowet , 2002 ) .
the explanatory memorandum states that this ban was based on the argument that sex selection for non - medical reasons reduces children to mere objects of the wishes and preferences of their parents ( embryowet , 2000 ) .
this forbids conducting research or treatments aimed at sex selection , with the exception of selection aimed at preventing sex - linked diseases ( embryowet , 2003 ) . according to a commentary by hansen et al . , the main motive for not allowing preconception sex selection was the conclusion of the belgian national consultative bioethics committee in its brief advisory report of 1997 , that the then only available method for preconception sex selection ( gradient method ) to be combined with artificial insemination , was unreliable ( hansen et al . , 2004 ) .
a later report from this committee contained a more comprehensive discussion of arguments both for and against allowing preconception sex selection for non - medical reasons , but without this leading to a consensus view or policy advice .
hfea ( 2003 ) and amended human fertilisation and embryology act ( 2008 ) . in the authority s
view , the likely benefits of permitting sex selection for non - medical reasons in the uk are at best debatable and certainly not great enough to sustain a policy to which the great majority of the public are strongly opposed .
accordingly , the authority decided that treatment services provided for the purpose of selecting the sex of children , by whatever means this is to be achieved , should be restricted under license to cases in which there is a clear and overriding medical justification ( hfea , 2003 ) . in the amended british human fertilisation and embryology ( hfe ) act of 2008 this is defined as cases where there is a particular risk that a woman will give birth to a child who will have or develop a
gender - related serious disease or other medical condition ( hfe act , 2008 ) .
the notion of a disease or condition being gender - related is further specified as either affecting only one sex or as affecting one sex significantly more than the other . whereas the corresponding provisions in the german , dutch and belgian embryos acts can be read as referring only to monogenetic sex - linked diseases ( the german act refers to duchenne muscular dystrophy or equally serious sex - linked diseases )
, the specification in the amended british act explicitly also includes non - mendelian disorders with unequal sex - incidence ( amor , 2008 ) . parents already having ( more than one ) children affected by disorders in this category , such as autism , are at a ( significantly ) higher risk that any further child will also be affected .
whether sex selection to avoid this risk would be legal in britain may still depend on the interpretation of whether the disease in question would satisfy the requirement of seriousness .
interestingly , the belgian act does not require the disease in question to be of a serious or severe nature , whereas the german , dutch and british acts do .
none of these acts , nor any of the reports we are aware of , refer to sex selection for intermediate reasons . on the basis of this short overview
, the following conclusions can be drawn : firstly , there is wide approval , in principle , of the application of preconception sex selection for medical reasons , although both the precise definition of this category and the conditions imposed differ .
secondly , opinions differ widely with regard to the moral acceptability of preconception sex selection for non - medical reasons .
in many countries this is categorically forbidden , although the reports from ethical committees give a more varied picture .
the ethical justification of the various positions regarding sex selection for non - medical reasons is often unclear .
indeed , some committees do not or hardly present any arguments in favor of their recommendations .
intermediate reasons has not yet arrived on the agenda of societal debate and policy making .
however , the often quite narrow definition of the category of non - prohibited medical reasons ( in terms of avoiding the birth of a child with a sex - linked disease ) and the rigid , binary distinction between medical and non - medical reasons , seems to rule out sex selection for intermediate reasons in most if not all jurisdictions with legislation on sex - selection . in the next section
, we will scrutinize the arguments for disallowing preconception sex selection for non - medical reasons .
where relevant , we will also discuss whether and how these arguments apply to the use of preconception sex selection for intermediate reasons .
for the sake of debate , we assume , for the moment , that preconception sex selection techniques are completely efficient and of proven safety . that this is not yet an established fact will be addressed in section 4 .
several types of moral objections to preconception sex selection for non - medical reasons need to be discerned ( warren , 1985 ) . on the one hand , so - called non - consequentialist or deontological objections refer to what those making such objections regard as morally problematic aspects of the act of sex selection for non - medical reasons taken in itself , that is to say : prior to or apart from any consequences such a choice may have .
non - consequentialist objections include the following : sex selection is unnatural. appealing to nature as a moral norm is quite common in societal debates but highly questionable . the argument that x is wrong because it is unnatural can only succeed if there is an interpretation of the term ( un)natural which enables us both to distinguish between natural and unnatural actions , and to understand what there is about the latter which is morally objectionable ( warren , 1985 ) .
interestingly , proponents of the argument from nature are highly selective in their moral reasoning ; they regularly object to sex selection for non - medical reasons in particular , but is nt sex selection for medical reasons likewise
if valid , therefore , the argument would apply to all forms of sex selection , including medical and what we have called intermediate reasons .
however , since it surely is an important part of human nature to be inventive , to intervene in nature , and thus to create culture , we must look elsewhere for criteria to ethically evaluate our actions .
so physicians should refrain from this type of sex selection. this objection implicitly refers to the traditional goals of medicine , namely the prevention of disease , curing the ill , and caring for sick people who can not be cured .
sex selection for non - medical reasons can not be subsumed under any of these traditional tasks of the physician , so there is no medical indication for this practice ( rco , 1993 ) .
this criticism seems to mistakenly suggest that the domain of medicine and health care is surrounded by clear and fixed borders . for a start
, one may ask how sex - selection for what we have called intermediate reasons would relate to the goals of medicine as traditionally understood . as in those cases sex selection will not change the health status of the child to be born , reasoning from a strict understanding of those goals would lead to the conclusion that sex selection for intermediate reasons is beyond the scope of medicine .
after all , the prospect that a daughter may have to face difficult reproductive decisions later in her life is not in itself a medical condition .
so understood , intermediate reasons seem closer to social than to medical reasons . on the other hand
, one may reason that what makes those reproductive decisions difficult are health related considerations and that , indirectly , the request for sex selection would still be motivated by the wish to prevent gender - related diseases in one s offspring . from this perspective , a case can be made for regarding those intermediate reasons for sex selection as
the further question here is why an appeal to the bounds of medicine as concerned with health related issues would imply that sex selection for what are clearly non - medical reasons is not acceptable .
in fact , there are various widely accepted medical solutions for non - medical problems .
classical examples are sterilisation for the purpose of family planning or cosmetic surgery . if these are acceptable practices , one may doubt as to whether the absence of a medical indication as such is a strong moral objection to assisting in preconception sex selection for non - medical reasons .
sex selection for non - medical reasons is inherently sexist. sexism can be defined as the wrongful discrimination between individuals on grounds of sex .
the current objection implies that sex selection for non - medical reasons is nothing but an expression of sex prejudice , reflecting an attitude that one sex is in general inferior. ( glover report , 1989 ) or even the original sexist sin ( powledge , 1983 ) .
it is obvious that a sexist motivation for sex choice is morally wrong , as it stands in denial of the principle that all human beings have the same worth .
this charge is clearly not applicable to sex selection for medical , nor for intermediate reasons , but what about sex selection for personal or cultural reasons ?
do motives in the non - medical category necessarily presuppose the view that the desired sex is superior or do they necessarily build on stereotyping views of differences between the sexes ?
if so , that would indeed amount to a strong argument for disallowing sex selection for such reasons . however , it is far from obvious that it is necessarily sexist for someone to want to select the sex of a child for a non - medical reason .
firstly , in some subcultures in some countries , a son is an economic asset ; a son will earn more money , and parents will have to provide their daughter(s ) with a dowry , which might almost ruin their own family . against the background of such conditions , son - preference is not necessarily a sign of sexism of the prospective parents even though their preference would be related to sexism at the socio - cultural level
. it may even be the case that couples prefer to have sons because they want to spare their daughters the plight of having to live in a sexist society .
of course , the fact that such choices may reinforce sexist structures and institutions is a relevant consideration where the possible social consequences of sex selection are concerned .
however , this does not mean that those choices are inherently sexist themselves ( warren 1985 ) .
are those who believe that family dynamics would be enhanced by having children of both sexes guilty of a sexist attitude ?
think of couples who have one or more sons and prefer their next child to be a girl , or vice versa .
although the preference for a mixed family does not presuppose the view that one of the sexes is superior , it may still be informed by a sexist stereotyping of gender differences . here again , the issue is that this would not necessarily be the case .
the preference for a mixed family is perfectly compatible with respecting and welcoming children as individuals whose worth does not depend on whether they fulfill some preconceived image of what a boy or girl should be ( robertson , 1994 ; health council , 1995 ) .
obviously , these examples do not suffice to take away concerns that motives for sex selection may still be sexist in individual cases ( cf .
the concerns of the glover - committee , as summarized in section 2 ) . however , the question is whether the fact that some applicants may have morally dubious motives should lead to a categorical rejection of sex - selection for non - medical reasons .
an interesting analogy is that some applicants for sex selection for medical reasons ( and the same would apply to what we have called intermediate reasons ) may be motivated by discriminatory motives regarding people with disabilities .
most commentators and regulators do not think that this possibility should be taken as a reason for prohibiting sex selection aimed at avoiding the birth of a child with a gender - related disease .
sex selection for non - medical reasons is an invasion of human dignity. another objection regards human dignity .
some critics , e.g. the ( canadian ) law reform commission ( see section 2 ) , suggest that non - medically motivated sex selection undermines human dignity .
this was also the view of the dutch government as laid down in the explanatory memorandum to the embryos act ( quoted in section 2 ) . in a related government document
it is said that sex selection for non - medical reasons runs counter to the general understanding that children are more than a means to satisfying parental preferences ( besluit geslachtskeuze , 1998 ) . in this connection
, the government also referred to the reasoning behind the relevant prohibition in the european convention on human rights and biomedicine .
implicit in this line of argument is the ( anthropological version of the ) categorical imperative of immanuel kant , which holds that a person should always be treated as an end in himself and never merely as a means to other ends ( kant , 1785 ; sullivan , 1989 ) .
no doubt , there might be parents who prefer to have a child of a particular sex as mere material to optimally fit their parental gendered expectations .
people who opt for sex selection for non - medical reasons may very well respect their future child as an autonomous agent , as an end in him-/herself .
consequentialist objections include the following : sex selection for non - medical reasons will distort the sex ratio. that sex selection for non - medical reasons may have adverse societal effects is shown by developments in some countries , including india ( notably the indian state punjab ) , where this practice has resulted in a substantial increase of males ( patel , 2007 ) . these
surplus males may not be able to find a mate and some commentators fear a ( further ) loss of status and rights of females by virtue of their being in the minority although the reverse might be possible as well ( warren , 1985 ) .
critics sometimes assume that sex selection for non - medical reasons will likewise distort the sex ratio in western countries ( cf both the glover report and the canadian law reform commission as summarized in section 2 ) . whether this scenario is realistic , depends on sex selection both becoming widespread here and being used to select mainly for males .
recent surveys in western countries ( canada , the usa , the uk , germany and the netherlands ) reveal that most people prefer a more or less balanced family ( health council of the netherlands , 1995 ; dahl , 2003 ; dahl et al . , 2006 ) .
insofar as there still is a preference for boys , this seems to be a marginal phenomenon .
perhaps even more instructive than surveys are data from so - called gender clinics offering sperm sorting and insemination to interested couples in several countries without prohibitive legislation .
apparently , a very large majority of applicants visiting gender clinics in the uk ( prior to a recent tightening of licensing requirements ; laurance , 2008 ) and the usa were seeking preconception sex selection for family balancing ( dahl , 2003 ) .
however , this may not apply to applicants from some ethnic minorities . of the more than 800 couples who visited the london gender clinic in the first two years of its existence ,
the large majority of these couples wanted a son ( liu , 1995 ) .
sex selection for non - medical reasons will have negative consequences for the psychosocial development of children. this argument refers to possible harm that may result from the feeling of being conditionally wanted .
however , the knowledge that one s parents have used sex selection is not enough to generate this feeling . this would seem to very much depend on whether and to what extent the way parents interact with their children is informed by stereotypical ideas about how girls or boys should behave .
it has been observed that sex selection may also have the positive effect of reducing the number of children having to bear the burden of being wanted less because of their sex ( health council , 1995 ) . without further data ,
the question whether allowing sex selecting for non - medical reasons would lead to exposing more children to harmful styles of parenting , remains speculative . sex selection for non - medical reasons will affect the position of women in society. the presupposition that in western societies sex selection for social reasons will not significantly affect the sex ratio does not rule out that there may still be effects on the social position of women .
it has been suggested that , given a preference for boys , men will profit more from the first - born advantage than women . the scenario ( not supported by the findings of more recent surveys in western countries ) that most people would want a boy as the first child is alarming to those who support the psychological theory that firstborns
are advantaged because parents are apt to concentrate their efforts at providing a stimulating environment on them : with sex selection , boys will monopolize the eldest - child bonuses in addition to other male privileges. ( holmes , 1985 ) .
the theory of the eldest - child bonuses , however , is controversial . in her overview
, warren concludes that the evidence available appears to support a sceptical view on the intrinsic importance of birth order ( warren , 1985 ) .
anyway , even if it were evidence - based , the current objection does not in itself imply that all forms of sex selection for non - medical reasons should be discouraged or banned .
indeed , it would only imply that selecting the sex of one s fist child should perhaps be prohibited .
as such , the argument can not serve as an objection against sex selection aimed at having a mixed family
the motive that the large majority of applicants of sex selection in gender clinics in western countries seem to have ( vide supra ) .
sex selection for non - medical reasons is a first step on a slippery slope towards morally unacceptable forms of reproductive selection. this objection holds that allowing sex selection for non - medical reasons will inevitably result in permitting prospective parents to design what they consider to be the perfect child , to conceive a designer baby , whether that involves high intelligence , high musical and/or athletic competencies , or any number of other special qualities .
the former ( american ) president s commission , for instance , argued that sex selection for non - medical reasons reflects a morally objectionable attitude to the future child , an attitude which , taken to an extreme , treats the child as an artifact and the reproductive process as a chance to design children according to parental standards of excellence ( president s commission , 1983 ; cf . section 2 ) .
as the latter is morally unacceptable , so the argument runs , we should not allow people to select the sex of their child , except for clear medical reasons .
the slippery slope argument can likewise be brought to bear upon sex selection for what we have called intermediate reasons .
one may argue that with this application , a crucial line is being crossed towards selecting children for characteristics unrelated to their own health .
the idea is that after accepting this first step away from a strictly medical justification , it will be impossible not to end up also accepting selection of children for all kinds of non - health related traits .
even if sex selection for non - medical or intermediate reasons need not in itself be morally problematic , it will be impossible not to slide down towards reprehensible practices of commodification and instrumentalisation .
let us first state that , from a scientific point of view , the feasibility of such reproductive selection or repro - designing is strongly exaggerated in the media and in science fiction ; in reality , the preferred characteristics are rather complex , co - determined by many genes and ( mostly poorly understood ) gene - environment interactions .
this makes the reprogenetic creation of the prefect child highly unlikely ( human genetics commission , 2006 ) . from an ethical point of view
, the question is why and when ( still futuristic ) designing our progeny would be morally wrong .
some seem to argue that any type of reproductive enhancement ( selecting for or designing special qualities ) would undermine the autonomy of the future child and would , therefore , constitute an unacceptable violation of the dignity of the child ( habermas , 2001 ) .
others may argue that such enhancement may be morally justified as long as the future child s autonomy is respected ( glover , 2006 ) .
whatever we think of this debate , the current objection is problematic as it presumes that sex selection for non - medical ( or indeed : intermediate ) reasons will automatically lead to reproductive selection procedures or interventions that are intrinsically wrong .
the presumed automatism is untenable : if selection for sex is morally acceptable , but selection for x , y and z is not , one may simply prohibit the latter and allow the former . in summary :
deontological arguments against allowing preconception sex selection for non - medical reasons ( including intermediate ones ) are either mistaken or unconvincing , whereas arguments referring to possible negative consequences are speculative at best . however , with regard to consequences , the analysis is not yet complete .
so far , we have presumed that for couples wanting to select the sex of their children a reliable and safe sperm sorting technique was available that as such would not give rise to further moral concerns .
whether this is the case , is a question that we will now go on to consider .
preconception sex selection involves sperm sorting , i.e. separating sperm cells that carry the x chromosome ( gynogenic sperm ) from sperm cells that carry the y chromosome ( androgenic sperm ) .
the enriched sperm sample may ideally be used in artificial insemination ( ai ) , which is less expensive and less invasive than ivf . while various methods of sperm sorting have been suggested , studied , and commercially offered , flow cytometry is currently the only option with a demonstrated efficacy . according to an overview of the hfea
, the pros of this technology include that it has a relatively high success rate and that sperm can indeed be used for ai rather than ivf , while its cons include that the technology is not completely reliable and that there are no conclusive studies yet confirming the safety for use in humans , although there are no contraindications from use in domestic cattle ( hfea , 2003 ) .
obviously , both the reliability and safety of the technology used for preconception sex selection is morally relevant , as these relate to the interests of prospective parents applying for this technology , future children thus conceived and society as a whole . in this section ,
we concentrate on the interests of the child . in a recent paper , karabinus ( 2009 )
provides the updated findings of the ongoing clinical trial performed at the genetics & ivf institute at fairfax , usa .
according to this study , the microsort sperm separa - tion technology effectively increases the percentage of x- or y - chromosome - bearing sperm in the sorted specimen .
consistent with this finding are the birth sex data : 92% of the babies were female after xsort and 83.6% of babies were male after ysort .
these data provide evidence , so karabinus concludes , of the efficacy of the method used for sex selection .
although one may admit that this method has a relatively high success rate , its efficacy is clearly suboptimal .
what happens if things go wrong , and the expected boy turns out to be a girl or vice versa ?
this is not a theoretical risk , at least for the moment : after using flow cytometry sorted sperm , in 8% of the cases where parents wish to have a girl they will have a boy and in approx .
16% of the cases where they would want to have a boy they will have a girl .
one hopes the parents will adjust in such cases , recover from the surprise or shock , and will welcome and love their child for its own sake .
but what if parents who have invested a lot in trying to select the sex of their child have difficulty in overcoming their disappointment ?
of course , the imperfection of the technology for preconception sex selection is also relevant for its application for medical reasons . where the use of flow - cytometry to avoid a the transmission of a serious sex - linked disease is concerned , a misdiagnosis rate of 8 - 16% rate may seem dramatic
. however , this may still be acceptable if regarded as a preselection step to be combined with further selective procedures , either postconceptionally ( through pgd and embryoselection ) , or indeed prenatally , through prenatal diagnosis and abortion . the combination of flow cytometry and pgd is explicitly envisaged by the hfea as a conceivable option for patients at a significant risk of passing on a serious sex - linked genetic condition to their offspring ( hfea , 2003 ) .
this would have as an advantage over direct pgd that a higher number of embryos of the desired sex will be available for postconception selection .
whether this step - wise approach should in principle also be available for couples at a higher risk of non - mendelian diseases with an unequal sex incidence ( and perhaps also for sex selection for intermediate reasons ) is a separate question that invokes considerations of proportionality in the context of the ethics of pgd and is outside the scope of this paper .
the same holds for whether these considerations rule out the use of this combined approach where non - medical reasons are concerned .
even is this need not necessarily be the case , the combined approach will as a matter of fact be out of reach for most applicants wanting to select the sex of their offspring for non - medical reasons .
this means that the suboptimal efficacy of flow cytometry remains a challenge given concerns about what it may mean for a child to be born to parents having gone so far in trying to have a child of the opposite sex .
ever since flow cytometry has been proposed as a method for preconception sex selection , there have been concerns about its safety , mainly because it requires the staining of spermatozoa with a fluorescent dye ( hoechst 33342 ) .
however , the updated results from the microsort trial show that the malformation rate among babies conceived using this technology is similar to that of the general population .
these data are now based on a total of 760 baby medical records ( karabinus , 2009 ) .
reassuring outcomes were also reported in a recent review of the safety aspects of the same procedure in a large variety of mammalian species , based on data of around a million offspring ( garner , 2009 ) .
this review led to the conclusion that there is no apparent genotoxic effect from exposure of sperm to hoechst 33342. however , the same review also concluded that embryonic development is slower in sorted than in non - sorted sperm and that fertility rates are also lower .
moreover , there are no published data about cross - generational effects on health and fertility ( garner , 2009 ) .
although there are different hypotheses for the effect on the lower fertility following insemination of mammalian females with sex sorted sperm ( grant , 2007 ) , the more likely explanation seems to be that sperm may be damaged by the sorting process , in which the cells are exposed to a number of procedures that may be harmful , including ( but not limited to ) the fluorescent staining ( bermejo - alvarez , 2008 ) .
one report also found different expression patterns of developmentally important genes in bovine embryos that have been derived from sorted and non - sorted sperm ( morton , 2007 ) .
in a recent paper , karabinus ( 2009 ) provides the updated findings of the ongoing clinical trial performed at the genetics & ivf institute at fairfax , usa .
according to this study , the microsort sperm separa - tion technology effectively increases the percentage of x- or y - chromosome - bearing sperm in the sorted specimen .
consistent with this finding are the birth sex data : 92% of the babies were female after xsort and 83.6% of babies were male after ysort .
these data provide evidence , so karabinus concludes , of the efficacy of the method used for sex selection .
although one may admit that this method has a relatively high success rate , its efficacy is clearly suboptimal . what happens if things go wrong , and the expected boy turns out to be a girl or vice versa ?
this is not a theoretical risk , at least for the moment : after using flow cytometry sorted sperm , in 8% of the cases where parents wish to have a girl they will have a boy and in approx .
16% of the cases where they would want to have a boy they will have a girl .
one hopes the parents will adjust in such cases , recover from the surprise or shock , and will welcome and love their child for its own sake .
but what if parents who have invested a lot in trying to select the sex of their child have difficulty in overcoming their disappointment ?
of course , the imperfection of the technology for preconception sex selection is also relevant for its application for medical reasons . where the use of flow - cytometry to avoid a the transmission of a serious sex - linked disease is concerned , a misdiagnosis rate of 8 - 16% rate may seem dramatic
. however , this may still be acceptable if regarded as a preselection step to be combined with further selective procedures , either postconceptionally ( through pgd and embryoselection ) , or indeed prenatally , through prenatal diagnosis and abortion . the combination of flow cytometry and pgd is explicitly envisaged by the hfea as a conceivable option for patients at a significant risk of passing on a serious sex - linked genetic condition to their offspring ( hfea , 2003 ) .
this would have as an advantage over direct pgd that a higher number of embryos of the desired sex will be available for postconception selection .
whether this step - wise approach should in principle also be available for couples at a higher risk of non - mendelian diseases with an unequal sex incidence ( and perhaps also for sex selection for intermediate reasons ) is a separate question that invokes considerations of proportionality in the context of the ethics of pgd and is outside the scope of this paper .
the same holds for whether these considerations rule out the use of this combined approach where non - medical reasons are concerned .
even is this need not necessarily be the case , the combined approach will as a matter of fact be out of reach for most applicants wanting to select the sex of their offspring for non - medical reasons .
this means that the suboptimal efficacy of flow cytometry remains a challenge given concerns about what it may mean for a child to be born to parents having gone so far in trying to have a child of the opposite sex .
ever since flow cytometry has been proposed as a method for preconception sex selection , there have been concerns about its safety , mainly because it requires the staining of spermatozoa with a fluorescent dye ( hoechst 33342 ) .
however , the updated results from the microsort trial show that the malformation rate among babies conceived using this technology is similar to that of the general population .
these data are now based on a total of 760 baby medical records ( karabinus , 2009 ) .
reassuring outcomes were also reported in a recent review of the safety aspects of the same procedure in a large variety of mammalian species , based on data of around a million offspring ( garner , 2009 ) .
this review led to the conclusion that there is no apparent genotoxic effect from exposure of sperm to hoechst 33342. however , the same review also concluded that embryonic development is slower in sorted than in non - sorted sperm and that fertility rates are also lower .
moreover , there are no published data about cross - generational effects on health and fertility ( garner , 2009 ) .
although there are different hypotheses for the effect on the lower fertility following insemination of mammalian females with sex sorted sperm ( grant , 2007 ) , the more likely explanation seems to be that sperm may be damaged by the sorting process , in which the cells are exposed to a number of procedures that may be harmful , including ( but not limited to ) the fluorescent staining ( bermejo - alvarez , 2008 ) .
one report also found different expression patterns of developmentally important genes in bovine embryos that have been derived from sorted and non - sorted sperm ( morton , 2007 ) .
we conclude that the grounds for the present ban on preconception sex selection for non - medical reasons need reconsidering .
first of all , the legislative delineation of what counts as a medical reason tends to be drawn too tightly .
this is often defined in terms of avoiding a sex - linked disorder , which is usually understood as referring to a specific category of monogenetic diseases .
this narrow definition is problematic , as it excludes sex selection aimed at avoiding the birth of a child with a non - mendelian disorder with an unequal sex incidence . in our view
, parents at a higher risk of having a child with such a disorder should be allowed to use preconception sex selection , as is also acknowledged in the new british act . the requirement that this should be a serious disease is too strict here , although it may be in place where postconceptional sex selection ( via pgd ) is at stake , as that would obviously change the proportionality of the procedure . moreover
, current regulations wrongly ignore the category of intermediate reasons : sex selection to avoid that one s offspring may have to face difficult reproductive decisions .
we contend that like medical reasons stricto sensu this is a morally unproblematic motive and that preconception sex selection for intermediate reasons should ( in principle ) be allowed . with regard to what are clearly non - medical ( personal , cultural ) reasons things
is not inherently sexist nor necessarily instrumentalising , and objections in terms of unnaturalness or tensions with the traditional goals of medicine are unconvincing , reasons for a ban must be based on robust indications ( rather than speculations ) regarding harmful effects that allowing the procedure would have on children , women or society at large . since
, at least in a european context , no such indications have been identified , a categorical prohibition seems difficult to reconcile with the principle of respect for reproductive autonomy ( warren , 1985 ; health council 1995 ; dahl , 2003 ; robertson , 2004 ) .
concerns about possible harmful effects of non - medical sex selection both on the psychosocial development of children and on society at large should be taken seriously ( mcmahon , 2004 ) . the obvious response to this
is indeed to allow preconception sex selection in a regulated setting aimed to strictly monitor its effects at all levels ( pennings , 2004 ) . whether this should include the condition that preconception sex selection may only be used to allow parents to enlarge their chances of having a mixed family ( requiring that they already have at least one child of the other sex ) is a matter for debate ( pennings , 1996 )
as has rightly been argued , it can not be maintained that setting this condition would rule out the use of sex selection for sexist motives ( including gender stereotyping ) that may be harmful for the development of children in those families ( wilkinson , 2008 ) .
even so , setting this condition might be a way of responding to societal concerns without making too large an inroad on the reproductive freedom of those who want to make use of preconception sex selection .
after all , research has shown that the larger majority of those couples would want to do so with the aim of creating a mixed family .
monitoring of effects should include long term follow - up of children born after preconception sex selection . in this connection , psychosocial research
is needed to clarify issues such as possible pressures on the child to conform to gender stereotypes , effects on family dynamics and child development , uptake in and effects on ethnic communities , the position of women , etc .
finally , a difficult set of questions is raised by concerns about the imperfect efficacy and unproven ( long - term ) safety of the technology .
how should these concerns affect the debate over the acceptability of preconception sex selection for non - medical reasons ? if the practice as such is acceptable in principle , is this still the case if the only technology flow cytometry that may presently work has these features ?
with regard to effects of failed sexing on the psychosocial development of the child : this is a further concern that we think should be closely monitored , but that as such is no less speculative than those listed before . whether the fate of children in this situation would be worse than that of children of non - congruent sex whose parents have not attempted sex selection
cases of which would be prevented by allowing sex selection for non - medical reasons is unclear ( health council , 1995 ; pennings , 2004 ) . but
although any remaining safety concerns are relevant for all clinical applications of the technology , accepting these is more difficult to justify where the procedure is carried out to satisfy a mere parental preference than where the aim is to avoid the birth of a child with a serious disease .
we see two possible lines of reasoning here . given the debate about the need for the field to proceed with greater care when introducing new reproductive technologies into clinical practice ( pennings et al .
, 2007 ) , one might argue that as long as safety concerns can not be fully answered , flow cytometry may indeed only be used for medical reasons .
the alternative is to say that this is too strict , given the extensive amount of reassuring data from the use of this same technology in cattle and other animals over more than 20 years , and that any remaining safety concerns should be addressed by careful monitoring rather than by imposing limitations on clinical use .
however , we do want to stress that this issue should be on the agenda of the urgently needed reopening of the debate about preconception sex selection for non - medical reasons
firstly , because unjustified limitations of reproductive freedom can not be accepted in a liberal society ( dahl , 2004 ) .
but there is a second reason that is both morally relevant and of practical importance . in the coming years
, new techniques allowing non - invasive prenatal diagnosis will become available for routine applications in early pregnancy ( wright , 2009 ) .
this will allow easy and risk - free testing for fetal aneuploidies , but also for fetal sex .
in the light of the current ban on preconception sex selection for non - medical reasons , a possible consequence of this development may well be that interested couples will use information on fetal sex obtained from early fetal diagnosis for sex selective abortion ( newson , 2008 ; de jong et al . , 2009 ) .
not only would this amount to a form of sex selection that ( unlike the use of a technique such as flow cytometry ) is impossible to regulate and monitor , but also would the use of abortion as a means be morally more problematic than the forms of sex selection that are currently prohibited . | sex selection for non - medical reasons is forbidden in many countries . focusing on preconception sex selection
, the authors first observe that it is unclear what should count as a medical reason in this context and argue for the existence of
intermediate reasons that do not fit well within the rigid distinction between
medicaland non - medical. the article further provides a critical review of the arguments for the prohibition of sex selection for non - medical reasons and finds that none of these are conclusive .
the authors conclude that the ban should be reconsidered , but also that existing societal concerns about possible harmful effects should be taken seriously .
measures to this effect may include limiting the practice to couples who already have at least one child of the sex opposite to that which they now want to select ( family balancing ) .
finally , a difficult set of questions is raised by concerns about the reliability and unproven ( long - term ) safety of the only technology ( flow cytometry ) proven to work . |
the accuracy of the measurement of the higgs - boson mass by the atlas and cms collaborations at the large hadron collider ( lhc ) has already reached the level of 300400 mev [ 1 , 2 ] and , being still dominated by statistics , is bound to improve further when the lhc restarts operations in 2015 .
this puts new emphasis on the need for high - precision calculations in those extensions of the standard model ( sm ) , such as the minimal supersymmetric standard model ( mssm ) , in which the higgs - boson mass can be predicted as a function of other physical observables .
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\begin{document}$${{m}_{h } } < |\cos 2\beta |\,{{m}_{{\scriptscriptstyle z}}}$$\end{document}mh<|cos2|mz applies . in a significant portion of the parameter space the lightest scalar \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh has long been disproved by the lep [ 3 , 4 ] .
however , radiative corrections can raise the prediction for the lightest - scalar mass up to the value \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}\approx 125$$\end{document}mh125 gev observed at the lhc , and they bring along a dependence on all mssm parameters . among the latter ,
particularly relevant are the masses and mixing of the scalar partners of the third - generation quarks , the stop and sbottom squarks . due to the crucial role of radiative corrections in pushing the prediction for the lightest - scalar mass above the tree - level bound ,
an impressive theoretical effort has been devoted over more than 20 years to the precise determination of the higgs sector of the mssm.1 after the early realization [ 59 ] of the importance of the one - loop \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t})$$\end{document}o(t ) corrections2 involving top and stop , full one - loop computations of the mssm higgs masses have been provided [ 1013 ] , leading logarithmic effects at two loops have been included via renormalization - group methods [ 1417 ] , and genuine two - loop corrections of \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { b}+ \alpha _ { b}^2)$$\end{document}o(tb+b2 ) have been evaluated in the limit of vanishing external momentum in the higgs self - energies .
all of these corrections have been implemented in widely used computer codes for the calculation of the mssm mass spectrum , such as feynhiggs , softsusy [ 31 , 32 ] , suspect and spheno [ 34 , 35 ] .
furthermore , a complete two - loop calculation of the mssm higgs masses in the effective potential approach ( i.e. , at zero external momentum ) , including also two - loop corrections controlled by the ew gauge couplings , has been presented in refs .
[ 36 , 37 ] . some of the dominant three - loop corrections to \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh have also been obtained , both via renormalization - group methods [ 3840 ] and by explicit calculation of the higgs self - energy at zero external momentum [ 41 , 42 ] . already at the two - loop level , going beyond the approximation of zero external momentum brings significant complications to the calculation of the higgs self - energies .
different algorithms for expressing all two - loop self - energy integrals with arbitrary external momentum in terms of a minimal set of master integrals ( mis ) were developed in refs .
however , explicit analytical formulas for the mis can be derived only for special values of the masses of the particles circulating in the loops , whereas in the general case a numerical calculation becomes unavoidable . a method to compute all the mis of ref . by numerically solving a system of differential equations in the external momentum
[ 4851 ] . a library of routines for the computation of the mis with the method of ref .
a calculation of the two - loop contributions to the higgs self - energies involving the strong gauge coupling or the third - family yukawa couplings , based on the methods of refs .
that calculation goes beyond the two - loop results implemented in public codes [ 1921 , 2529 ] in that it includes external - momentum effects , as well as contributions involving the d - term - induced ew interactions between higgs bosons and sfermions . when combined with the effective - potential results of refs .
provide an almost - complete two - loop calculation of the higgs masses in the mssm the only missing contributions being external - momentum effects that involve only the ew gauge couplings . however , no code for the calculation of the mssm mass spectrum implementing the results of refs .
[ 36 , 37 , 53 ] was ever made available , and the way those results are organized does not lend itself to a straightforward implementation in the existing public codes .
on one hand , the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization scheme adopted in refs .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme adopted in feynhiggs . on the other hand , implementation of the results of refs .
[ 36 , 37 , 53 ] in softsusy , suspect and spheno , which also adopt the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , is complicated by the fact that in refs . [ 36 , 37 , 53 ] the running masses of the higgs bosons entering the loop corrections are defined by the second derivatives of the tree - level potential .
while this choice amounts to a legitimate reshuffling of terms between different perturbative orders , it restricts the applicability of the calculation to rather specific ranges of renormalization scale where none of the running higgs masses as defined in refs .
[ 36 , 37 , 53 ] is tachyonic . perhaps as a consequence of these complications , a full decade after the publication of ref .
its results have yet to be included in phenomenological analyses of the mssm higgs sector . in this paper
we present a new calculation of the momentum - dependent part of the two - loop corrections to the neutral higgs masses of \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) , i.e. those involving both the top yukawa coupling and the strong gauge coupling .
we also compute mixed two - loop corrections that we denote by \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) : they involve both the strong gauge coupling and the ew gauge couplings , under the approximation that the only non - vanishing yukawa coupling is the top one .
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections ( in practice , we find that both classes of corrections are considerably more suppressed than that , but still comparable to each other in size ) . in our calculation
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[ 54 , 55 ] to express the momentum - dependent loop integrals in terms of the mis of ref . , which we evaluate by means of the package tsil .
we obtain results for both the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization schemes , organized in such a way that they can be directly implemented in the existing codes for the computation of the mssm mass spectrum .
we verify that our results are in full agreement with the ones of ref . where they overlap .
after describing our calculation in some detail , we briefly discuss the numerical impact of the momentum - dependent \documentclass[12pt]{minimal }
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while our paper was in preparation , an independent calculation of the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the neutral higgs masses in the mssm appeared , relying on the results of ref . for the decomposition of two - loop integrals into mis and on the package secdec [ 57 , 58 ] for the numerical evaluation of the latter .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , and they have been implemented in the latest version of feynhiggs . although we have verified that our results for the contributions of genuine two - loop diagrams involving the strong - gauge and top - yukawa couplings agree numerically with those of ref .
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the higgs masses .
we trace the reason for the discrepancy to an inconsistency in ref . concerning the definitions of the wave - function - renormalization ( wfr ) constants for the higgs fields and of the parameter \documentclass[12pt]{minimal }
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we outline here our calculation of the two - loop corrections to the masses of the neutral higgs bosons in the mssm with real parameters ( we do not consider the possibility of cp violation in the higgs sector ) .
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\begin{document}$$g^0$$\end{document}g0 .
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\begin{document}$$s_2$$\end{document}s2 combine into the scalars \documentclass[12pt]{minimal }
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\begin{document}$$h$$\end{document}h and \documentclass[12pt]{minimal }
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\begin{document}$$h$$\end{document}h .
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\begin{document}$$p^2$$\end{document}p2 of the equation2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathrm{det } \left [ \gamma _ s(p^2 ) \right ] = 0 , \end{aligned}$$\end{document}dets(p2)=0,where \documentclass[12pt]{minimal }
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\begin{document}$$\gamma _ s(p^2)$$\end{document}s(p2 ) denotes the \documentclass[12pt]{minimal }
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\begin{document}$$2\times 2$$\end{document}22 inverse - propagator matrix in the \documentclass[12pt]{minimal }
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\begin{document}$$(s_1,s_2)$$\end{document}(s1,s2 ) basis , \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}p being the external momentum flowing into the scalar self - energies . we can decompose \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \gamma _ s(p^2 ) = p^2 - \mathcal{m}_0 ^ 2 - \delta \mathcal{m}^2(p^2 ) , \end{aligned}$$\end{document}s(p2)=p2-m02-m2(p2),where \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 denotes the tree - level mass matrix written in terms of renormalized parameters , and \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2 ) collectively denotes the radiative corrections . at each order in the perturbative expansion ,
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\begin{document}$$\gamma _ s(p^2)$$\end{document}s(p2 ) .
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\begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 in terms of the parameter \documentclass[12pt]{minimal }
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\begin{document}$$\tan
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , and of the physical masses of the pseudoscalar and of the \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z boson4\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{m}_0 ^ 2 = \left ( \begin{array}{cc } c^2_\beta \,m_z^2 + s^2_\beta \,m_a^2 & { } \quad - { { s}_{\beta } } \ , { { c}_{\beta } } \left ( m_z^2 + m_a^2 \right ) \\ - { { s}_{\beta } } \ , { { c}_{\beta } } \left ( m_z^2 + m_a^2\right ) & { } \quad s^2_\beta \ , m_z^2 + c^2_\beta \ , m_a^2 \end{array}\right ) , \end{aligned}$$\end{document}m02=c2mz2+s2ma2-scmz2+ma2-scmz2+ma2s2mz2+c2ma2,using ( here and thereafter ) the shortcuts \documentclass[12pt]{minimal }
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\begin{document}$$c_\phi \equiv \cos \phi $ $ \end{document}ccos and \documentclass[12pt]{minimal }
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\begin{document}$$s_\phi \equiv \sin \phi $ $ \end{document}ssin for a generic angle
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\begin{document}$$\phi $ $ \end{document}. neglecting terms that do not contribute at \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) or \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{m}_0 ^ 2$$\end{document}m02 lead to the following expressions for the two - loop parts of the individual entries of \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2):5\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{11}&= s_\beta ^2\ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + c_\beta ^2\,\mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\nonumber \\&-\pi _ { 11}^{(2)}(p^2 ) -\delta \mathcal { z}_1^{(2 ) } \left ( p^2- c^2_\beta \ , m_z^2 - s^2_\beta \,m_a^2\right ) \nonumber \\&+ \left ( 1- { { s}_{\beta } } ^4\right ) \frac{~\,t_1^{(2)}}{v_1 } - s_\beta ^2 c_\beta ^2 \frac{~\,t_2^{(2)}}{v_2}\nonumber \\&-2\,s_\beta ^2 c_\beta ^2\ , \left ( m_z^2-m_a^2\right ) \frac{~\delta \tan \beta ^{(2)}}{\tan \beta } , \end{aligned}$$\end{document}m2(p2)11(2)=s2reaa(2)(ma2)+c2rezz(2)(mz2)-11(2)(p2)-z1(2)p2-c2mz2-s2ma2 + 1-s4t1(2)v1-s2c2t2(2)v2 - 2s2c2mz2-ma2tan(2)tan,6\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{12}&= - { { s}_{\beta } } { { c}_{\beta } } \left [ \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + \mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\right .
s_\beta ^2 \frac{~\,t_1^{(2)}}{v_1 } - c_\beta ^2 \frac{~\,t_2^{(2)}}{v_2 } \right ] -\pi _ { 12}^{(2)}(p^2)\nonumber \\&-\frac{1}{2}{{s}_{\beta } } { { c}_{\beta } } \left ( m_z^2 + m_a^2\right ) \nonumber \\&\times \left [ 2\ , ( c_\beta ^2-s_\beta ^2)\frac{~\delta \tan \beta ^{(2)}}{\tan \beta } \!+\ !
\delta \mathcal { z}_1^{(2)}\!+\!\delta \mathcal { z}_2^{(2)}\!\right ] , \nonumber \\ \end{aligned}$$\end{document}m2(p2)12(2)=-screaa(2)(ma2)+rezz(2)(mz2)-s2t1(2)v1-c2t2(2)v2-12(2)(p2)-12scmz2+ma22(c2-s2)tan(2)tan+z1(2)+z2(2),7\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left [ \delta \mathcal{m}^2(p^2)\right ] ^{(2)}_{22}&= c_\beta ^2\ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}(m_a^2 ) + s_\beta ^2 \ , \mathrm{re}\ , \pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}^{(2)}(m_z^2)\nonumber \\&-\pi _ { 22}^{(2)}(p^2 ) -\delta \mathcal { z}_2^{(2 ) } \left ( p^2- s^2_\beta \,m_z^2 - c^2_\beta \ ,
s_\beta ^2 c_\beta ^2 \frac{~\,t_1^{(2)}}{v_1 } + \left ( 1- { { c}_{\beta } } ^4\right ) \frac{~\,t_2^{(2)}}{v_2}\nonumber \\&+2\,s_\beta ^2c_\beta ^2\ , \left ( m_z^2-m_a^2\right ) \frac{~\delta \tan \beta ^{(2)}}{\tan \beta } .
\end{aligned}$$\end{document}m2(p2)22(2)=c2reaa(2)(ma2)+s2rezz(2)(mz2)-22(2)(p2)-z2(2)p2-s2mz2-c2ma2-s2c2t1(2)v1 + 1-c4t2(2)v2 + 2s2c2mz2-ma2tan(2)tan.in the equations above , \documentclass[12pt]{minimal }
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\begin{document}$$t^{(2)}_i$$\end{document}ti(2 ) and \documentclass[12pt]{minimal }
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\begin{document}$$i , j=1,2$$\end{document}i , j=1,2 ) denote the two - loop parts of tadpoles and self - energies , respectively , for the scalars \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z - boson self - energies .
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\begin{document}$$i=1,2$$\end{document}i=1,2 ) in eqs .
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\begin{document}$$\begin{aligned } h^0_i ~\longrightarrow ~\sqrt{\mathcal{z}_i}\,h^0_i ~\simeq ~ \left ( 1 + \frac{1}{2}\,\delta \mathcal{z}^{(1)}_i + \frac{1}{2}\,\delta \mathcal{z}^{(2)}_i \right ) \,h^0_i , \end{aligned}$$\end{document}hi0zihi01 + 12zi(1)+12zi(2)hi0,where in the expansion of the square root we have again neglected terms that do not contribute at \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for the \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{z}_i$$\end{document}zi , which can then be determined from the anomalous dimensions of the higgs fields and from the \documentclass[12pt]{minimal }
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\begin{document}$$\beta $ $ \end{document} functions of the couplings entering the anomalous dimensions . taking from the general formulas of refs .
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\begin{document}$$\begin{aligned } \delta \mathcal{z}^{(1)}_1&= \delta \mathcal{z}^{(2)}_1= 0,\quad \delta \mathcal{z}^{(1)}_2 = - \frac{\alpha _ { t}}{4\pi } \,n_c\,\cdot \,\frac{1}{\epsilon } , \nonumber \\
\delta \mathcal{z}^{(2)}_2&= \frac{\alpha _ { t}\alpha _ { s}}{(4\pi ) ^2}\,2\,n_c\,c_f\cdot \left ( \frac{1}{\epsilon ^2}-\frac{1}{\epsilon } \right ) , \end{aligned}$$\end{document}z1(1)=z1(2)=0,z2(1)=-t4nc1,z2(2)=ts(4)22nccf12 - 1,where \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{z}^{(1)}_2$$\end{document}z2(1 ) is in turn renormalized in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme .
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\begin{document}$$\delta \tan \beta ^{(2)}$$\end{document}tan(2 ) in eqs .
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\begin{document}$$\tan \beta $ $ \end{document}tan. the choice of a \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan implies that , in our approximation , its counterterm can be expressed via the wfr counterterms:10\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{~\delta \tan \beta ^{(\ell ) } } { \tan \beta } = \frac{1}{2}\,\left ( \delta \mathcal{z}^{(\ell ) } _ 2 -\delta \mathcal{z}^{(\ell ) } _
( 5)(7 ) include both 1pi two - loop contributions and non-1pi contributions arising from the renormalization of the parameters entering their one - loop counterparts .
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
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\begin{document}$$m_{\tilde{q}_i}$$\end{document}mq~i of all squarks other than the stops .
the latter enter the one - loop tadpoles and self - energies of the higgs bosons via d - term - induced ew couplings , and the one - loop self - energy of the \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z boson via the gauge interaction . in our calculation
we neglect all yukawa couplings ( and hence quark masses ) other than the top one,3 therefore none of the other squarks mix .
we obtained results for the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) and \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) contributions to tadpoles and self - energies assuming that the relevant quark / squark parameters are renormalized either in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr or in the os scheme .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dros shift of the parameters in the top / stop sector can be found , e.g. , in appendix b of ref . , while the shifts for the remaining squark masses can be obtained by setting \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{t}}=\theta _ t=0$$\end{document}mt=t=0 in the corresponding formulas for the stop masses .
( 5)(7 ) are constructed to give finite entries in the inverse - propagator matrix of the scalars .
the \documentclass[12pt]{minimal }
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\begin{document}$$1/\epsilon ^2$$\end{document}1/2 and \documentclass[12pt]{minimal }
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\begin{document}$$1/\epsilon $ $ \end{document}1/ poles in the right - hand sides of eqs .
( 5)(7 ) cancel out . in principle , the two - loop contributions to the higgs inverse propagator given in eqs .
( 5)(7 ) must be combined with a full calculation of the corresponding one - loop contributions , and used to determine the physical higgs masses by solving directly eq .
however , as will be discussed in the next section , the computing times required for the evaluation of momentum - dependent two - loop integrals are not negligible . a numerical search for the solutions of eq .
( 2 ) could significantly slow down the codes for the calculation of the higgs masses , making them unsuitable for extensive phenomenological analyses of the mssm parameter space .
it is therefore convenient to compute the higgs masses in two steps , with a procedure similar to the one discussed in refs .
( 2 ) including in \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{m}^2(p^2)$$\end{document}m2(p2 ) the full one - loop corrections plus the dominant two - loop corrections of \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) , \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { b}\alpha _ { s})$$\end{document}o(bs ) , and \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{o}(\alpha _ { t}^2+\alpha _ { t}\alpha _ { b}+\alpha _ { b}^2)$$\end{document}o(t2+tb+b2 ) computed in the approximation of vanishing external momentum . from feynhiggs we obtain the scalar masses \documentclass[12pt]{minimal }
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\begin{document}$$\overline{m}_h^2$$\end{document}mh2 and \documentclass[12pt]{minimal }
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\begin{document}$$\overline{m}_{{\scriptscriptstyle h}}^2$$\end{document}mh2 , and an effective mixing angle \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\alpha } $ $ \end{document} which diagonalizes the loop - corrected scalar mass matrix at vanishing external momentum .
our full results for the scalar masses are then obtained by adding to the results of feynhiggs the momentum - dependent parts of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the whole \documentclass[12pt]{minimal }
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{ s})$$\end{document}o(s ) corrections:11\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } m^2_{h,{\scriptscriptstyle h } } = \overline{m}^2_{h,{\scriptscriptstyle h } } ~+~ ( \delta m^2_{h,{\scriptscriptstyle h}})^{\alpha _ { t}\alpha _ { s},\,p^2 } ~+~ ( \delta m^2_{h,{\scriptscriptstyle h}})^{\alpha \alpha _ { s}}. \end{aligned}$$\end{document}mh , h2=mh , h2+(mh , h2)ts , p2+(mh , h2)s.concerning the former , we have12\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left ( \delta m^2_{h}\right ) ^{\alpha _ { t}\alpha _ { s},\,p^2}&= c^2_{\beta -\overline{\alpha } } \,\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}\left ( m_a^2\right ) - s^2_{\overline{\alpha } } \ , \delta \pi _ { 11}^{(2)}\left ( \overline{m}_h^2\right ) \nonumber \\&\quad + s_{2\overline{\alpha } } \,\delta \pi _ { 12}^{(2)}\left ( \overline{m}_h^2\right ) - c^2_{\overline{\alpha } } \,\delta \pi _ { 22}^{(2)}\left ( \overline{m}_h^2\right ) , \end{aligned}$$\end{document}mh2ts , p2=c-2aa(2)ma2-s211(2)mh2+s212(2)mh2-c222(2)mh2,13\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left ( \delta m^2_{{\scriptscriptstyle h}}\right ) ^{\alpha _ { t}\alpha _ { s},\,p^2}&= s^2_{\beta -\overline{\alpha } } \,\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}^{(2)}\left ( m_a^2\right ) - c^2_{\overline{\alpha } } \ , \delta \pi _ { 11}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \nonumber \\&\quad - s_{2\overline{\alpha } } \,\delta \pi _ { 12}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) - s^2_{\overline{\alpha } } \,\delta \pi _ { 22}^{(2)}\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) , \end{aligned}$$\end{document}mh2ts , p2=s-2aa(2)ma2-c211(2)mh2-s212(2)mh2-s222(2)mh2,where we define \documentclass[12pt]{minimal }
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\begin{document}$$\delta \pi ( p^2 ) \,\equiv \ , \pi ( p^2)-\pi ( 0)$$\end{document}(p2)(p2)-(0 ) , and retain only the real and finite part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) contributions to the two - loop self - energies . for what concerns the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
( 5)(7):14\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left ( \delta m^2_{h}\right ) ^{\alpha \alpha _ { s}}&= s^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{11}\nonumber \\&\quad - s_{2\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{12 } + c^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_h^2\right ) \right ] ^{\alpha \alpha _ { s}}_{22},\end{aligned}$$\end{document}mh2s = s2m2mh211s - s2m2mh212s+c2m2mh222s,15\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left ( \delta m^2_{{\scriptscriptstyle h}}\right ) ^{\alpha \alpha _ { s}}&= c^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{11}\nonumber \\&\quad + \ , s_{2\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{12 } + s^2_{\overline{\alpha } } \,\left [ \delta \mathcal{m}^2\left ( \overline{m}_{{\scriptscriptstyle h}}^2\right ) \right ] ^{\alpha \alpha _ { s}}_{22 } , \end{aligned}$$\end{document}mh2s = c2m2mh211s+s2m2mh212s+s2m2mh222s , where again we take the real part of all two - loop self - energies .
. proposes an alternative two - step procedure to include the momentum - dependent parts of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections in feynhiggs , differing from the one outlined above only by higher - order effects . finally , a comment is in order about the dependence of the corrections to the higgs masses on the wfr constants.4 in principle , the predictions for the physical higgs masses at a given order in the perturbative expansion should not depend directly on the wfr constants ( although they could still depend indirectly on them via the \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan counterterm ) . indeed ,
if the two - loop contributions to the inverse - propagator matrix are computed with \documentclass[12pt]{minimal }
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\begin{document}$$p^2$$\end{document}p2 equal to the tree - level scalar masses and then rotated to the mass - eigenstate basis via the tree - level mixing angle , so that the computation is performed strictly at the two - loop level , the terms in eqs .
( 5)(7 ) that depend explicitly on \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) drop out of the mass corrections \documentclass[12pt]{minimal }
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\begin{document}$$\delta m^2_{h,{\scriptscriptstyle h}}\,$$\end{document}mh , h2 . on the other hand ,
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\begin{document}$$\overline{m}_h^2$$\end{document}mh2 and \documentclass[12pt]{minimal }
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\begin{document}$$\overline{m}_{{\scriptscriptstyle h}}^2$$\end{document}mh2 and the effective mixing angle \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\alpha } $ $ \end{document} are used , as in eqs .
( 12)(15 ) above , or if the zeroes of the inverse - propagator matrix are determined numerically , the corrections to the scalar masses retain a dependence on the wfr counterterms . in our calculation
we adopt a \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition for the wfr ; therefore the terms involving \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) are purely divergent and cancel out against other divergent terms in the individual entries of the inverse - propagator matrix , hence they do not show up in eqs .
if , however , one adopts a non - minimal definition of the wfr , higgs - mass corrections computed as in eqs .
( 14 ) and ( 15 ) will contain non - vanishing terms that depend on the finite part of \documentclass[12pt]{minimal }
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\begin{document}$$\delta \mathcal{z}^{(2)}_i$$\end{document}zi(2 ) .
albeit formally of higher order in the perturbative expansion , these terms can be numerically relevant when the loop - corrected scalar masses differ significantly from their tree - level values ( as is the case for a sm - like scalar \documentclass[12pt]{minimal }
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\begin{document}$$h$$\end{document}h with mass around 125 gev ) .
the computation of the two - loop corrections to the neutral mssm higgs masses considered in this paper requires the knowledge of the tadpole and self - energy diagrams entering eqs .
while the strategy for the computation in the zero - momentum approximation is well known , the evaluation of the self - energies with arbitrary external momentum is more involved .
we illustrate in this section the details of our calculation , which we performed in a fully automated way .
the relevant diagrams are generated with feynarts , using a modified version of the original mssm model file that implements the qcd interactions in the background field gauge .
the diagrams contributing to the vacuum polarization of the \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z boson are contracted with a suitable projector in order to single out their transverse part .
the color factors are simplified with a private package and the dirac algebra is handled by form .
the computation is performed in dimensional reduction , which we can implement in this case by generating the diagrams in dimensional regularization and replacing , in each diagram involving an internal \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon $ $ \end{document}-scalar contribution .
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\begin{document}$$\begin{aligned } \int d^dk_1 d^dk_2 \frac{\left ( k_1 ^ 2\right ) ^\alpha \left ( k_2 ^ 2\right ) ^\beta ( k_1\cdot p)^\gamma ( k_2\cdot p)^\delta ( k_1\cdot k_2)^\eta } { d_1^{a_1 } d_2^{a_2 } d_3^{a_3 } d_4^{a_4 } d_5^{a_5 } } , \end{aligned}$$\end{document}ddk1ddk2k12k22(k1p)(k2p)(k1k2)d1a1d2a2d3a3d4a4d5a5,where \documentclass[12pt]{minimal }
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\begin{document}$$\alpha , \ldots , \eta , \ , a_1 , \ldots , a_5$$\end{document}, ,,a1, ,a5 are positive ( or zero ) integer exponents and the \documentclass[12pt]{minimal }
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\begin{document}$$d_i$$\end{document}di s are defined as\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } d_1&= k_1 ^ 2 - m_1 ^ 2 , \ , d_2 = ( k_1-p)^2 - m_2 ^ 2 , \ , d_3 = k_2 ^ 2 - m_3 ^ 2,\\ d_4&= ( k_2-p)^2 - m_4 ^ 2 , \ , d_5 = ( k_1-k_2)^2 - m_5 ^ 2 .
\end{aligned}$$\end{document}d1=k12-m12,d2=(k1-p)2-m22,d3=k22-m32,d4=(k2-p)2-m42,d5=(k1-k2)2-m52.integrals belonging to the class above are in general not linearly independent of each other .
when the scalar products in the numerator are expressed in terms of the denominators , powers of a \documentclass[12pt]{minimal }
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\begin{document}$$d_i$$\end{document}di present in the original integral might cancel against a \documentclass[12pt]{minimal }
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\begin{document}$$d_i$$\end{document}di in the numerator , possibly generating a feynman integral in which \documentclass[12pt]{minimal }
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\begin{document}$$d_i$$\end{document}di does not appear , i.e. in which the corresponding line has been shrunk to a point . for given \documentclass[12pt]{minimal }
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\begin{document}$$a_i$$\end{document}ai s and high enough \documentclass[12pt]{minimal }
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\begin{document}$$\alpha , \ldots , \eta $ $ \end{document},
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\begin{document}$$d_i$$\end{document}di s may acquire negative exponents .
( 16 ) therefore reduces to the evaluation of a number of integrals of the form17\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \int \frac{d^dk_1 d^dk_2}{d_1^{n_1 } d_2^{n_2 } d_3^{n_3 } d_4^{n_4 } d_5^{n_5 } } , \end{aligned}$$\end{document}ddk1ddk2d1n1d2n2d3n3d4n4d5n5,where the exponents \documentclass[12pt]{minimal }
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\begin{document}$$n_i\in \mathbb { z}$$\end{document}niz . in the present case
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\begin{document}$$\mathcal { o}(300)$$\end{document}o(300 ) different feynman integrals .
there exists a convenient procedure for dealing with such large numbers of different feynman integrals in a more efficient way than their direct evaluation .
dimensionally regularized integrals , at arbitrary loop order and with arbitrary number of external legs , satisfy identities of the ibp type [ 54 , 55 ] .
these identities are linear relations that connect integrals with different sets of exponents \documentclass[12pt]{minimal }
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\begin{document}$$\{n_1,\ldots , n_5\}$$\end{document}{n1, ,n5}. after a set of independent integrals , the mis , has been identified , all the remaining integrals can then be reduced to linear combinations of the mis , the coefficients being rational functions of the masses , the kinematic invariants and the space - time dimension \documentclass[12pt]{minimal }
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\begin{document}$$d$$\end{document}d .
one practical advantage of such a procedure is its divide and conquer spirit . on the one hand few mis
encode the analyticity properties ( singularities , thresholds , branch cuts ) of the problem under consideration . on the other hand ,
the evaluation of the large number of different feynman integrals entering a computation is reduced to a problem of linear algebra , which can easily be automated , if the mis are known .
we perform the reduction to mis with the public code reduze [ 70 , 71 ] , which implements the laporta algorithm and produces the ibp identities relevant to our case .
the evaluation of the mis is in general a complicated problem and can proceed via different techniques , like the integration in the feynman , schwinger or mellin
a remarkable consequence of the aforementioned ibp relations is that the mis obey linear systems of first - order differential equations ( des ) in the kinematic invariants , which provide an alternative means for their computation [ 73 , 74 ] .
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\begin{document}$$d$$\end{document}d is possible only in some simple cases . in more general cases
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\begin{document}$$\epsilon = ( 4-d)/2$$\end{document}=(4-d)/2 , giving rise to a ( generally coupled ) system of des for the expansion coefficients . in the limit of vanishing external momentum ,
two - loop self - energies become two - loop vacuum diagrams , which reduce via ibp to linear combinations of only one genuine two - loop mi and products of the one - loop one - propagator mi .
two - loop self - energies with arbitrary external momentum , in the general case with five different masses in the loops , reduce via ibp to linear combinations of 30 mis .
the finite part of such mis can be expressed in terms of four functions , in addition to the well - known one - loop mis.5 as already mentioned , analytic solutions for such functions have been derived only for special patterns of up to two internal masses ( only one function is known in a particular case with three different masses ) . on the other hand , the diagrams that in our approximation contribute to the self - energies entering eqs .
( 5)(7 ) require the knowledge of mis with up to four different masses , the most complicated ones being those involving simultaneously \documentclass[12pt]{minimal }
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\begin{document}$$m_t^2,{m}_{\tilde{t}_1}^{2},{m}_{\tilde{t}_2}^{2}$$\end{document}mt2,mt~12,mt~22 , and \documentclass[12pt]{minimal }
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\begin{document}$$m_{\tilde{g}}^{2}$$\end{document}mg~2 .
in our computation we rely on the package tsil , which implements ( besides all the analytically known cases ) the numerical solution of the des for the two - loop self - energy mis .
[ 4751 ] is based on the fact that the value at \documentclass[12pt]{minimal }
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\begin{document}$$p^2$$\end{document}p2 in the case of logarithmically divergent integrals ) is known for each function and can be used to build the set of initial conditions needed for the solution of the des . in the computation of the self - energies entering eqs .
( 5)(7 ) we need to evaluate the corresponding mis at \documentclass[12pt]{minimal }
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\begin{document}$$p^2=m_z^2,m_a^2,m_h^2,m_h^2$$\end{document}p2=mz2,ma2,mh2,mh2 .
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\begin{document}$$\pi _ { { \scriptscriptstyle z}{\scriptscriptstyle z}}$$\end{document}zz ( in the approximation \documentclass[12pt]{minimal }
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\begin{document}$$m_q=0$$\end{document}mq=0 ) and that \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma is a free parameter , it is clear that the way thresholds are handled in the numerical evaluation of the mis is of particular relevance . in the des approach ,
the physical two- and three - particle thresholds show up , together with the pseudothresholds , as poles in the coefficients of the des .
tsil overcomes the numerical instabilities related to such poles by displacing the \documentclass[12pt]{minimal }
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\begin{document}$$p^2$$\end{document}p2-integration contour in the upper half - plane when the momentum is above the smallest ( pseudo)threshold .
the evaluation at ( or very close to ) the ( pseudo)thresholds is performed through a variant of the algorithm , which is slightly less efficient but ensures reliable results in such critical cases . as an example , the time needed on an intel core i7 - 4650u cpu for the evaluation of the complete set of mis , for any of the mass patterns entering the self - energies , ranges between \documentclass[12pt]{minimal }
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\begin{document}$$8 \times 10^{-2}\,s$$\end{document}810 - 2s , the latter being the typical time for \documentclass[12pt]{minimal }
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\begin{document}$$\sqrt{p^2}$$\end{document}p2 close or equal to the heavy stop pair threshold and to the three - particle ( pseudo)thresholds \documentclass[12pt]{minimal }
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\begin{document}$$m_{\tilde{t}_i}+m_{\tilde{g}}\pm { { m}_{t}}$$\end{document}mt~i+mg~mt . in ref . the relative accuracy of tsil is claimed to be better than \documentclass[12pt]{minimal }
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\begin{document}$$10^{-10}$$\end{document}10 - 10 for generic cases , or worse in cases with large mass hierarchies .
being tsil a package dedicated to the evaluation of the mis for two - loop self - energy diagrams , it is not surprising that its speed and accuracy prove much better than those quoted in ref . , where the general - purpose package secdec is used and , in the most complicated case , \documentclass[12pt]{minimal }
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\begin{document}$$10^{-5}$$\end{document}10 - 5 close to a threshold .
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and of the whole \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections on the predictions for the neutral higgs - boson masses in the mssm .
we focus on six benchmark scenarios introduced in ref . , which identify regions in the mssm parameter space compatible with the current bounds from susy - particle searches and with the requirement that the predicted value of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh agrees , within the theoretical uncertainty of \documentclass[12pt]{minimal }
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\begin{document}$$\pm 3~\mathrm{gev}$$\end{document}3gev estimated in refs .
[ 78 , 79 ] , with the mass of the sm - like higgs boson discovered at the lhc.6 in our numerical examples we adopt the mixed os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme described in sect . 2 .
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\begin{document}$$m_t = 173.2~\mathrm{gev}$$\end{document}mt=173.2gev , the running bottom mass \documentclass[12pt]{minimal }
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\begin{document}$$g_f = 1.16639 \times 10^{-5}~\mathrm{gev}^{-2}$$\end{document}gf=1.1663910 - 5gev-2 , the strong gauge coupling \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ s({{m}_{{\scriptscriptstyle z } } } ) = 0.118$$\end{document}s(mz)=0.118 , and the pole gauge - boson masses \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle z}}}= 91.1876~\mathrm{gev}$$\end{document}mz=91.1876gev and \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle w}}}= 80.385~\mathrm{gev}$$\end{document}mw=80.385gev . to compute the scalar masses \documentclass[12pt]{minimal }
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\begin{document}$$\overline{m}^2_{h,{\scriptscriptstyle h}}$$\end{document}mh , h2 and the effective mixing angle \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\alpha } $ $ \end{document} entering the corrections in eqs .
we use default values for all settings with the exception of runningmt = 0 , i.e. the top mass in the radiative corrections is identified with the pole mass ( to match the renormalization conditions imposed both in our os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr calculation and in the one of ref . ) . by default , the renormalization scale associated to the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr definition of the higgs wfr and of \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan is fixed as \documentclass[12pt]{minimal }
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\begin{document}$$\mu _ { { \scriptscriptstyle r}}=m_t$$\end{document}r = mt . in fig . 1 we present our predictions for the lightest - scalar mass \documentclass[12pt]{minimal }
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\begin{document}$$m_h$$\end{document}mh in the six benchmark scenarios .
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}= 500~\mathrm{gev}$$\end{document}ma=500gev and \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 20$$\end{document}tan=20 , so that the lightest scalar \documentclass[12pt]{minimal }
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\begin{document}$$h$$\end{document}h is sm - like , the bound on its tree - level mass is saturated , and the corrections controlled by the bottom yukawa coupling , which we do not compute beyond the approximations of feynhiggs , are not expected to be particularly relevant . for each scenario
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\begin{document}$$\overline{m}_{h}$$\end{document}mh , obtained from feynhiggs without additional corrections ; the middle bar includes the effect of the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , i.e. the \documentclass[12pt]{minimal }
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\begin{document}$$m_h$$\end{document}mh , and includes the effects of both the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections , i.e. the \documentclass[12pt]{minimal }
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\begin{document}$$(\delta m_h^2)^{\alpha \alpha _ { s}}$$\end{document}(mh2)s defined in eq .
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\begin{document}$$h$$\end{document}h in the six benchmark scenarios of ref . , for \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}=500~\mathrm{gev}$$\end{document}ma=500gev and \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 20$$\end{document}tan=20 . for each scenario ,
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\begin{document}$$\overline{m}_h$$\end{document}mh computed with feynhiggs 2.10.2 ( upper ) , the inclusion of the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections ( middle ) and the additional inclusion of the whole \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections ( lower ) . from top to bottom , the considered scenarios are \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}^\mathrm{mod-}$$\end{document}mhmod- ( green ) , light stop ( turquoise ) , light stau ( purple ) , tau - phobic ( orange ) predictions for the mass of the lightest scalar \documentclass[12pt]{minimal }
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\begin{document}$$h$$\end{document}h in the six benchmark scenarios of ref . , for \documentclass[12pt]{minimal }
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\begin{document}$$\overline{m}_h$$\end{document}mh computed with feynhiggs 2.10.2 ( upper ) , the inclusion of the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections ( lower ) . from top to bottom , the considered scenarios are \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}^\mathrm{mod-}$$\end{document}mhmod- ( green ) , light stop ( turquoise ) , light stau ( purple ) , tau - phobic ( orange ) figure 1 shows that , in all considered scenarios , the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$$m_h$$\end{document}mh by several hundred mev each ( the largest shifts , of about \documentclass[12pt]{minimal }
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\begin{document}$$\pm 1$$\end{document}1 gev , occur in the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{mod-}$$\end{document}mhmod- scenario ) .
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections on the prediction for the lightest - scalar mass as a function of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , and in fig . 3 we do the same for the heaviest - scalar mass .
in each figure , the mssm parameters for the left plot are chosen as in the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax benchmark scenario of ref . , while for the right plot they are chosen as in the modified light - stop scenario . in each plot , the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , while the solid lines include both the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections .
the red lines were obtained with \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 5$$\end{document}tan=5 while the blue lines were obtained with \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 20$$\end{document}tan=20 .
2corrections to the lightest - scalar mass as a function of \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 5$$\end{document}tan=5 ( red ) and for \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 20$$\end{document}tan=20 ( blue ) .
the other mssm parameters are chosen as in the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario ( left ) or as in the light - stop scenario ( right ) .
the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , the solid lines include both the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) correctionsfig . 3same as fig . 2 for the corrections to the heaviest - scalar mass corrections to the lightest - scalar mass as a function of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , for \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 5$$\end{document}tan=5 ( red ) and for \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 20$$\end{document}tan=20 ( blue ) .
the other mssm parameters are chosen as in the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario ( left ) or as in the light - stop scenario ( right ) .
the dashed lines represent the contribution of the sole momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections , the solid lines include both the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
2 for the corrections to the heaviest - scalar mass figure 2 shows that the corrections to the lightest - scalar mass are negligible at low values of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , but they become larger and essentially independent of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma as the latter increases .
the transition to this decoupling regime where the lightest scalar has sm - like couplings and its mass is insensitive to the value of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma is sharper for larger values of \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan. in both the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax and light - stop scenarios , the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) effects decrease \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh by 300400 mev at large \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma .
however , as already seen in fig . 1 , the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
effects enter the prediction for \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh with comparable magnitude but opposite sign , significantly reducing the total size of the correction .
figure 3 shows that for low values of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma , where the heaviest scalar is the one with sm - like couplings , the corrections to its mass are comparable to the ones that affect the lightest - scalar mass in the decoupling region . on the other hand , for larger values of the pseudoscalar mass where \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}\approx { { m}_{{\scriptscriptstyle a}}}$$\end{document}mhma the corrections to the heaviest - scalar mass show a series of spikes , related to the opening of real - particle thresholds in diagrams that involve a virtual gluon .
the first spike is visible in correspondence with \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}= 2\,{{m}_{t}}$$\end{document}ma=2mt in the plot on the left for the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario .
more - pronounced spikes ( note the different scale on the \documentclass[12pt]{minimal }
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\begin{document}$$y$$\end{document}y axis ) are visible in correspondence with \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}= m_{\tilde{t}_1}+m_{\tilde{t}_2}\,$$\end{document}mh = mt~1+mt~2 , and \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}= 2\,m_{\tilde{t}_2}\,$$\end{document}mh=2mt~2 in the plot on the right for the light - stop scenario .
analogous spikes would appear at larger values of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma in the \documentclass[12pt]{minimal }
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\begin{document}$$m_h^\mathrm{max}$$\end{document}mhmax scenario , where the stops are heavier .
we stress that our results are not reliable in the vicinity of these thresholds : the two - loop correction to the heaviest - scalar mass is actually divergent there , and the height of the spikes in the plots carries no physical meaning
. a more sophisticated analysis , taking into account the widths of the virtual particles in the loops as well as non - perturbative qcd effects , would be necessary around the thresholds , but it is beyond the scope of our calculation .
figure 3 also shows that , in the decoupling region and away from the thresholds , the corrections to the heaviest - scalar mass amount at most to a few hundred mev , and they decrease in size with increasing \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan. moreover , the effect of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections is negligible ( the dashed and solid lines are practically overlapping in the plots ) .
inspection of our analytic formulae shows that , in the decoupling limit , the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh are suppressed by one or two powers of \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan , whereas the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections contain unsuppressed contributions proportional to the square of the superpotential higgs - mass parameter \documentclass[12pt]{minimal }
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\begin{document}$$\mu $ $ \end{document}. while corrections of this size might be considered negligible in comparison with the value of \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh itself , they are not entirely irrelevant when compared to the difference \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}-{{m}_{{\scriptscriptstyle a}}}$$\end{document}mh - ma , which can be of the order of a few gev and is the quantity of interest when a large physical mass for the pseudoscalar is taken as input in the calculation .
the way we compute the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
corrections to the entries of the inverse - propagator matrix for the neutral scalars allows for a relatively easy comparison with earlier calculations .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , i.e. we introduce minimal counterterms that , by definition , subtract only powers of \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr-renormalized masses and couplings . in a second step ,
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr top / stop parameters entering the one - loop part of the corrections with the corresponding os parameters plus the finite one - loop shifts given in ref . . as a first obvious check
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and we compared our results with those in ref . , finding full agreement .
we also successfully compared the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections at vanishing external momentum with the results of an independent calculation based on the effective - potential techniques of ref . .
note , however , that this comparison does not cover the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) contributions to the \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z - boson self - energy .
concerning the latter , we checked that we can reproduce the result of ref .
for the subset of two - loop diagrams that involve only quarks and a gluon , taking into account the fact that ref .
we then compared our results for the momentum - dependent corrections with those of ref . , where the two - loop calculation of the higgs masses was performed entirely in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . as mentioned in sect . 1 , the higgs - mass corrections in ref
are organized in a different way with respect to our calculation , therefore we could compare only at the level of individual two - loop self - energies for scalars and pseudoscalars ( the two - loop self - energy for the \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z boson was not computed in ref . ) .
rotating our scalar self - energies from the \documentclass[12pt]{minimal }
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\begin{document}$$(s_1,s_2)$$\end{document}(s1,s2 ) basis to the \documentclass[12pt]{minimal }
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\begin{document}$$(h , h)$$\end{document}(h , h ) basis with the tree - level mixing angle defined in ref .
, we reproduce perfectly the results for the top / gluon and top / stop / gluino contributions to \documentclass[12pt]{minimal }
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\begin{document}$$\delta \pi _ { hh}$$\end{document}hh shown in figure 2 of that paper . this provides a full cross - check of the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) contribution to the self - energy , as well as a partial check of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s )
contribution , restricted to diagrams involving the stop squarks ( the diagrams involving the other squarks are included in the others line in the above - mentioned figure ) .
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\begin{document}$$\delta \pi _ { { \scriptscriptstyle a}{\scriptscriptstyle a}}$$\end{document}aa against results provided by the author of ref . , finding again perfect numerical agreement .
although our calculation and the one in ref . both use tsil to compute the mis , and thus can not be considered entirely independent , the agreement in the results for the self - energies gives us confidence that the computation of two - loop feynman diagrams in terms of mis and the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr subtraction of their divergences are correct in both papers .
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections in the mixed os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme can in turn be compared with those of ref . .
to start with , we compared our two - loop 1pi contributions to the scalar and pseudoscalar self - energies with analogous results provided by the authors of ref . , and we found agreement within the accuracy of the sector - decomposition procedure used to compute the loop integrals in that paper .
the successful comparison between two sets of self - energies in which the loop integrals were evaluated with tsil and secdec , respectively , validates the results for the two - loop mis , thus reinforcing our cross - check of ref . .
on the other hand , our results for the momentum - dependent \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections to the higgs masses , obtained by combining the 1pi diagrams with all the necessary counterterm contributions , differ significantly from the ones in ref . .
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\begin{document}$$m_h^{\mathrm{max}}$$\end{document}mhmax scenario discussed in the previous section , we find that for large \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle a}}}$$\end{document}ma the lightest - scalar mass is subject to a negative correction of about 350400 mev ( depending on \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan ; see the left plot in fig .
is also negative but quite smaller , about 5060 mev ( see the upper plot in figure 7 of that paper ) .
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\begin{document}$$\tan \beta $ $ \end{document}tan. in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , the wfr counterterm for each field \documentclass[12pt]{minimal }
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\begin{document}$$h^0_i$$\end{document}hi0 can be related to the divergent part of the derivative of the scalar self - energy with respect to the external momentum:18\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta \mathcal{z}^{(\ell ) } _ i = - \left [ \frac{d\ , \mathrm{re}\,\pi ^{(\ell ) } _ { ii}(p^2)}{dp^2 } \right ] ^\mathrm{div}\quad ( \ell = 1,2 ) .
\end{aligned}$$\end{document}zi()=-dreii()(p2)dp2div(=1,2).indeed , when all parameters entering the one - loop part of the scalar self - energies are renormalized in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , eq .
( 18 ) leads to the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr wfr counterterms given in eq .
( 9 ) , in accordance with the anomalous dimensions of the higgs fields given in refs .
however , in the mixed os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme of ref .
the top / stop parameters in the one - loop self - energies are renormalized os . in that case , the use of eq .
( 18 ) to determine the wfr counterterms leads to19\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{z}_2^{}&= 1 - \frac{~\alpha _ { t}^{{\mathrm{os}}}}{4\pi } \,n_c\,\cdot \,\frac{1}{\epsilon } ~+~ \frac{\alpha _ { t}\alpha _ { s}}{(4\pi ) ^2}\,2\,n_c\,c_f\nonumber \\&\cdot \left ( \frac{1}{\epsilon ^2}-\frac{1}{\epsilon } \right ) - \frac{\alpha _ { t}}{2\pi } \,n_c\ , \frac{\delta m_t}{m_t}\,\cdot \,\frac{1}{\epsilon } , \end{aligned}$$\end{document}z2=1-tos4nc1+ts(4)22nccf12 - 1-t2ncmtmt1,where \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ { t}^{{\mathrm{os}}}$$\end{document}tos is a scale - independent coupling extracted from the pole top mass , and \documentclass[12pt]{minimal }
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\begin{document}$$\delta m_t$$\end{document}mt is the finite one - loop shift for the top mass given in eq . ( b2 ) of ref . .
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\begin{document}$$\alpha _ { t}^{{\mathrm{os}}}$$\end{document}tos in the one - loop term of eq .
( 19 ) into the corresponding \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr coupling , it is easy to see that \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{z}_2^{}$$\end{document}z2 differs from the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr wfr constant in eq .
( 9 ) by a finite two - loop term:20\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{z}_2^ { } = \mathcal{z}_2^{\,{\overline{\mathrm{dr } } } } ~+~ \frac{\alpha _ { t}}{2\pi } \,n_c\ , \frac{\delta _
\epsilon m_t}{m_t } , \end{aligned}$$\end{document}z2=z2dr+t2ncmtmt , where \documentclass[12pt]{minimal }
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\epsilon m_t$$\end{document}mt denotes the part proportional to \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon $ $ \end{document} in the top self - energy regularized with dimensional reduction:21\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{\delta _
s}{4 \pi } \,c_f\ , \left\ { -\frac{3}{2}\,\ln ^2 \frac{{{m}_{t}}^2}{\mu _ { { \scriptscriptstyle r}}^2 } + 5\,\ln \frac{{{m}_{t}}^2}{\mu _ { { \scriptscriptstyle r}}^2 } - \frac{~\pi ^2}{4 } - 9 - \frac{m_{\tilde{g}}^{2}}{{{m}_{t}}^2}\right .
\left ( \frac{1}{2 } \ , \ln ^2 \frac{m_{\tilde{g}}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } - \ln \frac{m_{\tilde{g}}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } + \frac{~\pi ^2}{12 } + 1 \right ) \right .
\nonumber \\&+ \frac{1}{2}\left [ ~ \frac{m_{\tilde{g}}^{2}+{{m}_{t}}^2 - { m}_{\tilde{t}_1}^{2}- 2 \,{s}_{2\theta _ { t}}\,m_{\tilde{g}}\ , { { m}_{t}}}{{{m}_{t}}^2 } \,b_\epsilon ( { { m}_{t}}^2,m_{\tilde{g}}^{2},{m}_{\tilde{t}_1}^{2 } ) \right . \nonumber \\&+\left . \left . \,\frac{{m}_{\tilde{t}_1}^{2}}{{{m}_{t}}^2 } \left ( \frac{1}{2 } \ , \ln ^2 \frac{{m}_{\tilde{t}_1}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } - \ln \frac{{m}_{\tilde{t}_1}^{2}}{\mu _ { { \scriptscriptstyle r}}^2 } + \frac{~\pi ^2}{12 } + 1 \right ) \right . \right .
( \tilde{t}_1 \rightarrow \tilde{t}_2 , ~~ { s}_{2\theta _ { t}}\rightarrow -{s}_{2\theta _ { t } } ) \right ] \right\ } .
\end{aligned}$$\end{document}mtmt=s4cf-32ln2mt2r2 + 5lnmt2r2-24 - 9-mg~2mt212ln2mg~2r2-lnmg~2r2+212 + 1 + 12mg~2+mt2-mt~12 - 2s2tmg~mtmt2b(mt2,mg~2,mt~12)+mt~12mt212ln2mt~12r2-lnmt~12r2+212 + 1+(t~1t~2,s2t-s2t).in the equation above \documentclass[12pt]{minimal }
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\begin{document}$$\mu _ { { \scriptscriptstyle r}}$$\end{document}r is the renormalization scale associated to the higgs wfr and to \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan , while \documentclass[12pt]{minimal }
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\begin{document}$$b_\epsilon ( s , x , y)$$\end{document}b(s , x , y ) denotes the coefficient of \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon $ $ \end{document} in the expansion of the passarino
veltman function \documentclass[12pt]{minimal }
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\begin{document}$$b_0$$\end{document}b0 .
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\begin{document}$$b_\epsilon $ $ \end{document}b can be found , e.g. , in eq .
( 2.31 ) of the tsil manual . in the calculation of ref . , where the top / stop parameters entering the one - loop part of the corrections are directly renormalized os instead of being first renormalized in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme and then converted to the os scheme via a finite shift , the two - loop self - energies and tadpoles contain terms proportional to \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ \epsilon m_t$$\end{document}mt .
such terms would drop out of the final result for the renormalized inverse - propagator matrix if eq .
( 20 ) was used to obtain the correct \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{z}_2^{\,{\overline{\mathrm{dr}}}}$$\end{document}z2dr , and consequently for \documentclass[12pt]{minimal }
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\begin{document}$$\delta \tan \beta $ $ \end{document}tan , but they survive if the wfr constant is defined as in eq .
( 19 ) . to prove that these terms are indeed at the root of the observed discrepancies
, we modified our own calculation , using \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{z}_2^{}$$\end{document}z2 as obtained from eq .
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\begin{document}$$\mathcal{z}_2^{\,{\overline{\mathrm{dr}}}}$$\end{document}z2dr and then computing a non - minimal counterterm for \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan via eq .
we checked that , with this modification , we reproduce exactly the corrections to the renormalized inverse propagator shown in figures 5 and 10 of ref . .
we also reproduce the corrections to the scalar masses shown in figures 7 , 8 , 12 and 13 of that paper , although small discrepancies persist in the case of the heaviest scalar when its mass is above the threshold \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}=2\,m_t$$\end{document}mh=2mt .
these residual discrepancies are formally of higher order in the perturbative expansion , and they result from different approximations in the two - step procedure for the determination of the poles of the propagator ( namely , we drop the imaginary parts of the two - loop self - energies , while ref
the two - loop renormalization of the higgs fields and of the parameter \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan is not performed in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme as claimed in the paper , but rather in some non - minimal scheme where the wfr counterterms and \documentclass[12pt]{minimal }
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\begin{document}$$\delta \tan \beta $ $ \end{document}tan differ from their \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr counterparts by finite , non - polynomial terms , and neither the higgs fields nor \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan obey their usual renormalization - group equations ( because of the explicit scale dependence of the additional terms ) .
this inconsistency should be taken into account when comparing the results of ref . with those of calculations that actually employ \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan. first of all , to account for the difference in \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan should be converted to the corresponding value in the non - minimal scheme of ref . , according to22\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \tan \beta ^ { } = \tan \beta ^{\,{\overline{\mathrm{dr } } } } - \frac{\alpha _ { t}}{4\pi } \,n_c\,\tan \beta ~ \frac{\delta _
( 5)(7 ) show that the contributions of \documentclass[12pt]{minimal }
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\begin{document}$$\delta \tan \beta ^{(2)}$$\end{document}tan(2 ) to the entries of the higgs mass matrix are suppressed by powers of \documentclass[12pt]{minimal }
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\begin{document}$$\cos \beta $ $ \end{document}cos. consequently , the effect on the higgs masses arising from a two - loop difference in \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta = 5$$\end{document}tan=5 .
in fact , the bulk of the numerical discrepancy between our results and those of ref . is due to higher - order effects that are directly related to the finite shift in the wfr . as discussed at the end of sect . 2 , such effects
are included in the higgs - mass corrections when the latter are computed in terms of loop - corrected higgs masses and mixing , and can become numerically relevant when the loop - corrected masses differ significantly from their tree - level values .
we computed the two - loop corrections to the neutral mssm higgs masses of \documentclass[12pt]{minimal }
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\begin{document}$$h_t$$\end{document}ht including the effect of non - vanishing external momenta in the self - energies .
we relied on an ibps technique to express the momentum - dependent loop integrals in terms of a minimal set of master integrals , and we used the public code tsil to evaluate the latter .
we obtained results for the higgs - mass corrections valid when all parameters in the one - loop part of the corrections are renormalized in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme , as well as results valid in a mixed os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme where the top / stop parameters are renormalized on - shell .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme confirm the results of an earlier calculation , ref .
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\begin{document}$$z$$\end{document}z - boson self - energy that involve the strong gauge coupling .
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections to the higgs masses when the tree - level mass matrix of the scalars is expressed in terms of the physical \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z - boson mass .
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme with those of a recent calculation of the same corrections , ref . , and found disagreement .
we traced the reason for the discrepancy to the fact that , contrary to what stated in ref .
, in that calculation the higgs fields and the parameter \documentclass[12pt]{minimal }
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\begin{document}$$\tan \beta $ $ \end{document}tan are renormalized in a non - minimal scheme instead of the usual \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme . when this difference is taken into account
, we reproduce the results of ref . , providing in passing a cross - check of the codes used to evaluate the loop integrals in the two calculations ( i.e. , tsil and secdec , respectively ) . however , we noticed that tsil , which implements dedicated algorithms for two - loop self - energy integrals , can be a thousand times faster than a multi - purpose code such as secdec in the computation of the higgs - mass corrections .
this should be taken into consideration when including the momentum - dependent corrections in phenomenological analyses of the mssm parameter space .
as to the numerical impact of the corrections computed in this paper , it could at best be described as moderate .
we considered six benchmark scenarios compatible with the results of higgs and susy searches at the lhc , and we found that both the momentum - dependent part of the \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha _ { t}\alpha _ { s})$$\end{document}o(ts ) corrections and the whole \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) corrections can shift the prediction for the lightest - scalar mass \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh by several hundred mev .
however , we noticed that at least in the considered scenarios the two classes of corrections enter the prediction for \documentclass[12pt]{minimal }
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\begin{document}$${{m}_{h}}$$\end{document}mh with opposite sign , and they compensate each other to a good extent .
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\begin{document}$${{m}_{{\scriptscriptstyle h}}}$$\end{document}mh , the impact of the new corrections is also modest , with the exception of regions around real - particle thresholds where a fixed - order calculation is not reliable anyway .
the predictions for the lightest - scalar mass , as obtained from popular codes for the determination of the mssm mass spectrum , carry a theoretical uncertainty that has been estimated to be ( at least ) of the order of \documentclass[12pt]{minimal }
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\begin{document}$$\pm 3$$\end{document}3 gev see , e.g. , refs . [ 78 , 79 ] and the more recent discussion in ref . . against this backdrop ,
the corrections presented in this paper can be considered sub - dominant , and their inclusion in public codes might seem less urgent than , e.g. , the inclusion of the dominant three - loop effects [ 41 , 42 ] or the proper resummation of large logarithms in scenarios with multi - tev stop masses [ 39 , 40 , 82 ] , both of which can shift the prediction for the lightest - scalar mass by several gev . nevertheless , one should not forget that the accuracy of the measurement of the higgs mass at the lhc has already reached the level of a few hundred mev
i.e. , comparable to our sub - dominant corrections and will improve further when more data become available .
if susy shows up at last when the lhc operates at 1314 tev , the higgs mass will serve as a precision observable to constrain mssm parameters that might not be directly accessible by experiment . to this purpose
, the accuracy of the theoretical prediction will have to match the experimental one , making a full inclusion of the two - loop corrections to the higgs masses unavoidable . | we compute the two - loop qcd corrections to the neutral higgs - boson masses in the minimal supersymmetric standard model , including the effect of non - vanishing external momenta in the self - energies .
we obtain corrections of \documentclass[12pt]{minimal }
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\begin{document}$${\mathcal { o}}(\alpha \alpha _ { s})$$\end{document}o(s ) , i.e. , all two - loop corrections that involve the strong gauge coupling when the only non - vanishing yukawa coupling is the top one .
we adopt either the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr renormalization scheme or a mixed on - shell ( os)\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme where the top / stop parameters are renormalized on - shell .
we compare our results with those of earlier calculations , pointing out an inconsistency in a recent result obtained in the mixed os\documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{dr}}$$\end{document}dr scheme .
the numerical impact of the new corrections on the prediction for the lightest - scalar mass is moderate , but already comparable to the accuracy of the higgs - mass measurement at the large hadron collider . |
dynamic magnetic resonance imaging ( mri ) of the breast was reported to be a highly sensitive method for detection and further evaluation of clinically , mammographically , and sonographically occult cancers
two different approaches are mentioned in literature aiming to improve the specificity : ( 1 ) single - breast imaging protocols with high spatial resolution offer a meticulous analysis of the lesion 's structure and internal architecture and are able to distinguish between benign and malignant lesions and ( 2 ) lesion differential diagnosis in dynamic protocols is based on the assumption that benign and malignant lesions exhibit different enhancement kinetics . in , it was shown that the shape of the time - signal intensity curve represents an important criterion in differentiating benign and malignant enhancing lesions in dynamic breast mr imaging .
the results indicate that the enhancement kinetics , as represented by the time - signal intensity curves visualized in figure 1 , differ significantly for benign and malignant enhancing lesions and thus represent a basis for differential diagnosis . in breast cancers , plateau or washout - time courses ( type ii or iii ) prevail . steadily progressive signal intensity time courses ( type i )
are exhibited by benign enhancing lesions . also , these enhancement kinetics are shared not only by benign tumors but also by fibrocystic changes .
the success of cad in conventional x - ray mammography motivated the research of automated diagnosis techniques in breast mri to expedite diagnostic and screening activities .
a standard multilayer perceptron ( mlp ) was applied to the classification of signal - time curves from dynamic breast mri in .
breast mr segmentation and lesion detection is accomplished based on cellular neural networks in and a 100% detection sensitivity is reported . in
, the performance of a backpropagation neural network based on kinetic , morphologic , and combined mr features was shown to be comparable to that of an expert radiologist .
the same type of neural networks was used for breast mri lesion classification in . as inputs ,
a subset of 13 features out of a total of 42 features describing lesion shape , texture , and enhancement kinetics was selected .
the main result was that the performance of the human readers significantly improved when aided by a cad system .
it could be shown that specificity at a sensitivity of 90% was 0.505 for lesion classification without cad assistance and 0.807 for classification with cad assistance .
mean shift clustering in connection with automated selection of the most suspicious cluster resulted in accurate rois in breast mri lesions , as shown in .
in , a fuzzy c - means clustering - based technique was tested for automatically identifying characteristic kinetic from breast lesions . by using four features extracted from these curves ( maximum contrast enhancement , time to peak , uptake rate , and washout rate of the lesion kinetics )
, it was demonstrated that the prototype curves determined by the fuzzy classifier outperform those determined based on averaging over an roi determined by an experienced radiologist .
three different classifiers , a multilayer perceptron , a threshold classifier , and a nearest - neighbor classifier , were employed for the classification of signal - time curves in . as feature vectors , both the complete signal - time curve and
it was shown that the quantitative classifiers can support the radiologist in the diagnosis of breast lesions .
a major disadvantage of most supervised techniques is the fixed number of input nodes which imposes the constraint of a fixed imaging protocol , as shown in .
delayed administration of the contrast agent or a different temporal resolution has a negative effect on the classification and segmentation capabilities .
thus , a change in the mr imaging protocol requires a new training of the cad system .
a signal time - series is obtained for every pixel and has to be interpreted given the experimental conditions .
thus , supervised and unsupervised represent two different techniques when it comes to biomedical signal analysis .
the supervised technique represents a model - driven approach that tries to interpret time - series as a result of dynamically changing input parameters , given a specific model that defines the pattern of interaction between the underlying physiological processes and the observed signal dynamics .
however , this model - based , supervised technique is based on the underlying knowledge of experimental conditions and model assumptions .
this aspect makes it difficult or impossible and therefore unsupervised techniques represent better training approaches for expert advanced cad systems since they fit the model to the observations and analyze and visualize the data in an exploratory manner .
an important aspect remains the fact that many of these techniques were applied on a database of predominantly tumors of a size larger than 2 cm . in these cases
, mri reaches a very high sensitivity in the detection of invasive breast cancer due to both morphological criteria as well as characteristic time - signal intensity curves .
however , the value of dynamic mri and of automatic identification and classification of characteristic kinetic curves is not well established in small lesions when clinical findings , mammography , and ultrasound are unclear . in the present study , we design and evaluate an unsupervised cad system for the diagnosis of small breast masses with a diameter of only a few mm .
for those lesions containing only a small number of voxels , morphologic criteria can hardly be evaluated . to overcome the above mentioned problems
, we employ a neural - gas network as a quantization method that focuses strictly on the observed complete mri signal time - series , and enables a self - organized data - driven segmentation of dynamic contrast - enhanced breast mri time - series w.r.t .
fine - grained differences of signal amplitude , and dynamics , such as focal enhancement in patients with indeterminate breast lesions .
this method is developed , tested , and evaluated for functional and structural segmentation , visualization , and classification of dynamic contrast - enhanced breast mri data .
in addition , a comparison with another unsupervised method , the minimal free - energy vector quantization , and the conventional segmentation method is performed .
we will show that the inspection of the clustering results represents a unique practical tool for the radiologists enabling a fast scan of the data set for regional differences or abnormalities of contrast - agent uptake .
the proposed technique contributes to the diagnosis of indeterminate breast lesions by non - invasive imaging .
a total of 40 patients , all female and age range 4861 , with indeterminate small mammographic breast lesions were examined .
all patients were consecutively selected after clinical examinations , mammography in standard projections ( craniocaudal and oblique mediolateral projections ) , and ultrasound .
only lesions birads 3 and 4 were selected where at least one of the following criteria was present : nonpalpable lesion , previous surgery with intense scarring , or location difficult for biopsy ( close to chest wall ) .
all patients had histopathologically confirmed diagnosis from needle aspiration / excision biopsy and surgical removal .
the size of malignant tumors ranged from 0.5 mm to 2.0 mm , average size was 1.06 mm , the size of benign tumors ranged from 0.8 mm to 1.8 mm , and average size was 1.2 mm .
mri was performed with a 1.5 t system ( magnetom vision , siemens , erlangen , germany ) equipped with a dedicated surface coil to enable simultaneous imaging of both breasts .
first , transversal images were acquired with an stir ( short ti inversion recovery ) sequence ( tr = 5600 ms , te = 60 ms , fa = 90 , it = 150 ms , matrix size 256 256 pixels , slice thickness 4 mm ) . then a dynamic t1 weighted gradient echo sequence ( 3d fast low angle shot sequence ) was performed ( tr = 12 ms , te = 5 ms , fa = 25 ) in transversal slice orientation with a matrix size of 256 256 pixels and an effective slice thickness of 4 mm . the dynamic study consisted of 6 measurements with an interval of 83 seconds .
the first frame was acquired before injection of paramagnetic contrast agent ( gadopentatate dimeglumine , 0.1 mmol / kg body weight , magnevist , schering , berlin , germany ) immediately followed by the 5 other measurements .
the initial localization of suspicious breast lesions was performed by computing difference images , that is , subtracting the image data of the first from the fourth acquisition . as a preprocessing step to clustering , each raw gray level time - series s( ) , { 1 , , 6 }
was transformed into a signal time - series of relative signal enhancement x( ) for each voxel , the precontrast scan at = 1 serving as reference .
thus , we ensure that the proposed method is less sensitive to changing between different mr scanners and/or protocols . the small lesion evaluation is performed by an automated computer - aided diagnosis system based on preprocessing of the signal - intensity time - courses , segmentation of signal - intensity time - courses , and then automated evaluation of the time - signal intensity curve based on an unsupervised classifier .
the flow diagram of the cad system is shown in figure 2 . in the present study
, we selected only lesions with an initial contrast enhancement 50% for a comparative analysis between the conventional segmentation analysis method and the unsupervised classifier .
the employed classifier the neural - gas network is based on grouping image voxels together based on the similarity of their intensity profile in time ( i.e. , their time courses )
. another important feature of the presented algorithm compared to som is that it does not require a prespecified graph ( network ) . in addition
, it can produce topologically preserving maps , which is only possible if the topological structure of the graph matches the topological structure of the data manifold . in cases
, however , where it is not possible to a priori determine an appropriate graph , for example , in cases where the topological structure of the data manifold is not known a priori or is too complicated to be specified , kohonen 's algorithm necessarily fails in providing perfectly topology preserving maps .
let n denote the number of subsequent scans in a dynamic contrast - enhanced breast mri study , and let k be the number of voxels in each scan .
the dynamics of each voxel { 1 , , k } , that is , the sequence of signal values { x(1 ) , , x(n ) } can be interpreted as a vector x(i ) r in the n - dimensional feature space of possible signal time - series at each voxel .
cluster analysis groups image voxels together based on the similarity of their intensity profile in time . in the clustering process , a time course with n points is represented by one point in an n - dimensional euclidean space which is subsequently partitioned into clusters based on the proximity of the input data .
these groups or clusters are represented by prototypical time - series called codebook vectors ( cvs ) located at the center of the corresponding clusters .
vector quantization ( vq ) represents a fast clustering technique for feature vectors describing pixel time courses in breast mri .
vq approaches determine the cluster centers wi by an iterative adaptive update based on the following equation :
( 1)wi(t+1)=wi(t)+(t)ai(x(t),c(t),)(x(t)wi(t ) ) ,
where (t ) represents the learning parameter , ai a codebook c(t ) dependent cooperativity function , a cooperativity parameter , and x a randomly chosen feature vector .
algorithm is an efficient clustering approach which , applied to the task of vector quantization , ( 1 ) converges quickly to low distortion errors , ( 2 ) reaches a distortion error , ( which measures the fidelity of data encoding and is given by the squared euclidean distance between the data vectors and the corresponding approximating reference vectors)lower than that from self - organizing map ( som ) , and ( 3 ) at the same time obeys a gradient descent on an energy surface representing the error function of the learning algorithm . instead of using the distance ||x
wi|| or of using the arrangement of the ||wi|| within an external lattice , it utilizes a neighborhood - ranking of the reference vectors wi for the given data vector x. the learning rule for the neural - gas network is
( 2)wi(t+1)=wi(t)+(t)exp { ki(x , wi)}(x(t)wi(t ) ) ,
where ki = 0 , , n 1 represents the rank index describing the neighborhood - ranking of the reference vectors wi to the data vector x in a decreasing order , n is the number of units in the network , and determines the number of neural units changing their synapses with every iteration .
it was shown by martinetz et al . that the average change of the reference vectors corresponds to an overdamped motion of particles in a potential that is given by the negative data point density .
superimposed on the gradient of this potential is a force , which points toward the direction of the space where the particle density is low .
force is the result of a repulsive coupling between the particles ( reference vectors ) . in its form
it resembles an entropic force and tends to homogeneously distribute the particles ( reference vectors ) over the input space , like the case of a diffusing gas .
it is interesting also to mention that the reference vectors wi change their locations slowly but permanently , and wi that are neighboring at an early stage of the adaptation procedure might not be neighboring anymore at a more advanced stage .
another important clustering method represents the minimal free energy vector quantization [ 14 , 15 ] .
it is a divisive procedure starting first with one cluster containing the whole data set . during the learning process , the number of clusters increases and their size decreases as they specialize on specific data distributions .
other than fuzzy c - means clustering , this algorithm does not operate with prespecified cluster centers [ 13 , 16 ] .
the adaptation paradigm for this technique is
( 3)wi(t+1)=wi(t)+(t)exp ||x(t)wi(t)||2/22iexp ||x(t)wi(t)||2/22(x(t)wi(t ) ) ,
where is the fuzzy range of the model and defines a length scale in data space and is annealed to repeatedly smaller values in the vq approach .
||x(t ) wi(t)||/2 ) represents the so - called softmax activation function , and accordingly the outputs lie in the interval and they sum up to one .
the main advantages of fuzzy clustering based on deterministic annealing over som were pointed out in : ( 1 ) the divisive and multiresolution aspect of data analysis , ( 2 ) different control parameters ( free energy , entropy ) control and enable a direct cluster splitting , and ( 3 ) the gradient descent type of the learning rule monitored by an explicitly given error function . in the following , we will present the two employed segmentation methods for the evaluation of signal intensity time courses for the differential diagnosis of enhancing lesions in breast mri . the clinical standard method to analyze dynamic mri of the breast
is based on carefully choosing a region of interest ( roi ) surrounding the contrast enhancing lesion .
lesion segmentation was performed automatically by a region growing algorithm after positioning a seed within the lesion .
for all the voxels belonging to this roi , an average signal intensity ( si ) time curve was computed . in the present study
, we selected only lesions with an initial contrast enhancement 50% for comparative analysis between the standard evaluation method and unsupervised clustering . thus , we use a semiautomatic segmentation method to determine the roi including all voxels of a lesion with an initial contrast enhancement of 50% .
the initial contrast enhancement was calculated according to the following equation :
( 4)sai=(si2nd frame post - contrastsipre - contrast)sipre - contrast100% .
the postinitial signal course was calculated based on the following equation :
( 5)svp=(si5th frame post - contrastsimaximum 1st to 2nd frame post - contrast)simaximum 1st to 2nd frame post - contrast100% .
for all voxels belonging to this roi an average time - signal intensity curve is computed for both initial signal increase and postinitial signal course .
the contrast - enhanced pixels are shown in figure 3(b ) . based on a region - growing method
this method uses the roi of the previous segmentation method while all the voxels within this roi are subject to cluster analysis .
this allows the clinical radiologist to be a more detailed view of the signal curves by partitioning the roi .
this segmentation method reveals regional properties of contrast - agent uptake characterized by subtle differences of signal amplitude and dynamics as it is demonstrated by our results in figures 6 , 7 , and 8 . as a result
, we obtain both a set of prototypical time - series and a corresponding set of cluster assignment maps which further provides a segmentation with regard to identification and regional subclassification of pathological breast tissue lesions . for every single cluster a mean percentage signal intensity change- ( psic- ) curve
is computed which contains only the signal information of the signal time curves that are assigned to this particular cluster .
for example , if we perform clustering for n = 4 clusters , then we obtain 4 cluster - specific psic - curves .
the obtained time - signal intensity curves of enhancing lesions were plotted and presented to two experienced radiologists who were blinded to any clinical or mammographic information of the patients .
the radiologists were asked to rate the time courses as having a steady , plateau , or washout shape
the cluster vector of each lesion with the highest classification level was used for further statistical evaluation .
all lesions ( n = 40 ) with an initial signal increase 50% after contrast injection were included in the comparative analysis of the conventional segmentation method and cluster analysis .
lesion size was determined as the number of voxels with an initial si increase of 50% .
clustering results were evaluated by ( i ) qualitative visual inspection of cluster assignment maps , that is , cluster membership maps according to a minimal distance criterion in the metric of the pixel - time course ( ptc ) feature space shown for the neural - gas network , ( ii ) qualitative visual inspection of corresponding cluster - specific time - signal intensity curves for the neural - gas network , ( iii ) optimal parameter combination , ( iv ) optimal number of clusters , ( v ) comparison between neural - gas network , minimal free energy vector quantization and conventional segmentation method , and ( vi ) receiver operating characteristic ( roc ) analysis . the unsupervised classifier based on the neural - gas network , as shown in ( 2 ) , has three free parameters : the initial step size , the number of epochs being equal to the number of the training steps multiplied by the number of training data vectors , and the initial decay constant . we analyzed the whole possible range of parameters , corresponding thus to the largest range : was varied between 0.1 and 1 , the number of epochs and the initial decay constant were also varied according to .
we calculated the variations of parameters in terms of the initial and postinitial contrast enhancement according to ( 4 ) and ( 5 ) as these parameters are crucial for the correct detection .
we tested 30 different parameter combinations with : ( a ) being varied between 0.1 and 1 , ( b ) number of epochs between 1 and 10 , and ( c ) between n/6 and n with n being the number of clusters and additionally , = 3n/2,4n/2,5n/2,6n/2 .
the optimal combination of these three parameters is important to achieve stable clustering results , in the sense that the same clustering results are obtained regardless the initialization .
our simulation results showed that an optimal parameter combination for small lesion detection is characterized by a small standard deviation of the minimum and maximum values of the percentage of the initial signal increase sai and the percentage of the postinitial signal intensity svp .
exemplary , we show in figures 4 and 5 the minimal and maximal values for sai and svp for the variation of the initial step size . small variances indicate a stable clustering result . as an optimal value ,
the optimal parameter combination achieved for data set # 1 is shown in table 1 .
an important aspect is to define the number of cvs prior to clustering . the exact number of clusters is usually determined by cluster validity techniques . in general , the higher the number , the finer grained the analyzed roi is partitioned , however at the expense of an increase in signal noise susceptibility , while a lower number leads to overlooking of pertinent information . in , the number of clusters is determined as the number of voxels in the 3d breast lesion is divided by 80 . in our study
the distribution of different cluster numbers to the four kuhl classes is shown in table 2 .
the mean and standard deviation of the clusters ' assignments to the kuhl classes is represented in function of the cluster number .
the results are for 20 runs performed on the actual kinetic curves using the same parameters for the neural network but with different codebook initializations and can be interpreted as follows : the smaller the value of the standard deviation , the better the reproducibility and the higher the significance of the simulation experiments .
it becomes quickly evident that the optimal representation is achieved for four clusters : a zero standard deviation means a correct identification of the time - signal intensity curve types .
figures 6 to 8 visualize the cluster distribution and the representative time - signal intensity time curves for two , four , and six clusters for data set no .
1 . all lesions with an initial signal increase 50% after contrast injection were included in the comparative analysis of the conventional segmentation method and the two cluster analysis methods .
histological findings were malignant in 31 and benign in 9 lesions . in this study ,
the results of the conventional segmentation method compared with the two unsupervised clustering methods were correlated with the histological findings and represented in tables 3 and 4 .
senstitivity increased with unsupervised clustering in dcis , in invasive ductal , and especially in lobular carcinoma while specificity remained constant .
clustering is done for 20 different runs using the same parameters but different algorithms ' initializations .
the tables show an increased sensitivity of segmentation method based on both vector quantization methods compared to the conventional segmentation analysis in dcis , lobular and invasive ductal carcinoma in comparison to the mean signal intensity time curve of the undivided roi .
neural - gas shows an increase in sensitivity versus mfe for the two latter carcinoma .
specificity remained constant for all benign lesions for the neural - gas , however decreased based on mfe for the benign lymph node . in summary ,
the use of clustering allows a more differentiated view of the hidden structure of the analyzed dataset .
this method reveals information , else not evident . on the other hand , the
clustering results were also evaluated by an roc analysis . in the roc curve , we compare both segmentation methods with the gold standard ( histology ) . for both segmentation methods ,
we obtain for each of the 40 small lesions a value between 1 and 4 showing to each of the four curves from figure 1 it belongs .
for the benign lesions we have the following : type ia has value 1 and type ib has value 2 .
similarly , for malignant lesions , we obtain the following : type ii has value 3 and type iii has value 4 . to determine the roc curves
, we have to vary the threshold based on which a lesion is considered malignant .
each roc curve has 6 parameters : 4 from the different classes and 2 corresponding to sensitivity = 1 , specificity = 0 , and sensitivity = 0 , specificity = 1 . from table 5
, we see that the second segmentation method yields better results than the first one : in other words , the unsupervised classification outperforms the conventional segmentation method based on averaged above - threshold classification .
the goal of the presented study was the introduction of new approaches for the evaluation of dynamic mr mammography in small lesions and is motivated by the conceptual weaknesses of the conventional technique .
we presented two different segmentation methods for the evaluation of signal intensity time courses for the differential diagnosis of small enhancing lesions in breast mri .
it is important to mention that the interpretation of kinetic curves is not standardized in the current clinical practice .
a manually predefined roi is substantially impacting the differential diagnosis in breast mri by being both time - consuming and significantly suffering from inter- and intraobserver variability . on the other hand ,
the conventional method based on threshold segmentation allows a differentiation between contrast - enhanced lesions and surrounding tissue .
a fusion of the techniques of threshold segmentation and cluster analysis combines the advantages of these single methods .
thus , a fast segmentation method is obtained which carefully discriminates between regions with different lesion enhancement kinetics .
we have determined the optimal number of clusters and thus the optimal number of characteristic curves for lesion segmentation and classification .
a small number will lead to a characteristic curve being very close to the average curve , and thus not able to subdifferentiate the lesion .
the performed roc - analysis shows that the unsupervised clustering technique represents a valuable tool for supporting radiological diagnosis in dynamic breast mr imaging .
however , the most important advantage lies in the potential of increasing the diagnostic accuracy of mri mammography by improving the sensitivity without reduction of specificity for the data sets examined . | an application of an unsupervised neural network - based computer - aided diagnosis ( cad ) system is reported for the detection
and characterization of small indeterminate breast lesions , average size 1.1 mm , in dynamic contrast - enhanced mri .
this system
enables the extraction of spatial and temporal features of dynamic mri data and additionally provides a segmentation with regard
to identification and regional subclassification of pathological breast tissue lesions .
lesions with an initial contrast enhancement
50% were selected with semiautomatic segmentation . this conventional segmentation analysis is based on the mean initial signal
increase and postinitial course of all voxels included in the lesion . in this paper
, we compare the conventional segmentation analysis
with unsupervised classification for the evaluation of signal intensity time courses for the differential diagnosis of enhancing
lesions in breast mri .
the results suggest that the computerized analysis system based on unsupervised clustering has the potential to
increase the diagnostic accuracy of mri mammography for small lesions and can be used as a basis for computer - aided diagnosis
of breast cancer with mr mammography . |
the past three decades of studies have unveiled some of the genetic underpinnings of human disease . for complex diseases , those with obscure genetic roots , discoveries have accelerated recently owing to a bloom of genome - wide association studies ( gwass ) . nevertheless , even for the most successful cases ( such as inflammatory and ulcerative bowel disease )
, discoveries account for only a fraction , often small , of the disease 's heritability .
these yet to be discovered genetic variants comprise the ' missing heritability ' or the genetic ' dark matter ' for disease .
heritability , the proportion of trait variability explained by genetic factors , has two somewhat different meanings .
if genetic variation were all to act additively , the best predictor of an offspring 's trait value would be the average of his / her parents ' values .
hunting for dark matter for a trait such as human height will be more straightforward than for a disease such as schizophrenia , for which the evidence for substantial gene - gene interaction is compelling .
yet when researchers refer to heritability of human height , they implicitly mean narrow - sense heritability ; for schizophrenia , it is heritability in a much broader sense .
greater understanding of the genetics equals greater understanding of molecular etiology and , with it , eventually more cogent treatments .
however , the origins of some human diseases , especially those of the mind , can be mysterious . for diseases of the mind ,
few environmental or genetic risk factors are understood ; instead the hope is that identified genetic factors will lead to a subtler understanding of why diseases such as schizophrenia arise and how they can be treated effectively . even for cardiovascular disease , for which environmental risk factors are well characterized , new insights into its genetics could produce more targeted treatment
this leads to the other expectation - that greater genetic knowledge will pave the way for ' personalized ' medicine .
the rapid technological advances in genomics will soon make it feasible to sequence whole genomes at relatively low cost .
the idea that each individual will have meaningful sequence variation in their medical records and will have interventions tailored to their risk profile and likely treatment response is quite appealing .
the goal of personalized medicine , however , is hindered because so much molecular etiology remains in the dark .
one way to explain more of the dark matter is to develop more efficient ways to use existing data .
they apply these ideas to a gwas of obesity , as measured by body mass index ( bmi ) .
studies estimate bmi 's heritability at 45 to 85% , but identified genetic variants explain about 1% of the total variance . to discover more variants , the authors examined gene expression of adipose tissue in a sample of monozygotic ( mz ) twins discordant for bmi and in a sample of unrelated individuals . because mz twins are genetically identical , or nearly so , the authors reasoned that genes showing expression differences between twins are ' reactive ' genes with differences that are due to regulatory or epigenetic changes in response to environmental factors .
by contrast , genes uncovered in unrelated individuals are a combination of reactive and genetically ' causal ' genes . by contrasting results from the unrelated sample and discordant mz twins , the authors identified 27 causal genes that were differentially regulated .
they then tested 197 single nucleotide polymorphisms ( snps ) falling in and around these genes in a sample of 21,000 subjects .
they discovered a significant excess of small p - values in this set of snps . neither the set of snps defined by reactive genes nor the individual snps in the reactive set were associated with bmi .
notably , this work identifies a new gene , f13a1 , which encodes the coagulation factor xiii a chain , with variation that affects bmi .
this gene has also been identified by meta - analysis of 12 studies of venous thromboembolism .
obesity is well known to predispose to vein thromboses ; however , the study of naukkarinen et al
. reveals a potential biological pathway for the relationship between obesity , thrombosis and cardiovascular risk .
the methods advanced by naukkarinen and colleagues require discordant mz twins , which were available for bmi .
this experimental design could prove highly informative for similar quantitative traits , for which extremes are easily identified and by which the pathology or phenotype of interest is defined . for some diseases , especially diseases of the brain , quantitative traits that map precisely onto risk are not yet available .
in addition , because reactive genes are environment - dependent , successful implementation of this design might require a sample exposed to a homogeneous environment , limiting its generality .
regardless , this study shows how innovative research can cast more light on dark matter .
moreover , the study design could also inform us about pathways of correlated gene expression and how much these correlations are influenced by genetic and environmental variation .
many other methods and designs are available to illuminate dark matter [ 8 - 15 ] .
one appealing approach teams gene - expression results with genome - wide association data to produce targeted hypothesis tests .
one possibility is to organize tests by expression quantitative trait loci affecting genes in pathways meaningful for the disease .
statistical methods for targeted testing are available , whether on the basis of prior information of the likelihood of an association between a snp and the phenotype or on the basis of plausible disease pathways .
genetic variants with parental origin effects , or whose effects depend on the parent from whom they were inherited , could be part of the dark matter ; methods are now available to determine the parental origin of alleles and haplotypes even in the absence of genotyped parents .
studies of copy number variants and their inheritance in families could also reveal insight into plausible biological pathways for disease .
it is also safe to say that rare variants account for some of the dark matter , possibly the majority of it in some cases .
next - generation sequencing promises to fill some of our void in knowledge by identifying more penetrant but rarer variants .
let 's reconsider human height . we know numerous rare variants and about 50 common variants that have an impact on height .
thus far , known genetics account for roughly 5% of the variance . using many snps from gwas analysis that are not significantly associated with height , yang et al .
estimated the proportion of variance in height explained by snps as 0.45 and even got close to the heritability estimate of 0.84 after correcting for incomplete linkage disequilibrium between snps genotyped and causal variants . in spirit ,
this approach is similar to the allele score method , which seeks a predictive model for disease status on the basis of thousands of snps with modest evidence for association .
if their results are correct , both studies suggest that the effects of snps are small and will be difficult or impossible to detect from simple analyses of gwass , at least for current sample sizes .
these intriguing approaches have some drawbacks : they shed no new light on the molecular etiology of phenotype ; and inherent in the calculations are assumptions that could prove difficult to validate .
there are other complex pathways for the transmission of a phenotype across generations without the transmission of a specific common or rare variant , namely through epigenetic factors that can result in the inheritance of gene expression patterns without an alteration of the dna sequence .
gene - environment interactions could also affect the estimates of heritability and when they are in play , they can explain as much of the variance in the phenotype as genetic factors .
concerted effort will almost surely be required to understand the genetic architecture of most complex diseases .
's novel study design illustrates the impact that concerted effort can have in advancing our knowledge of the genetic etiology of such diseases .
there remains ample room for novel analytic methods and study designs to shed light on the genetic dark matter of disease .
it is entirely possible , 10 years hence , that we will realize that much of the missing heritability was hiding in plain sight in common variants .
gwas : genome - wide association study ; mz : monozygotic ; snp : single nucleotide polymorphism
bd is associate professor of psychiatry and human genetics , university of pittsburgh school of medicine , pittsburgh .
nm is assistant professor of psychiatry , university of pittsburgh school of medicine , pittsburgh .
this work was supported by r01 grants ( mh057881 , bd ) and a k01 grant ( mh077930 , nm ) from the national institute of mental health .
the national institute of mental health did not participate in the preparation , review or approval of the manuscript . | discoveries from genome - wide association studies have contributed to our knowledge of the genetic etiology of many complex diseases . however , these account for only a small fraction of each disease 's heritability . here ,
we comment on approaches currently available to uncover more of the genetic ' dark matter , ' including an approach introduced recently by naukkarinen and colleagues .
these authors propose a method for distinguishing between gene expression driven by genetic variation and that driven by non - genetic factors .
this dichotomy allows investigators to focus statistical tests and further molecular analyses on a smaller set of genes , thereby discovering new genetic variation affecting risk for disease .
we need more methods like this one if we are to shed a powerful light on dark matter . by enhancing our understanding of molecular genetic etiology
, such methods will help us to understand disease processes better and will advance the promise of personalized medicine . |
the dominant role in muscle homeostasis and regeneration is played by satellite cells , which reside between the basal lamina and sarcolemma of myofibers .
satellite cells can be activated in response to both physiological stimuli , such as exercise , and under pathological conditions , such as injury and degenerative diseases , to generate a committed population of myoblasts that are capable of fusion and differentiation .
satellite cells are able to fuse with existing myofibers , repairing damaged muscle fibers , or alternatively fuse to each other to form new myofibers .
rt - pcr analysis , gene targeting strategies , and molecular imaging revealed that satellite cells present a heterogeneous profile of gene expression depending on the functional stage of the myogenic program .
it has been reported that quiescent satellite cells express several relevant markers such as c - met , m - cadherin , foxk , pax-7 , ncam , syndecan 3 and 4 , cd34 , caveolin-1 , sox 8 , sox 15 , and vcam-1 ( figure 1b ) .
pax3 , the paralog ( pair of genes that derive from the same ancestral gene ) of pax7 , is also expressed in quiescent muscle satellite cells in a subset of muscles and it plays an important role in regulating the entry of satellite cells into the myogenic programme . once activated , satellite cells up - regulate c - met , pax-7 , and m - cadherin and activate the expression factors involved in the specification of the myogenic program such as myf-5 and myod ( figure 1b ) .
the activated satellite cells proliferate as indicated by the expression of factors involved in cell cycle progression ( figure 1b ) and by incorporation of brdu and [ 3h ] thimidine .
ultimately the committed satellite cells fuse each other , or to the existing fibers , to form new muscle fibers , which are characterized by small size and by the expression of the embryonic / neonatal isoform of myhc ( figure 1b ) .
stem cell populations can participate to muscle regeneration , contributing to maintain the pool of satellite cells .
these stem cell populations could either reside within muscle , or recruited via circulation in response to homing signals secreted by injured skeletal muscle .
these populations include endothelial - associated cells , interstitial cells , and bone marrow - derived cells . at first sight
the origin of non muscle - derived stem cells appears to be mainly restricted to the hemo - vascular system ( hematopoietic , endothelial , pericytes ) .
a question relevant to muscle regeneration is whether there is any lineage relationship between one or more types of mesoderm stem cells and muscle satellite cells .
although there is not a conclusive answer to this question , it is possible that any of these cells may leave the vessel wall , enter the interstitial space and produce factors that stimulate the activity of satellite cells .
one of the critical points that remain to be addressed is the following : if skeletal muscle possesses a stem cell compartment , it is not clear why skeletal muscle fails to regenerate under pathological conditions .
either the resident muscle stem cells drastically decrease during aging and in several degenerative diseases or perhaps the pathological muscle is a prohibitive environment for stem cells activation and function .
although we lack definitive answers , several evidences suggested that with age or under pathological conditions , the systemic environment impinges the activity of satellite cells and stimulates fibrotic accumulation .
in particular , recent reports described the identification of muscle - derived interstitial cells , referred to as fibro - adipocyte progenitors ( faps ) . under physiologic regenerative stimuli ,
in contrast , in degenerating muscles , such as muscular dystrophy , these cells turn into fibro - adipocytes , which mediate fat deposition and fibrosis . the environmental niche that nurtures and maintains the stem cells at different anatomical sites and influences stem cell proliferation and differentiation represents a critical component of muscle regeneration .
indeed , heterochronic experiments demonstrated that old muscle successfully regenerates when transplanted in a young animal , whereas the regeneration of young muscle transplanted in an old host was impaired .
this hypothesis has been clearly validated by parabiotic experiments , the union of two organisms that share the circulatory system , demonstrating the rejuvenation of aged progenitor cells by exposure to a young systemic environment .
similarly , we also demonstrated that human satellite cells fail to differentiate when cultured in isochronic conditions .
immunofluorescence analysis for the expression of myhc revealed that aged satellite cells did not display major defect in the propensity to fuse when differentiated under standard conditions , namely in dmem supplemented with 5% horse serum .
of note , we observed that autologous serum ( isochronic culture conditions ) dramatically reduced muscle differentiation , which was partially rescued when aged satellite cells were differentiated in heterologous/ heterochronic serum ( from young donors ) .
these data suggest that with age , the systemic environment is less effective in maintaining the myogenic fate of muscle stem cells and , instead , facilitates conversion to a fibrogenic fate .
the modifications with aging in the secretome of the different cell types present at different phases of regeneration is probably then crucial , as is expression of relevant cell surface receptors , which can also change with age . among potential factors , notch , wnt , and insulin - like growth factor-1 ( igf-1 )
notch signaling is one of the major pathway that regulates the activation and expansion of the satellite cell lineage during embryogenesis , and in the adult .
the decline of notch signaling with age is thought to be one of the causes of the decreased regenerative potential of aged satellite cells . indeed forced activation of notch signaling in injured muscle of aged mice restores regenerative potential to that tissue . in addition , the temporal balance between notch and wnt signaling orchestrates the precise progression of muscle precursor cells along the myogenic lineage pathway , during postnatal myogenesis .
nevertheless , the wnt signal must be finely modulated , since a persistence of wnt signal , as , for example , observed in aged mice , can be associated with an increase in tissue fibrosis .
conversely , the myogenic - to - fibrogenic conversion has been abrogated by wnt inhibitors . among growth factors ,
insulin - like growth factor-1 ( igf-1 ) has been implicated in many anabolic pathways in skeletal muscle and it plays a central role during muscle regeneration . unlike other growth factors , the local isoform of igf-1 ( migf-1 ) also stimulates myogenic differentiation , generates a pronounced muscle hypertrophy , modulates the inflammatory response , and accelerates the functional rescue of injured skeletal muscle .
in addition , we observed that forced expression of migf-1 improves muscle mass and strength , decreases myonecrosis , and reduces fibrosis in dystrophic mice .
this is of particular relevance to the human dystrophic condition where virtually all skeletal muscles succumb to fibrosis .
in addition we recently demonstrated that muscle - specific expression of migf-1 can counter aspects of the muscular dystrophy associated with the loss of dystrophin , modulating relevant molecules of the genetic and epigenetic circuitries in the mdx dystrophic mouse model .
our work is consistent with a model in which overexpression of migf-1 confers robustness to dystrophic muscle , impedes the activation of a chronic inflammatory response , activates the circuitry of muscle differentiation and maturation .
another study also demonstrated that co - injection of the raav - microdystrophin and raav - migf-1 vectors resulted in increased muscle mass and strength , reduced myofiber degeneration , and increased protection against contraction - induced injury .
these results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin .
in addition , localized expression of migf-1 delays the onset and progression of disease in amyotrophic lateral sclerosis ( als ) mice , ameliorates muscle pathology and reduces motor neuron loss in a mouse model of spinal and bulbar muscular atrophy ( sbma ) , and improves muscle mass and extends the survival of severe spinal muscular atrophy ( sma ) mice .
interestingly , we recently demonstrated that igf-1 isoforms are also up - regulated in senior patients treated with electrical stimulation ( es ) , promoting satellite cells activation , reduction in the expression of muscle - specific atrophy - related ubiquitin ligase genes , and promoting the remodeling of extracellular matrix .
another potential factor that , similarly to igf-1 , plays important physiological roles in skeletal muscle development and homeostasis is the neurohypophyseal hormone vasopressin ( avp ) . in a recent study
, we demonstrated that muscle - specific stimulation of avp induces acceleration of satellite cell activation and earlier stimulation of specific pathways involved in muscle regeneration .
these findings demonstrate that while stem cells represent an important determinant for tissue regeneration , a qualified
thus , in several muscle pathologies the systemic environment is less effective in maintaining the myogenic fate of muscle stem cells and , instead , facilitates conversion to a fibrogenic fate . in this context
, therapeutic applications of adult stem cells to aged or pathological tissue repair , in the context of regenerative medicine , will require an increased understanding of stem - cell biology , environment of pathological tissue and interaction between the two . | capacity of adult muscle to regenerate in response to injury stimuli represents an important homeostatic process .
regeneration is a highly coordinated program that partially recapitulates the embryonic developmental program .
however , muscle regeneration is severely compromised in several pathological conditions .
it is likely that the restricted tissue repair program under pathological conditions is due to either progressive loss of stem cell populations or to missing signals that limit the damaged tissues to efficiently activate a regenerative program .
it is therefore plausible that loss of control over these cell fates might lead to a pathological cell transdifferentiation , limiting the ability of a pathological muscle to sustain an efficient regenerative process .
the critical role of microenvironment on stem cells activity and muscle regeneration is discussed . |
the quality of life is concerned with the degree to which a person enjoys the important possibilities of life. oral health has a major impact on the general quality of life .
people 's oral health status can affect them physically and psychologically and influence how they enjoy life ; how they look , speak , chew , taste , enjoy food , and socialize .
it also affects their self - esteem , self - image , and feelings of social well - being .
successful aging is related to maintaining one 's quality of life , which in turn is dependent on how well individuals can fulfill these day to day activities .
oral diseases seriously impair the quality of life in a large number of individuals and they may affect various aspects of their life , including function , appearance , interpersonal relationships , and career opportunities .
poor oral health not only has an effect on the general health , but also might restrict various day - to - day activities leading to loss of working hours .
oral health is an integral part of general health and health of the police personnel is of utmost importance , as they are personnel who work against crime and facilitate the people to live in peace and harmony with each other , and thus help the growth of the nation .
the mission of the police of a state is to help the common man , to provide security , and to create a peaceful and law - abiding community with his cooperation .
police personnel are a group of professionals , who have an altogether different working environment , with 24 hours duty , often being exposed to the highest form of physical strain and mental stress . on account of their odd working pattern ,
they often miss timely food , sleep , rest , recreation , and family contact . due to these factors , police personnel
this complicates their general as well as oral health , which in turn has an influence on their life and level of living . in india
, few studies have been carried out to assess the oral health status of police personnel , but very scanty literature exists on the impact of oral health on the oral health - related quality of life in police personnel .
the objective of the study was to assess the impact of caries prevalence on the oral health - related quality of life among police personnel in virajpet , south india .
prior permission was taken from the deputy superintendent of police ( dysp ) , virajpet , and the police sub inspector ( psi ) of the respective police station to conduct the study .
the virajpet taluk consists of six police stations , with a total of 296 police personnel .
the sampling technique used was simple random sampling , without replacement , for a sample size n = 296 .
a sample size of n = 172 was obtained by using the formula , where , e was the desired level of precision , which we had selected to be 5% .
the police personnel were explained about the study and informed consent was obtained from the study participants .
the questionnaire was typed both in the local language ( kannada ) and english , to ensure comprehensibility by all police personnel .
the internal reliability ( cronbach alpha ) of the questionnaire was tested in a pilot study done and was found to be acceptable ( 0.7 ) .
the first part of the questionnaire consisted of questions on the sociodemographic factors , which recorded the sex and age of the police personnel .
the second part of questionnaire was pertaining to the impact of oral health on the quality of life , which was measured by the oral health impact profile ( ohip-14 ) questionnaire .
each question in the ohip-14 questionnaire was answered on a five - point likert scale , with answers ranging from very often to never, and the possible scores ranged from 14 to 70 .
the questionnaires were distributed to the police personnel personally and they were given sufficient time to answer the questions , and the questionnaire was collected back on the same day . after receiving the filled questionnaires from the participants ,
a clinical examination was carried out by a trained , calibrated , single examiner and the caries experiences of the participants were recorded as per the who criteria ( 1997 ) .
the ethical approval for the study was obtained from the institutional ethical committee of the coorg institute of dental sciences , virajpet , south india .
the spss version 20.0 software for windows was employed for statistical analysis . the frequency distributions of the responses to the questions were produced .
in addition to descriptive statistics , pearson 's correlation was used to assess the relationship between the variables .
anova was used to compare the within - group differences of the selected sociodemographic factors in the study .
most of them were male ( n = 154 , 89.5% ) and only 18 participants ( 10.5% ) were females .
their mean age was 38.02 9.08 years and their age ranged from 20 to greater than 50 years .
as shown in tables 1 and 2 there were no significant differences between the within - group comparison of age , gender or oral health - related quality of life .
sixty - eight ( 39.5% ) of the study participants were happy with their teeth and 66 ( 38.4% ) participants were happy with the appearance of their teeth .
ninety - eight ( 57% ) of the study subjects responded that they never avoided smiling and laughing and 90 ( 52.3% ) never felt embarrassed due to their oral health . between and within - group comparison of age and oral health - related quality of life between and within - group comparison of gender and oral health - related quality of life distribution of respondents by question and score other questions , such as ,
any difficulty in pronouncing words , avoiding certain types of food , headaches , problems in any other part of their body because of problems with their mouth or teeth were asked , to which 115 ( 66.9% ) , 80 ( 46.5% ) , 91 ( 52.9% ) , and 109 ( 63.4% ) of the participants replied negatively .
fifty - five ( 32% ) participants answered that they could have meals properly and 51 ( 29.7% ) replied to being satisfied with their regular food habits .
out of 172 police personnel 128 ( 74.4% ) , 111 ( 64.5% ) , 117 68% ) , and 118 ( 68.6% ) acknowledged that they never avoided other people , oral health never affected their ability to work or their everyday activities , never felt that life has been less satisfactory , and they had never been totally incapable of functioning , respectively .
it is evident from the table 4 that oral health - related quality of life scores were negatively correlated with the caries prevalence score ( r = -0.173 ) and it was statistically significant .
correlation of total oral health - related quality of life scores of the subjects with caries prevalence ( dmft ) it can be observed from table 5 that there was no significant association between caries prevalence decayed , missing and filled teeth ( dmft ) and the oral health - related quality of life scores .
association between caries prevalence ( dmft ) and oral health - related quality of life scores
the fact that the police personnel are quite often engaged in 24-hour duty , puts them under a lot of physical as well as mental strain .
the present cross - sectional , self - administered questionnaire study was conducted in order to obtain data regarding the oral health of the police personnel of the virajpet taluk , as well as , the impact of this on their oral health - related quality of life .
the reliability score of the questionnaire was assessed from a pilot study and was found to be 0.7 , which is considered good .
the high prevalence of caries in the present study may be because police personnel stay for a long time in their working environment , thus giving less priority for oral health .
not many studies have been published for comparison with this population group . however , a study done on police personnel in haryana showed a lower caries prevalence among the police personnel in that region . in the present study , between and within - group comparison of age and oral health - related quality of life was not statistically significant , which was similar to the other studies . moreover , the comparison of gender and oral health - related quality of life was not statistically significant .
this result was similar to the other studies and was in contrast to the other studies . in the present study
the total women police personnel were less , and therefore , a difference may not have been found .
women and younger age groups , in particular , perceived oral health as impacting more strongly on their life quality , as compared to men and older people .
it is important to highlight the gender effect , as women have specific oral health needs and these must be addressed by the oral healthcare services .
conflicting evidence on the influence of sex on the perceptions of how oral health impacts the quality of life has been reported .
discerning sex differences in oral health is important and may facilitate understanding the differences in oral health practices , such as , service utilization , which requires further investigation .
police personnel are exposed to many different types of stress that may affect the sleep quality and physical health , such as physical injury , injuring others in the line of duty , and witnessing death or injuries to other officers and civilians .
the impact of this high rate of exposure on their emotional health has long been a significant public health concern .
in addition , police officers are exposed to chronic non - traumatic stress arising from the demands of their work environment .
therefore , it can be hypothesized that police officers engaged in operational activities , which include exposure to danger and risk of life , would be more stressed .
police officers carrying out operational duties , being more exposed to confrontation , violence , and day - to - day involvement in a variety of traumatic incidents , would present a greater prevalence of emotional stress - and consequently bruxism - than those who do organizational work and are less subject to risks .
it can be observed that oral health - related quality of life was negatively correlated with caries prevalence ( r = 0.173 ) .
however , no significant association was observed between the prevalence of dental caries and quality of life scores among the police personnel .
this is because , although they had high caries prevalence , probably the extent of the caries was less severe , so the impact was less on their oral health - related quality of life .
therefore , further studies have to be carried out for a better understanding of the impact of dental caries on the quality of life .
furthermore , the very cross - sectional design of the study limits the validity of the study . keeping this in view
, further longitudinal studies have to designed and carried out for a better understanding of the impact of stress on dental caries in this population group .
the present study showed that there was no association with oral health - related quality of life and caries prevalence among police personnel in virajpet .
the sample of police personnel provided a unique opportunity to study a large population from diverse socioeconomic and geographical backgrounds .
hence , their health was of utmost importance , not only for them and their families , but also for the entire nation .
prevention oriented health education lectures should be delivered , and possibly , should also form part of police training curriculum .
prevention oriented health education lectures should be delivered , and possibly , should also form part of police training curriculum . | objective : the objective of this study is to assess the impact of caries prevalence on oral health - related quality of life among police personnel in virajpet , south india.materials and methods : police personnel were randomly selected from the 296 police staff working in virajpet , india . they were invited for a dental examination and a questionnaire survey .
a self - administered questionnaire was used to collect their demographic information , and to determine the oral health - related quality of life ( ohrqol ) .
caries experiences of the participants were recorded as per the world health organization ( who ) criteria ( 1997 ) .
the analysis of variance ( anova ) was used to compare within - group differences of the selected sociodemographic factors and the chi - square analysis was used to explore the association between the variables.results:all the 172 invited participants joined this study .
their mean age was 38.02 9.08 years .
there was no significant difference in oral health - related quality of life scores according to gender and age .
the prevalence of dental caries was found to be 78% in the current study .
data analysis showed that there was no statistically significant association between the oral health - related quality of life scores and caries prevalence.conclusion:the present study showed that there was no association between the oral health - related quality of life and caries prevalence among the police personnel in virajpet . |
primary acquired nasolacrimal duct obstruction ( pando ) describes a condition of nasolacrimal duct obstruction caused by inflammation or idiopathic fibrosis.1
distal obstruction causes stagnation of lacrimal secretion , which may promote bacterial colonization and lacrimal sac infection .
the clinical spectrum of nasolacrimal duct obstruction ranges from simple epiphora to acute or chronic dacryocystitis .
acute dacryocystitis is the acute inflammation of lacrimal sac with localized erythema and tenderness of the skin overlying lacrimal sac area .
chronic dacryocystitis contains purulent or mucoid material in the lacrimal sac , which regurgitates on irrigation or pressure over sac area .
there have been several previous studies regarding the microbiology of lacrimal duct obstruction causing chronic and acute dacryocystitis.24 the objective of this study was to describe the microbiology of nasolacrimal duct obstruction and to determine the susceptibility of the isolated organisms to common antimicrobial agents .
ninety - three patients ( 100 eyes ) diagnosed with pando between august 2008 and april 2011 were prospectively enrolled .
all patients underwent a complete ophthalmic examination with lacrimal drainage system irrigation and intranasal examination .
patients younger than 20 years , patients with canalicular obstruction , secondary nasolacrimal duct obstruction , ocular , and adnexal infection other than dacryocystitis ( such as canaliculitis , hordeolum , blepharitis , conjunctivitis , keratitis , and endophthalmitis ) , and patients with a history of topical or systemic antibiotic usage within 1 week were excluded from the study . written informed consent
patients were categorized into acute dacryocystitis , chronic dacryocystitis , and simple epiphora subgroups based on the following clinical findings .
acute dacryocystitis was diagnosed in patients with tenderness , erythema , and swelling in the lacrimal sac area .
chronic dacryocystitis was diagnosed in patients with regurgitation of mucoid or mucopurulent discharge on the application of pressure over the lacrimal sac area or on the irrigation of lacrimal drainage system .
simple epiphora was diagnosed if the patient had pando without any clinical signs of infection of the lacrimal drainage system and no discharge on the application of pressure over the lacrimal sac area or on lacrimal irrigation .
lacrimal sac contents of all 100 eyes were examined for the presence of bacteria and fungus using culture - based techniques . in patients with acute dacryocystitis who required incision and drainage , the contents of the lacrimal sac
were directly collected using sterile cotton wool swabs during the incision and drainage procedure , taking care not to touch the skin and wound margin . in the remaining patients ,
the samples were intraoperatively collected using sterile technique prior to dacryocystorhinostomy by irrigating the lacrimal drainage system with sterile saline and aspirating the contents from the lacrimal sac via the punctum .
each sample was inoculated into 5% sheep blood agar and incubated in an anaerobic jar .
samples were also inoculated into a second plate of 5% sheep blood agar , a chocolate agar plate , and a thioglycollate broth and incubated aerobically .
fungi were cultured on sabouraud s agar , with and without chloramphenicol , at 25c for 14 days .
antimicrobial susceptibility of bacteria was tested by the disk diffusion method according to the clinical and laboratory standards institute guidelines.5 this study followed the tenets of the declarations of helsinki and was carried out with approval from the siriraj institutional review board .
the continuous data were presented by mean and range . the chi - square test and fisher s exact test
were used to compare the differences in categorical data between subgroups . the p - value < 0.05 was considered statistical significance for all statistical tests .
the continuous data were presented by mean and range . the chi - square test and fisher s exact test
were used to compare the differences in categorical data between subgroups . the p - value < 0.05 was considered statistical significance for all statistical tests .
cultures were obtained from 93 patients ( 100 eyes in total ) diagnosed with pando .
the mean age was 59.2 years ( range 2089 years ) , and the majority of patients were female ( 81/93 , 87% ) .
fifty and 36 patients had right- and left - side infections , respectively , whereas seven patients had bilateral nasolacrimal duct obstruction .
the mean duration of symptoms was 26.0 months ( range 5 days to 22 years ) . among the100 samples
, positive cultures were obtained from 79 samples ( 79% ) . of the culture - positive samples , 47 ( 59.5% ) yielded a single organism , while 32 ( 40.5% ) had two to five mixed organisms . in total , 127 organisms were isolated , and 29 different species were identified .
the majority of microorganisms were gram - positive bacteria ( 45 samples , 57.0% of all positive culture samples ) , whereas gram - negative bacteria , anaerobic bacteria , and fungi were found in 39 ( 49.4% ) , 24 ( 30.4% ) , and four samples ( 5.1% ) , respectively .
the most commonly isolated microorganisms were coagulase - negative staphylococci ( 22 samples , 27.8% ) .
nonspore - forming gram - positive anaerobic rods , pseudomonas aeruginosa , and haemophilus influenzae were isolated from 14 ( 17.7% ) , 12 ( 15.2% ) , and eleven samples ( 13.9% ) , respectively .
the most frequent gram - positive isolate was a group of coagulase - negative staphylococci , which represented 17.3% of all isolates and 43.1% of the gram - positive group .
the two most frequent gram - negative isolates were p. aeruginosa and h. influenzae , which represented 9.4% and 8.7% of all organisms and 27.3% and 25.0% of the gram - negative group , respectively .
nonspore - forming gram - positive rods were the most frequent anaerobic bacteria and represented 50.0% of the anaerobe group and 11% of the total number of isolates .
patients were divided into three subgroups ( acute dacryocystitis , chronic dacryocystitis , and simple epiphora ) . of all 100 eyes ,
five , 45 , and 50 had acute dacryocystitis , chronic dacryocystitis , and simple epiphora , respectively .
the number of samples in the acute dacryocystitis subgroup was too small to provide adequate statistical power ; therefore , we analyzed this subgroup separately and compared the distribution of microorganisms between the chronic dacryocystitis and simple epiphora subgroups only .
gram - positive ( three samples ) , gram - negative ( three samples ) , and anaerobic ( three samples ) bacteria were cultured , but no fungi were detected in this subgroup .
the most commonly isolated bacterium was h. influenzae , which was isolated from two samples and represented 40% of the culture samples in the acute dacryocystitis subgroup .
the types of organisms isolated in each subgroup in the comparison between the simple epiphora and chronic dacryocystitis subgroups are presented in table 2 and figure 1 .
gram - negative organisms in the chronic dacryocystitis subgroup were isolated significantly more frequently than in the simple epiphora subgroup ( p=0.012 ) .
ciprofloxacin was the most effective agent ( 93.8% ) against all gram - positive and gram - negative organisms .
the most effective agent against gram - positive organisms was vancomycin ( 100% of isolates susceptible ) , followed by ciprofloxacin and chloramphenicol ( 93.3% ) .
all staphylococcus aureus isolates were susceptible to oxacillin , ciprofloxacin , chloramphenicol , co - trimoxazole , clindamycin , and vancomycin .
all streptococcus pneumoniae isolates were susceptible to vancomycin , while 85.7% , 85.7% , 71.4% , and 28.6% of these isolates were susceptible to ciprofloxacin , chloramphenicol , penicillin , and co - trimoxazole , respectively .
all gram - negative bacteria were susceptible to ceftazidime , and 93.9% were susceptible to ciprofloxacin .
all p. aeruginosa isolates were susceptible to ceftazidime , ciprofloxacin , gentamicin , and meropenem .
all h. influenzae isolates were susceptible to ceftazidime , chloramphenicol , and ciprofloxacin , whereas 90.9% were susceptible to ampicillin and amoxicillin / clavulanate , and 54.5% were susceptible to co - trimoxazole .
cultures were obtained from 93 patients ( 100 eyes in total ) diagnosed with pando .
the mean age was 59.2 years ( range 2089 years ) , and the majority of patients were female ( 81/93 , 87% ) .
fifty and 36 patients had right- and left - side infections , respectively , whereas seven patients had bilateral nasolacrimal duct obstruction .
the mean duration of symptoms was 26.0 months ( range 5 days to 22 years ) .
among the100 samples , positive cultures were obtained from 79 samples ( 79% ) . of the culture - positive samples , 47 ( 59.5% ) yielded a single organism , while 32 ( 40.5% ) had two to five mixed organisms . in total , 127 organisms were isolated , and 29 different species were identified .
the majority of microorganisms were gram - positive bacteria ( 45 samples , 57.0% of all positive culture samples ) , whereas gram - negative bacteria , anaerobic bacteria , and fungi were found in 39 ( 49.4% ) , 24 ( 30.4% ) , and four samples ( 5.1% ) , respectively .
the most commonly isolated microorganisms were coagulase - negative staphylococci ( 22 samples , 27.8% ) .
nonspore - forming gram - positive anaerobic rods , pseudomonas aeruginosa , and haemophilus influenzae were isolated from 14 ( 17.7% ) , 12 ( 15.2% ) , and eleven samples ( 13.9% ) , respectively .
the most frequent gram - positive isolate was a group of coagulase - negative staphylococci , which represented 17.3% of all isolates and 43.1% of the gram - positive group .
the two most frequent gram - negative isolates were p. aeruginosa and h. influenzae , which represented 9.4% and 8.7% of all organisms and 27.3% and 25.0% of the gram - negative group , respectively .
nonspore - forming gram - positive rods were the most frequent anaerobic bacteria and represented 50.0% of the anaerobe group and 11% of the total number of isolates .
patients were divided into three subgroups ( acute dacryocystitis , chronic dacryocystitis , and simple epiphora ) . of all 100 eyes ,
five , 45 , and 50 had acute dacryocystitis , chronic dacryocystitis , and simple epiphora , respectively .
the number of samples in the acute dacryocystitis subgroup was too small to provide adequate statistical power ; therefore , we analyzed this subgroup separately and compared the distribution of microorganisms between the chronic dacryocystitis and simple epiphora subgroups only .
gram - positive ( three samples ) , gram - negative ( three samples ) , and anaerobic ( three samples ) bacteria were cultured , but no fungi were detected in this subgroup .
the most commonly isolated bacterium was h. influenzae , which was isolated from two samples and represented 40% of the culture samples in the acute dacryocystitis subgroup .
the types of organisms isolated in each subgroup in the comparison between the simple epiphora and chronic dacryocystitis subgroups are presented in table 2 and figure 1 .
gram - negative organisms in the chronic dacryocystitis subgroup were isolated significantly more frequently than in the simple epiphora subgroup ( p=0.012 ) .
ciprofloxacin was the most effective agent ( 93.8% ) against all gram - positive and gram - negative organisms .
the most effective agent against gram - positive organisms was vancomycin ( 100% of isolates susceptible ) , followed by ciprofloxacin and chloramphenicol ( 93.3% ) .
all staphylococcus aureus isolates were susceptible to oxacillin , ciprofloxacin , chloramphenicol , co - trimoxazole , clindamycin , and vancomycin .
all streptococcus pneumoniae isolates were susceptible to vancomycin , while 85.7% , 85.7% , 71.4% , and 28.6% of these isolates were susceptible to ciprofloxacin , chloramphenicol , penicillin , and co - trimoxazole , respectively .
all gram - negative bacteria were susceptible to ceftazidime , and 93.9% were susceptible to ciprofloxacin .
all p. aeruginosa isolates were susceptible to ceftazidime , ciprofloxacin , gentamicin , and meropenem .
all h. influenzae isolates were susceptible to ceftazidime , chloramphenicol , and ciprofloxacin , whereas 90.9% were susceptible to ampicillin and amoxicillin / clavulanate , and 54.5% were susceptible to co - trimoxazole .
pando affects women more frequently than men , with a 3:1 female preponderance.6 in this study , 81 patients were female and 12 were male ( 6.7:1 ) .
measurement of the bony nasolacrimal duct system has revealed that women have significantly smaller lower nasolacrimal fossa and middle nasolacrimal ducts , which might explain the higher prevalence of pando in women.7 sample collection techniques vary between published studies , which might influence the levels of overall culture positivity .
hartikainen et al collected material refluxed through the lacrimal punctum , or by wiping a broth - moistened swab across the lower conjunctival cul - de - sac , and reported positive cultures in 84% of 127 samples.3 deangelis et al analyzed the posterior lacrimal flap and found only 41.7% of the 132 samples were culture positive.4 in the present study , 79% of the samples were culture positive .
these samples were obtained directly from the lacrimal sac through the irrigating syringe without a lid , or conjunctival contamination under sterile surgical technique in the simple epiphora and chronic dacryocystitis subgroups .
five samples in the acute dacryocystitis subgroup were obtained by using a sterile cotton swab to collect content during the incision and drainage procedure .
deangelis et al reported that 78.5% and 21.5% of the organisms from 132 samples were gram - positive and gram - negative bacteria , respectively , and 76.5% of the gram - positive bacteria were staphylococci.4 hartikainen et al found that 69% and 17% of all organisms isolated from 127 samples were gram - positive and gram - negative bacteria , respectively and that the most frequently cultured gram - positive and gram - negative species were staphylococcus epidermidis and h. influenzae , respectively.3 in this study , although gram - positive bacteria were the most common microorganisms ( 40.2% of all organisms ) , gram - negative bacteria were isolated in almost equal proportions ( 34.6% ) , which is in agreement with the study of pinar - sueiro et al who found 49.1% of gram - positive and 43.9% of gram - negative bacteria.8 these findings may show an increasing trend toward gram - negative bacteria in nasolacrimal duct obstruction .
patients in this study were clinically categorized into three subgroups : simple epiphora , chronic dacryocystitis , and acute dacryocystitis .
all samples in the acute dacryocystitis subgroup were culture positive ; however , the sample size was too small ( five samples ) for accurate statistical analysis .
there was no statistically significant difference in the proportion of positive cultures obtained from the simple epiphora ( 74% ) and chronic dacryocystitis subgroups ( 82.2% ) .
this is consistent with the report of deangelis et al , which showed that the presence of a positive culture result was independent of a history of dacryocystitis or the presence of a mucocele.4 owji and khalili also reported that the mean colony count of microbial organisms was significantly higher in lacrimal sac and conjunctival culture of the obstructed side than the normal side .
therefore , this study confirms the importance of routine preoperative irrigation before intraocular surgery to avoid potentially pathogenic bacteria.9 interestingly , in this study , we demonstrated a predominance of gram - negative bacteria in the chronic dacryocystitis subgroup ( 46.6% of isolates ) , in contrast to the previous studies of chronic dacryocystitis , which showed that most microorganisms were gram - positive bacteria.2,1013 however , briscoe et al demonstrated a higher incidence of gram - negative bacteria , particularly pseudomonas , in patients with purulent dacryocystitis.14 in this study , the prevalence of gram - negative organisms was significantly higher in the chronic dacryocystitis subgroup than in the simple epiphora subgroup ( p=0.012 ) . this result is consistent with the studies that have been described by hartikainen et al and amin et al.3,15 therefore , antimicrobial drugs against gram - positive and gram - negative bacteria should be administered in chronic dacryocystitis . in this study ,
anaerobic bacteria were found in all subgroups ( 22% of all isolates ) and were represented mostly by nonspore - forming gram - positive rods and propionibacterium acnes .
anaerobic bacteria were present in 13%19% of samples in previous studies , and propionibacterium species were predominant.3,13 in contrast , blicker and buffam did not detect any anaerobic bacteria in patients with chronic dacryocystitis.16 all gram - positive bacteria were sensitive to vancomycin , and 93.3% were sensitive to ciprofloxacin and chloramphenicol .
in contrast , all gram - negative bacteria were sensitive to ceftazidime , and 93.9% were sensitive to ciprofloxacin .
thus , ciprofloxacin was the most effective agent ( 93.8% ) against both gram - positive and gram - negative bacteria .
these findings suggest that ciprofloxacin was effective in vitro against both gram - positive and gram - negative bacteria in this study and may be an initial treatment for chronic dacryocystitis because of its relatively high activity for most commonly found gram - positive and gram - negative bacteria .
most patients with pando , with or without clinical signs of lacrimal infection , were culture positive .
the proportion of gram - negative organisms was high in patients with nasolacrimal duct obstruction , especially in the chronic dacryocystitis subgroup ; this may show an increasing trend toward gram - negative bacteria in nasolacrimal duct obstruction or reflect our geographic environment . | purposethe aim of this study was to determine the microbiology of primary acquired nasolacrimal duct obstruction ( pando ) and its antimicrobial susceptibilities.methodsninety-three patients ( 100 eyes ) diagnosed with pando , categorized as acute , chronic dacryocystitis , or simple epiphora , were prospectively enrolled .
lacrimal sac contents were cultured for aerobic and anaerobic bacteria and fungi .
cultured organisms were identified , and antimicrobial susceptibility testing was performed for aerobic bacteria.resultsseventy-nine of the 100 samples were culture positive .
one hundred twenty - seven organisms were isolated , and 29 different species were identified .
most microorganisms were gram - positive bacteria ( 45 samples or 57.0% of all positive culture samples ) , whereas gram - negative bacteria , anaerobic bacteria , and fungi were found in 39 ( 49.4% ) , 24 ( 30.4% ) , and four samples ( 5.1% ) , respectively .
the most frequently isolated group was coagulase - negative staphylococci ( 27.8% ) , followed by nonspore - forming gram - positive rods ( anaerobe ) ( 17.7% ) and pseudomonas aeruginosa ( 15.2% ) . of the 100 samples , five , 45 , and 50
samples were obtained from patients with acute dacryocystitis , chronic dacryocystitis , and simple epiphora , respectively .
subgroup analysis showed that gram - negative organisms were isolated more frequently from the chronic dacryocystitis subgroup than from the simple epiphora subgroup ( p=0.012 ) .
antimicrobial susceptibility testing demonstrated that ciprofloxacin was the most effective drug against all gram - positive and gram - negative organisms.conclusionpatients with pando , with or without clinical signs of lacrimal infection , were culture positive .
gram - negative organisms were frequently isolated , which were different from previous studies .
ciprofloxacin was the most effective agent against all gram - positive and gram - negative organisms . |
primary hepatic lymphoma ( phl ) refers to tumor confined in the liver at the early stage of lymphoma without infiltration of other locations .
phl is a rare disease , with an incidence of only 0.1% for malignant liver tumors .
this disease also accounts for 0.4% of all primary extranodal lymphoma and 0.016% of all non - hodgkin 's lymphoma .
phl of diffuse large b - cell lymphoma ( phl - dlbcl ) is more infrequent .
hence , the condition is difficult to distinguish from primary liver cancer , liver metastases , granulomatous pseudotumor , and other diseases and is thus easily misdiagnosed . to improve clinicians ' understanding of this disease ,
the diagnosis and treatment course of a case of phl in our hospital were discussed in detail .
a 61-year - old male was admitted because of fatigue and abdominal distension for over 50 days .
the patient presented with a 6 kg weight loss ( 12% ) and experienced fatigue and abdominal distension from an unknown cause since 50 days prior to admission .
the patient showed no signs of skin pruritus or yellow discoloration of the skin or eyes . despite his symptoms ,
he was then admitted in a local hospital from june 6 to june 12 , 2014 because of fever .
the patient suffered from chills and sweating , and his body temperature fluctuated at 37.438.9c .
he was then treated with cefoperazone / sulbactam sodium for the inflammation , pantoprazole to relieve hyperacidity , and polyene phosphatidylcholine to protect the liver , all of which afforded the patient with no improvement of condition .
the patient sought consultation in one of the top three local hospitals on june 19 .
his signs and symptoms of fatigue , abdominal distension , and yellow urine aggravated , along with the decrease in urine amount . at the time
, the patient presented with a urine output of less than 500 ml/24 h , with concurrent skin pruritus , icteric skin and sclerae , occasional cough , and severe bilateral lower extremities edema .
he also experienced slight chest tightness and hence was admitted to the hospital . upon admission
, routine blood examination obtained the following results : white blood cell count , 9.3 10/l ; neutrophil absolute value , 4.9 10/l ; hemoglobin , 128
g / l ; alt , 128 u / l ; ast , 109 u / l ; total bilirubin , 12.5 mol / l ; direct bilirubin , 2.7 mol / l ; indirect bilirubin , 125 mol / l ; hydroxybutyrate dehydrogenase , 1093 u / l ; lactate dehydrogenase , 1387 u / l ; and total bile acids ( tba ) , 2.461 mol / l .
the liver fibrosis index is as follows : hyaluronic acid , 1162.2 g / l ; type iv collagen , 279.4 g / l ; type iii collagen , 659.8 g / l ; and laminin , 149 g / l .
mol / l , whereas that of c - reactive protein was 136.4 mg /
hepatitis b screening tested positive for three components : hbv - dna , 3.1e + 02 copy / ml .
chest computed tomography ( ct ) scan revealed the following findings : ( 1 ) small fibrotic foci at the middle lobe of the right lung and lingular segment of the superior lobe of the left lung and ( 2 ) emphysema in the superior lobe of the right lung and calcified foci at the superior lobe of the left lung .
no swollen lymph nodes were found by ultrasound on the bilateral neck , supraclavicular , subaxillary , and inguinal regions . abdominal ct ( july 11 , 2014 ) displayed ( 1 ) hepatosplenomegaly , ascites , patchy low - density shadows in the liver ( especially in the left liver ) , and multiple low - density lesions in the right lobe of liver ; ( 2 ) gallbladder wall thickening ; and ( 3 ) prostate calcification ( figure 1 ) .
the patient was treated with entecavir dispersible tablet 0.5 mg qd , oral administration of antiviral drugs , and drugs for liver protection and diuresis .
pathological examination showed the following results : heteromorphic lymphocytes were observed in the hepatic sinusoid and portal area .
for immunohistochemistry , lca , cd20 , and bcl-6 were diffusely strongly positive ; cd79a and mum-1 were locally weakly positive ; the ki-67 labeling index was about 60% ; and ck , cd68 , cd34 , hepaar-1 , cd3 , cd43 , cd10 , and alk were negative ; hbs - ag and hbc - ag were negative ( figure 3 ) .
bone marrow morphology and biopsy showed no signs of lymphoma infiltration . on the basis of clinical , laboratory , and pathological examinations ,
the patient was diagnosed with primary hepatic non - hodgkin 's lymphoma staged iva ( dlbcl , non - gcb ) , chronic viral hepatitis ( b ) , and decompensated cirrhosis .
chop ( cyclophosphamide , doxorubicin , vincristine , and prednisone ) scheme chemotherapy was then initiated on july 17 with the following agents : ifo , 2 g ( days 1 and 8) ; thp , 60 mg ( day 1 ) ; dxm , 10 mg ( days 1 to 5 ) ; and vds , 4 mg ( days 1 , 8 , and 15 ) .
the ct review after the first chemotherapy ( august 11 ) showed that the liver significantly decreased in size , and liver density became uniform ( figure 2 ) .
the r - chop scheme was adopted for six cycles ( chop with rituximab injection , 500 mg [ day 0 ] ) ; the other drugs included were the same as those mentioned above ) .
no new lesion was noted , and the patient has lived for nearly 2 years till now of disease - free - survival .
primary hepatic lymphoma ( phl ) refers to the lesion only confined to the liver at the early stage of lymphoma .
infiltrations of lymph nodes , spleen , bone marrow , and other organs must be excluded at the onset .
the patient 's clinical characteristics include abdominal distension , fatigue , abnormal liver function , and progressive hepatomegaly .
the involvement of other locations was excluded by superficial lymph node examination , bone marrow morphology and biopsy , and radiologic examinations .
phl may occur at any age but more commonly in males of about 50 years old .
the male - to - female ratio for the disease incidence is about 3 : 1 .
however , primary liver non - hodgkin 's lymphoma is extremely rare , accounting for 0.4% of all primary extranodal lymphoma and 0.016% of all cases of non - hodgkin 's lymphoma .
the etiology of phl is uncertain and may include virus infection ( hiv , aids , hbv , hcv , and ebv ) , autoimmune diseases , and immune inhibitor application .
its mechanism potentially involves t lymphocyte loss of inherent immune surveillance and function after viral infection or application of immunosuppressive agents .
most patients seek consultation because of fatigue , loss of appetite , night sweats , low - grade fever , and weight reduction .
several patients suffer from hepatic encephalopathy early , resulting in coma and even death [ 2 , 4 ] . in our case ,
the patient experienced abdominal distension and fatigue and sustained low fever , hepatomegaly , and progressive increase of bilirubin , which were rapidly relieved by treatment .
the patient 's clinical characteristics were consistent with that of primary hepatic non - hodgkin 's lymphoma . for laboratory indices ,
most phl patients show elevated alt and ast levels by two to three times of the normal value , which is also significantly increased with the increase in bilirubin and hdl
. however , afp and cea levels are usually normal , in contrast to those in primary liver cancer and liver metastasis .
some patients sustain diffuse liver lesions , displayed in radiologic examination as hepatomegaly without liver space - occupying lesion .
the central mass mainly shows low - density shadows in ct and sometimes multilobar or patchy low - density shadows , which are peripherally intensified after enhancement .
this result may be explained by the lesser vascularization of the lymphoma compared with other tumors , resulting in tissue necrosis during the disease process .
the radiologic performance of the patient showed that the liver was obviously enlarged and the liver texture was not uniform , combined with patchy low - density shadows , which was consistent with literature reports [ 5 , 6 ] .
most hepatic lymphomas are derived from b - lymphocyte cells , whereas the minority originates from t lymphocytes .
phl patients with liver function abnormality , hepatomegaly , or space - occupying lesions but with normal afp and cea must be suspected of liver lymphoma .
however , tissue necrosis is often associated with liver lymphoma ; hence , fine needle aspiration biopsy may provide false negative results .
phl was often misdiagnosed as other conditions , such as embryonic sarcoma , inflammatory pseudotumor , or granulomatous hepatitis .
cellular immunology , gene rearrangement , cell genetics , and molecular biology related to phl should be explored to assist diagnosis and determine prognosis .
however , with the continuous development of chemotherapeutic regimens , especially the application of targeted therapeutic drugs , chemotherapy or combined local radiotherapy has become the first - line treatment of phl .
our patient was diagnosed with phl - dlbcl . the single - course treatment with chop combined with rituximab was significant .
the liver was significantly reduced in size , and the low - density lesions were significantly decreased . in a previous study , the follow - up visits of 24 phl cases after 20 years by the anderson cancer center in the united states revealed a complete remission rate of 85% through chemotherapy and an event - free five - year survival of 70% .
a considerable number of prospective , randomized controlled clinical trials are lacking and must be further conducted . | objective .
primary hepatic lymphoma is a rare disease . and the clinical manifestations of this disease are nonspecific .
the objective of this paper is to improve clinicians ' understanding of this disease
. methods .
we analyzed the clinical characteristics of a case of primary hepatic lymphoma in association with hepatitis b virus infection and reviewed the literature .
conclusion .
the clinical manifestations of primary hepatic lymphoma are nonspecific . and it is easily misdiagnosed .
postoperative radiotherapy of patients with early stage was previously speculated to achieve favorable improvement .
the application of targeted therapeutic drugs , chemotherapy , or combined local radiotherapy has become the first - line treatment strategy . |
estimating the total species richness within large communities of species , using only samplings limited in sizes , is a common , long standing challenge which has elicited numerous procedures of estimations .
for a few decades , a series of so - called nonparametricestimators provide elegant and convenient solutions to this question .
these new estimators are ordinarily simple format formulations which , moreover , require no specific assumption regarding the statistical distribution of the abundances of species ( which , thus , makes these formulations nonparametric ) . within this category of formulations ,
anne chao proposed a very popular nonparametric expression , which actually stands among the most commonly used estimators of the total species richness of a sampled community .
let be the number of species that are missed by ( i.e. , unrecorded within ) the limited sampling of a large community of species . then , according to chao 's formulation ,
( 1)=f122f2
with f1 and f2 as the numbers of species encountered only once and twice , respectively , in the sample .
this expression was later generalised as
( 2)=f1x(x!fx)(1/(x1 ) )
with fx standing for the number of species recorded x times in the sample .
these expressions , derived on a statistical basis , provide a lower - bound estimate of the number of missing species in a sampled community [ 1 , 2 ] .
that is , chao 's formulation is expected to provide only a minimal threshold ( in the statistical sense ) for the estimated species richness . yet , several decades of practice ( especially in the field of ecological purposes and biodiversity surveys ) call for placing chao 's expression among the most valuable and reliable estimators [ 37 ] since , in many occasions , this expression may well appear to provide approximatelycentered rather than only lower - bound estimates . in short , although designed conceptually as a lower - bound evaluation , chao 's formulation may , nonetheless , fairly often provide rather centered estimates in the common practice . hereafter
, we address this apparent paradox and propose new insights and argumentations , issued from an alternative , algebraic derivation of the originally statistically derived formulation by chao .
we demonstrate ( see appendix a for mathematical details ) that the general expression of chao 's estimator , established originally on a statistical basis , may also admit , alternatively , an algebraic derivation , leading to exactly the same expression as the statistically derived formulation = [ f1/(x!fx ) ] . yet , while the statistical derivation of chao 's formulation requires no particular restriction ( nonparametric ) , the algebraic derivation , implies a particular shape for the expected decrease of the proportion /s of missing ( i.e. , unrecorded ) species when the sample size n increases .
in fact , as demonstrated in appendix a , this asymptotic decrease should conform to a negative exponential :
( 3)s = exp(kn )
with(i)s as the species richness , that is , the total ( unknown ) number of species of the community,(ii)k as a constant,(iii)n as the sample size , that is , the number of individuals recorded in the sample .
s as the species richness , that is , the total ( unknown ) number of species of the community ,
n as the sample size , that is , the number of individuals recorded in the sample .
in turn , this particular form of the decrease of the number of missing species with enlarging sample sizes constrains the shape of the species abundance distribution ( the sad , that is , the distribution of species abundances when species are conventionally ranked by decreasing order of abundance ) . according to ( 3 ) , the number
r of recorded species in the sample is
( 4)r = s=s1expkn .
let k be the number of individuals belonging to the less abundant species among the r species recorded in the sample ( i.e. , the species of rank r when species are ranked by decreasing order of abundance ) .
then , the relative abundance ar of the species of rank r is expected to be inversely proportional to n , as
( 5)ar = kn .
according to ( 4 ) and ( 5 ) ,
( 6)r = s1expkkar .
according to the sample size n
, every species may be called to play the role of the less abundant species within the sample ( since , by continuously decreasing the size n of the sample , each species of the community [ including , at last , the most common ] would successively play the role of the least abundant species in the sample ) .
this equation thus describes the shape of the species abundance distribution ( ai = f(i ) ) when the proportion of unrecorded species in a sample is exponentially decreasing with the sample size , that is , the shape of the species abundance distribution which conditions the validity of the algebraic derivation of chao estimator .
as the algebraic derivation is deterministic by essence , it therefore provides a centered estimate of and of the resulting total species richness s , instead of being only a lower - bound estimate , as is the case in the nonparametric context .
as mentioned above , the algebraic derivation of chao 's formulation requires that the sampled community satisfies , at least approximately , the particular type of sad defined by ( 7 ) and illustrated at figure 1 .
this restrictive condition is the price to be paid for the more accurate estimate , namely , the loss of the strict nonparametric character of the statistically based conception . yet , this condition assigned to the shape of the distribution of species abundances might not be so restrictive in practice , at least as a first approximation .
reasons for this may be as follows : ( a)an asymptotic decrease to zero of with n ( equation ( 3 ) ) seems logical and intuitive , because aiming to estimate the total number of species in a community implicitly requires that this number does exist and might be actually reached progressively with sampling size n increasing continuously ; ( b)among the different types of accumulation curves with such an asymptotic evolution , the negative exponential answer of to increasing sampling size is , admittedly , one among the most simple , robust , and seemingly common [ 8 , 9 ] ; ( c)the sigmoidal shape of the prescribed sad ( equation ( 7 ) and figure 1 ) is not so far from the most classically referred empirical types , broken - stick and log - normal distributions . yet
for example , the equation i = f(ai ) for the sad corresponding to a broken - stick distribution is i s exp(s ai ) , which is formally different from ( 7 ) .
an asymptotic decrease to zero of with n ( equation ( 3 ) ) seems logical and intuitive , because aiming to estimate the total number of species in a community implicitly requires that this number does exist and might be actually reached progressively with sampling size n increasing continuously ; among the different types of accumulation curves with such an asymptotic evolution , the negative exponential answer of to increasing sampling size is , admittedly , one among the most simple , robust , and seemingly common [ 8 , 9 ] ; the sigmoidal shape of the prescribed sad ( equation ( 7 ) and figure 1 ) is not so far from the most classically referred empirical types , broken - stick and log - normal distributions .
for example , the equation i = f(ai ) for the sad corresponding to a broken - stick distribution is i s exp(s ai ) , which is formally different from ( 7 ) .
accordingly , it is no real surprise that chao 's formula often approaches a strictly centred estimate , in spite of being only a lower - bound estimate in all generality .
this would explain why , in ecological practice in particular , chao 's formulation is yet considered one of the more accurate and reliable estimators of the total species richness within partially sampled communities . as mentioned in particular by gotelli ( personal communication ) ,
a trend would remain for chao 's estimates to increase somehow when a series of sampling of growing sizes are extracted from the same community instead of remaining ideally stable on average .
this , however , is not necessarily contradictory to preceding arguments but should certainly result from residual discrepancy between the real sad and the ideal model described by ( 7 ) and exemplified at figure 1 . | anne chao proposed a very popular , nonparametric estimator of the species richness of a community , on the basis of a limited size sampling of this community .
this expression was originally derived on a statistical basis as a lower - bound estimate of the number of missing species in the sample and provides accordingly a minimal threshold for the estimation of the total species richness of the community .
hereafter , we propose an alternative , algebraic derivation of chao 's estimator , demonstrating thereby that chao 's formulation may also provide centered estimates ( and not only a lower bound threshold ) , provided that the sampled communities satisfy a specific type of sad ( species abundance distribution ) .
this particular sad corresponds to the case when the number of unrecorded species in the sample tends to decrease exponentially with increasing sampling size .
it turns out that the shape of this ideal sad often conforms approximately to the usually recorded types in nature , such as log - normal or broken - stick .
.
accordingly , this may explain why chao 's formulation is generally recognized as a particularly satisfying nonparametric estimator . |
burrata is a traditional cheese produced in the apulia region ( southern italy ) , particularly in bari , barletta - andria - trani and foggia provinces , as well as in martina franca , a town in the province of taranto .
the product is a pasta filata ( stretched curd ) cheese consisting of a filling made of cream mixed with mozzarella cheese strips ( usually leftovers from mozzarella production ) inside an envelope made of mozzarella .
this product came probably from the idea of reusing the scrap from mozzarella cheese production , and mixing it with cream to be used as filling of a cheese envelope shaped like a caciocavallo cheese ( salvadori del prato , 2001 ) .
although burrata cheese is not included in the list of eu protected agricultural products , it is listed in the register of traditional foodstuffs of the apulia region and is known worldwide ( lo russo , 2008 ) . its production technology is far from being standardised , having not yet achieved a legal protection .
moreover , different raw materials can be used depending on the manufacturer [ i.e. whey cream obtained by spontaneous rising or ultra high temperature ( uht ) centrifuged cream for the stuffing ] .
these could result in products different in shape , sensory traits , or safety aspects .
the absence of product and production standards in burrata cheesemaking and consequently the presence of different products on the market ( tantillo , 2007 ) can also influence the duration of their shelf life .
for these reasons data on microbial , physical and chemical characteristics of burrata cheese are necessary for dairy producers to achieve a sort of standardisation that may contribute to improve the quality and safety of such a product .
the aim of the present study was to evaluate the physical , chemical and hygienic characteristics of burrata cheese manufactured in two dairy plants that used different raw materials and production techniques .
samples of milk , intermediate products ( cream , pasta filata strips , and envelope ) throughout the cheesemaking process , and final products ( burrata cheeses ) were collected in two different sessions from an artisanal ( ap ) and an industrial ( ip ) dairy plant , both located in the foggia province ( apulia region , southern italy ) .
the burrata cheeses were produced according to two different production techniques . according to the tradition ,
the artisanal process was entirely carried out manually starting from raw milk and whey cream obtained by spontaneous rising from the water used for the curd stretching , collected from the previous production cycles .
the industrial cheesemaking used pasteurised milk and uht cream obtained by centrifugation and all the steps were performed using specific equipment , typical of most mozzarella production lines .
briefly , for both productions , the milk was added with natural whey starter ( 3/100 l ) and , after a partial acidification ( ph 6.1 ) , heated to 37c .
then , calf rennet was added ( 15 - 30 ml/100 l ) and the coagulation took place after 30 min .
the curd was cut and kept under whey for maturation ( acidification ) for 30 min .
later the curd was kneaded under hot salted water ( 85 - 90c ) and stretched to produce pasta filata strips and envelope .
two sampling sessions were performed in each plant . in each sampling session from the same batch , 5 samples were collected at the ip and 5 samples at the ap : the raw material ( milk ) , the semi - finished ( cream , pasta filata used for the filling and the envelope , and whole filling made with cheese strips and cream ) , and the finished product ( burrata ready for consumption ) .
the collection was performed under sterile conditions : samples were packaged in sterile plastic bags , refrigerated and sent to the laboratory of the school of biosciences and veterinary medicine , university of camerino [ matelica ( mc ) , italy ] . for the physical and chemical traits , activity water [ aw ; using a btrs1 rotronic hygroscope ( pbi international , milan , italy ) ] ,
ph [ according to bendall ( 1975 ) measured using a beckman 310 phmeter ( beckman , galway , ireland ) ] , moisture , and nacl content ( aoac , 1990 ) were determined .
milk samples were analysed only for ph and protein content ( aoac , 1990 ) .
as for the microbiological evaluation , total viable count ( tvc ) , different foodborne pathogens ( listeria monocytogenes , salmonella spp . , yersinia enterocolitica , bacillus cereus ) and process hygiene criteria ( escherichia coli , coagulase - positive staphylococci ) were considered .
these analyses were performed on all samples in duplicate except for the filling , whose overall microbiological characteristics were obtained from the analysis of the cream and the pasta filata strips used for its preparation . the quantitative determination of microbiological parameters was carried out by inoculating the media with specific scalar dilutions of 10 g ( or ml ) of sample homogenised in 90 ml of saline solution with peptone , while for the qualitative evidence , 25 g ( or ml ) of the sample were diluted 1:10 in specific enrichment broths , properly incubated and then inoculated on specific media .
tvc on plate count agar ( pca ) ( biolife , milan , italy ) and only milk samples on plate count agar wide skim milk ( biolife ) , in both cases incubated at 30c for 72 h. e. coli on selective e. coli / coliform medium ( oxoid , basingstoke , uk ) , incubated at 37c for 24 h , and subsequently in tbx medium ( oxoid ) at 44c for 18 - 24 h for confirmation .
coagulasepositive staphylococci on baird parker medium ( biolife ) , incubated at 37c for 24 - 48 h , followed by confirmation of the typical colonies using staphylase test ( oxoid )
. b. cereus on b. cereus agar base ( pemba ) ( biolife ) , added with b. cereus antimicrobic supplement ( biolife ) , incubated at 30c for 24 - 48 h , and subsequently on blood agar at 30c for additional 24 h. salmonella spp . with a pre - enrichment in buffered peptone water ( oxoid ) , followed by selective enrichment on rappaport vassiliadis soya peptone ( rvs ) broth ( oxoid ) at 42c for 24 h , and then on chromogenic salmonella agar base ( biolife ) and brilliant green agar ( biolife ) , at 37c for 24 h. the identification was performed using the api 20e biochemical test ( biomrieux , italy ) , as specified in the manufacturer s guide . l. monocytogenes on listeria enrichment broth low acriflavine ( lebb ) ( biolife ) incubated at 30c for 48 h , and subsequently on listeria oxford agar base ( biolife ) and aloa agar ottaviani agosti ( biolife ) at 37c for 24 - 48 h. the identification was made through the biochemical test api listeria ( biomrieux , marcy letoile , france ) , as specified in the manufacturer s guide .
y. enterocolitica on selective enrichment medium yersinia psb broth ( biolife ) incubated at 25c for 5 days , and subsequently on yersinia selective agar base ( oxoid ) , supplemented with yersinia selective supplement ( oxoid ) incubated at 25c for 24 - 48 h , after treatment in a solution of koh 0.5% .
the suspect colonies were identified using the api 20e biochemical test ( biomrieux ) , according to the manufacturer s guide , except for the incubation temperature , which was 30c instead of 37c .
all quantitative microbiological data were reported as colony forming unit ( cfu)/g or ml and converted to log10 .
data derived from the different sampling sessions were aggregated for statistical analyses . the physical , chemical and microbiological data obtained in all the different samples
were analysed with an unpaired t test ( statview sas , cary , nc , usa ) to detect differences between products from ap and ip .
samples of milk , intermediate products ( cream , pasta filata strips , and envelope ) throughout the cheesemaking process , and final products ( burrata cheeses ) were collected in two different sessions from an artisanal ( ap ) and an industrial ( ip ) dairy plant , both located in the foggia province ( apulia region , southern italy ) .
the burrata cheeses were produced according to two different production techniques . according to the tradition ,
the artisanal process was entirely carried out manually starting from raw milk and whey cream obtained by spontaneous rising from the water used for the curd stretching , collected from the previous production cycles .
the industrial cheesemaking used pasteurised milk and uht cream obtained by centrifugation and all the steps were performed using specific equipment , typical of most mozzarella production lines .
briefly , for both productions , the milk was added with natural whey starter ( 3/100 l ) and , after a partial acidification ( ph 6.1 ) , heated to 37c .
then , calf rennet was added ( 15 - 30 ml/100 l ) and the coagulation took place after 30 min .
the curd was cut and kept under whey for maturation ( acidification ) for 30 min .
later the curd was kneaded under hot salted water ( 85 - 90c ) and stretched to produce pasta filata strips and envelope .
two sampling sessions were performed in each plant . in each sampling session from the same batch , 5 samples were collected at the ip and 5 samples at the ap : the raw material ( milk ) , the semi - finished ( cream , pasta filata used for the filling and the envelope , and whole filling made with cheese strips and cream ) , and the finished product ( burrata ready for consumption ) .
the collection was performed under sterile conditions : samples were packaged in sterile plastic bags , refrigerated and sent to the laboratory of the school of biosciences and veterinary medicine , university of camerino [ matelica ( mc ) , italy ] .
for the physical and chemical traits , activity water [ aw ; using a btrs1 rotronic hygroscope ( pbi international , milan , italy ) ] , ph [ according to bendall ( 1975 ) measured using a beckman 310 phmeter ( beckman , galway , ireland ) ] , moisture , and nacl content ( aoac , 1990 ) were determined .
milk samples were analysed only for ph and protein content ( aoac , 1990 ) . as for the microbiological evaluation , total viable count ( tvc ) , different foodborne pathogens ( listeria monocytogenes , salmonella spp . , yersinia enterocolitica , bacillus cereus ) and process hygiene criteria ( escherichia coli , coagulase - positive staphylococci ) were considered .
these analyses were performed on all samples in duplicate except for the filling , whose overall microbiological characteristics were obtained from the analysis of the cream and the pasta filata strips used for its preparation . the quantitative determination of microbiological parameters was carried out by inoculating the media with specific scalar dilutions of 10 g ( or ml ) of sample homogenised in 90 ml of saline solution with peptone , while for the qualitative evidence , 25 g ( or ml ) of the sample were diluted 1:10 in specific enrichment broths , properly incubated and then inoculated on specific media .
tvc on plate count agar ( pca ) ( biolife , milan , italy ) and only milk samples on plate count agar wide skim milk ( biolife ) , in both cases incubated at 30c for 72 h. e. coli on selective e. coli / coliform medium ( oxoid , basingstoke , uk ) , incubated at 37c for 24 h , and subsequently in tbx medium ( oxoid ) at 44c for 18 - 24 h for confirmation .
coagulasepositive staphylococci on baird parker medium ( biolife ) , incubated at 37c for 24 - 48 h , followed by confirmation of the typical colonies using staphylase test ( oxoid )
. b. cereus on b. cereus agar base ( pemba ) ( biolife ) , added with b. cereus antimicrobic supplement ( biolife ) , incubated at 30c for 24 - 48 h , and subsequently on blood agar at 30c for additional 24 h. salmonella spp . with a pre - enrichment in buffered peptone water ( oxoid ) , followed by selective enrichment on rappaport vassiliadis soya peptone ( rvs ) broth ( oxoid ) at 42c for 24 h , and then on chromogenic salmonella agar base ( biolife ) and brilliant green agar ( biolife ) , at 37c for 24 h. the identification was performed using the api 20e biochemical test ( biomrieux , italy ) , as specified in the manufacturer s guide .
l. monocytogenes on listeria enrichment broth low acriflavine ( lebb ) ( biolife ) incubated at 30c for 48 h , and subsequently on listeria oxford agar base ( biolife ) and aloa agar ottaviani agosti ( biolife ) at 37c for 24 - 48 h. the identification was made through the biochemical test api listeria ( biomrieux , marcy letoile , france ) , as specified in the manufacturer s guide .
y. enterocolitica on selective enrichment medium yersinia psb broth ( biolife ) incubated at 25c for 5 days , and subsequently on yersinia selective agar base ( oxoid ) , supplemented with yersinia selective supplement ( oxoid ) incubated at 25c for 24 - 48 h , after treatment in a solution of koh 0.5% .
the suspect colonies were identified using the api 20e biochemical test ( biomrieux ) , according to the manufacturer s guide , except for the incubation temperature , which was 30c instead of 37c .
all quantitative microbiological data were reported as colony forming unit ( cfu)/g or ml and converted to log10 .
the physical , chemical and microbiological data obtained in all the different samples were analysed with an unpaired t test ( statview sas , cary , nc , usa ) to detect differences between products from ap and ip .
as for the milk , no significant difference ( p=0.872 ) was recorded for ph values between ap ( 6.750.06 ) and ip ( 6.700.01 ) ; protein content differed significantly ( p=0.002 ) between ap ( 3.000.06 ) and ip ( 3.360.06 ) ( data not shown ) .
as for the semifinished and finished products , the physical and chemical characteristics are reported in table 1 and the microbiological data are shown in table 2 .
differences in ph values were recorded in all the semi - finished samples , with a constantly lower value in ap than in ip , but not in the final products .
differences in the cream used , and therefore in the filling , were recorded also for moisture content ( higher in industrial than in artisanal cream ) and for nacl concentration ( lower in industrial than in artisanal cream ) .
the microbiological counts revealed higher values of tvc and e. coli in cream , mozzarella strips and final products from ap than those from ip .
coagulase - positive staphylococci were detected in all the artisanal samples collected but the counts were always well below the limit set by the european commission regulation n. 1441/2007 ( european commission , 2007 ) .
y. enterocolitica was qualitatively detected in samples obtained in the second sampling session from ap mozzarella strips and burrata cheese and from ip pasteurised milk ( data not shown ) .
the different cheesemaking technologies , mainly consisting in the cream used but also in the pasta filata envelope and strips , could be responsible for the chemical and physical differences recorded between ap and ip samples .
the values of nacl detected in the cream are quite interesting : the significantly ( p=0.003 ) higher value in ap samples is strictly related to the traditional cheesemaking technology that provides for the addition of hot water with 10% nacl used for curd stretching , from which the artisanal cream is recovered by spontaneous raising , as previously described in the cheesemaking process .
the spontaneous rising can even explain the much lower ( p<0.001 ) ph observed in the artisanal cream .
moreover , the proportion of the cream and mozzarella strips used for preparing the filling ( generally not standardised in the artisanal cheesemaking ) could have affected the final products that differed only for aw and nacl values .
the aw parameter is relevant for microbial growth and the values recorded in both types of burrata cheese were high enough to allow almost all pathogenic microorganisms to grow if other hurdles are not provided in the production process .
furthermore , in these fresh cheeses the salt content has to be low for improving the taste perception by the consumer .
the effect of salt in prolonging the shelf life of mozzarella cheese is almost contradictory , as longer durations were obtained for mozzarella without or with low ( 0.23/100 g ) salt concentration ( faccia et al.,2012 ) . from a microbiological perspective
moreover , coagulase - positive staphylococci and y. enterocolitica were found in samples from ap , even though at low concentration or as not pathogenic strains , respectively ( data not reported ) .
the tvc , considered as a process hygiene parameter in some foods , could be strongly affected by the number of lactobacillus and lactococcus ( lab ) in cheeses , where they play an important role as hurdle against microbial growth .
higher tvc values were constantly recorded in all products from ap , which showed lower ph values in the intermediate samples ( cream , pasta filata strips and filling ) .
the production procedure of whey cream obtained by spontaneous rising ( 24 h at room temperature ) used in the ap can explain the growth of a much higher number of lab , as well as other bacteria , in products from ap than those from ip .
as emphasised by other authors ( de candia et al . , 2007 ) , the lab present in the whey used as starter for making pasta filata ( strips and envelope ) may also have influenced the characteristics of the products . nonetheless ,
much higher values of e. coli were recorded in cream , mozzarella strips and final products from ap than those from ip .
the whey cream obtained by spontaneous rising and the whey used as starter may be responsible for the high amount off .
coli in the products , and even the high temperature ( 85 - 90c ) used in the kneading / stretching step of pasta filata production is not sufficient to reduce the e. coli load to an acceptable level .
( 2003 ) reported that the stretching of curd at temperatures over 80c should be able to eliminate up to 5 log of e. coli 0157:h7 , but time / temperature curve has to be carefully taken into consideration to obtain effective results ( trevisani et al . , 2014 ) .
increase in the final product was observed , with average values higher than the legal limit of acceptance set by the european commission regulation n. 1441/2007 ( european commission , 2007 ) .
this suggests that e. coli contamination and/or growth during the last phases of the cheesemaking could occur , i.e. during filling / closing , immersion for hardening , and packaging steps .
( 2013 ) , who did not observe burrata samples exceeding the limit of e. coli set by the ec regulation , while 3.7% of samples were positive for coagulase - positive staphylococci with values greater than 3 log cfu / g . no relevant pathogens were recorded in products from ap and ip , and the level of coagulase - positive staphylococci was almost low , in contrast with other studies on mozzarella cheese , where high levels of such bacteria were reported ( de luca et al . , 1997 ) .
the presence of virulent y. enterocolitica is reported in different cheeses ( hanifian and khani , 2012 ; kowalich and lobacz , 2015 ) but no data on mozzarella or burrata cheese is available from the literature .
the milk is reported as the major source of such a contamination ( efsa , 2013 ) and even pasteurised milk was found to be positive to y. enterocolitica ( gupta et al . , 2015 ) .
even if not detected in pathogenic form , the necessity of improving the control on this microorganism should be emphasised .
the differences observed in burrata cheesemaking have to be taken into consideration when a proper risk analysis is performed .
the bacterial contamination of the product could be reduced only if an appropriate temperature / time ratio , ensured by the curd kneading at 85 - 90c during the stretching step , is reached .
control strategies to avoid e. coli proliferation are further needed , especially in the artisanal process and in particular during the preparation of whey cream obtained by spontaneous rising and used in the filling . | the burrata cheese is a traditional product from southern italy , consisting of an envelope of pasta filata ( stretched curd ) filled with cream and pasta filata strips ( usually leftovers from mozzarella production ) .
physical [ water activity ( aw ) , ph ] , chemical ( moisture , nacl content ) and microbiological [ total viable count ( tvc ) , listeria monocytogenes , salmonella spp . , yersinia enterocolitica , bacillus cereus , escherichia coli , enterobacteriaceae , coagulase - positive staphylococci ] characteristics of burrata cheeses manufactured in artisanal and industrial plants were evaluated .
the artisanal burrata showed lower aw values in the filling and the final product .
the same was recorded in the filling for the moisture , probably due to differences between the types of cream used in the artisanal and the industrial cheesemaking .
the ph value of the filling differed between the two groups but no difference was recorded in the final product .
microbiological differences were also recorded , with higher values for tvc and e. coli in artisanal than industrial burrata .
all samples were negative for the other microbial determinations , with the exception of coagulase - positive staphylococci and y. enterocolitica , which were detected in artisanal burrata .
differences in cheesemaking process were probably responsible for the strong variability of the physical and chemical data between the two cheeses ; furthermore , differences in the hygienic features were also recorded .
even though artisanal products showed lower aw and ph values and higher nacl concentration , the higher e. coli loads highlighted the need for a more accurate compliance with hygienic procedures along the artisanal cheesemaking process . |
evidence for beneficial effects of solar ultraviolet - b ( uvb ) irradiance and vitamin d in reducing the risk of adverse health outcomes comes from a variety of study types : ecological , cross - sectional , case - control , cohort , and intervention .
however , others have conducted systematic reviews that find the evidence lacking partly because at the time of the reviews , few well - conducted randomized controlled trials ( rcts ) with vitamin d existed to analyze its nonskeletal effects . also , findings from cohort studiessometimes disagree with results from ecological studies .
a recent paper argued that prospective studies with long follow - up times lead to errors because the single serum 25-hydroxyvitamin d [ 25(oh)d ] concentration measurement taken at study enrollment loses prognostic value over time . for breast cancer ,
the linear regression line fit to the relative risk increased from 0.61 for case control studies with no follow - up period to 0.95 for a 7-y follow - up , whereas for colorectal cancer , the regression line fit increased from 0.48 for case - control studies with no follow - up to 0.72 for a 14-y follow - up . for prostate cancer , no statistically significant correlation emerged with respect to 25(oh)d concentration for any follow - up time between 4 and 28 y. further support for this assertion is that a prospective study of breast cancer incidence found a strong inverse correlation with high vitamin d intake in the first 5 y after baseline dietary assessment ( relative risk = 0.66 ; 95% ci , 0.460.94 compared with lowest - intake group ) , with the association diminishing over time . also , for a nested case - control study of lymphoma in finland , the odds ratio for chronic lymphocytic leukemia or small lymphocytic lymphoma was 0.41 ( 95% ci , 0.151.09 ) for follow - up time shorter than 7 y but was 1.15 ( 95% ci , 0.443.01 ) for follow - up longer than 7 y. a recent meta - analysis of all - cause mortality rate with respect to prediagnostic serum 25(oh)d concentration mentioned that the cohort studies with shorter follow - up times had a stronger association than those with longer follow - up .
this paper uses the data in that study to extend that analysis of follow - up time s role in cohort studies of health outcomes with respect to prediagnostic serum 25(oh)d concentration .
figure 1 is a scatterplot of the correlation coefficient for serum 25(oh)d concentration measurements vs. interval .
the value changes from a zero intercept of 0.7 to 0.42 for 14 y. the square of the correlation coefficient gives the fraction of the variance explained by the model .
correlation coefficient of serum 25(oh)d concentration measured in two periods vs. follow - up period .
the regression fit to the hazard ratio per 20-nmol / l increase in serum 25(oh)d concentration vs. time increased from 0.82 ( 95% ci , 0.671.02 ) for 6 y to 0.96 ( 95% ci , 0.901.01 ) for 14 y. the zero follow - up time value is 0.72 ( 95% ci , 0.501.03 ) .
these values differ considerably from the values in the meta - analysis by schttker and colleagues of 0.92 ( 95% ci , 0.890.95 ) , which corresponds to a value for 12 y for the regression fit in figure 2 .
however , the mean follow - up time of all 12 studies weighted by the relative weights in schttker s figure 3 is 9.6 y. thus , the results based on extrapolation to zero follow - up time find 3.5 times as great a risk reduction , whereas the ratio for 6 y is 2.3 times as great .
although the extrapolated 95% ci values for zero follow - up time are large , the values would be much smaller if the values were averaged , say , for each three values .
hazard ratios ( hrs ) for all - cause mortality rate per 20-nmol / l increase in serum 25(oh)d concentration , using data from figure 3 in schttker et al.13 vs. follow - up period .
the equations for the regression fits are : hr = 0.72 + 0.017 t ; hr [ lower confidence interval ( ci ) = 0.50 + 0.029 t ] ; hr ( upper ci ) = 1.03 0.0009 t , where t = time ( years ) .
the results in figure 2 suggest that a 20-nmol / l increase in vitamin d serum level reduces the risk of all - cause mortality rate by 1828% .
the 18% is similar to the 717% reduction achieved by increasing serum 25(oh)d concentration from 54 to 110 nmol / l , as estimated in a recent study based on the serum 25(oh)d concentration disease outcome relation for diseases contributing the most to all - cause mortality rate : cancer , cardiovascular disease , diabetes mellitus , respiratory diseases , respiratory infections , and tuberculosis .
this reduction in mortality rate would increase life expectancy by an estimated 2 y. however , because the 25(oh)d concentration mortality rate relation is nonlinear , changing rapidly for low serum 25(oh)d concentrations and very little above 80 nmol / l , determining a more precise estimate would require a more careful analysis . on the basis of figure 4 in zittermann and colleagues , the 8% reduction in schttker et al .
is consistent with an increase of serum 25(oh)d from 50 to 70 nmol / l .
these results further support the assertion that the apparent risk of adverse health outcomes decreases with longer follow - up time .
however , in nested case - control studies , shorter follow - up times have fewer cases and thus wider 95% cis .
the rationale for conducting nested case - control studies instead of case - control studies with no follow - up interval includes concerns about bias in selecting controls and that the disease may affect the health outcome . for mortality rates ,
death often comes after long - term illness , which can affect serum 25(oh)d concentrations . for cancer incidence rates ,
this concern is not as well founded : people who have cancer often do not find out until diagnosis through screening ( for breast and colorectal cancer ) or until they notice persistent pain for a short time . for breast cancer
, case - control studies found much greater inverse correlations between serum 25(oh)d concentration and incidence than did nested case - control studies .
this study recommends that meta - analyses of cohort study findings incorporate follow - up time , as demonstrated here .
it also suggests comparing case - control study results with those of cohort studies by plotting the results vs. follow - up interval ; if the data from both types of studies can be modeled with a linear or second - order regression fit without large deviations , the case - control results should be afforded more credibility .
this study also recommends that prospective cohort studies regularly measure serum 25(oh)d concentration , perhaps every 24 y. doing so would add additional costs to the studies but would yield more accurate results .
thus , in studies such as the vitamin d pooling project of rarer cancers , the disagreement with ecological studies may be due partly to the long mean follow - up time of 6.63 y. some of the disagreement can also be due to the few cases for each type of cancer . also , the role of solar uvb and vitamin d appears to be stronger for mortality rates than for incidence rates in ecological studies.ecological studies of the seven types of cancers in the vitamin d pooling project of rarer cancers [ endometrial , esophageal , gastric , kidney , ovarian , pancreatic cancer and non - hodgkin lymphoma ( nhl ) ] strongly support beneficial effects of solar uvb in reducing mortality rates .
the evidence of beneficial effects for incidence rates for these types of cancer in ecological studies is weaker.results from prospective cohort studies also support a protective role of uvb irradiance for nhland 25(oh)d concentration for gastric and pancreatic cancer , with moderate support for a role of serum 25(oh)d concentration in ovarian cancerbut no support for endometrial cancer or nhl .
however , long follow - up times could adversely affect some observational studies for endometrial and ovarian cancer and nhl .
for one , there are risk - modifying factors that are not included in the analyses , such as physical activity , obesity rates , and immigration .
for example , in the united states , there was considerable migration from the northeast to the south and west in the second half of the twentieth century .
the strength of the ecological approach is demonstrated in the fact that the results for many types of cancer are often repeated for different populations .
this study offers additional support for the thesis that long follow - up times adversely affect nested case - control studies from prospective cohort studies regarding the role of prediagnostic serum 25(oh)d concentration in health outcomes such as all - cause mortality rate and many types of cancer .
it is hoped that the research communities and health policy makers will take this thesis into account when analyzing epidemiological studies and making recommendations regarding vitamin d. this study also indicates that observational studies can strongly support the uvb - vitamin d - disease reduction hypothesis if the existing studies in the journal literature are carefully assessed and systemic biases in the interpretation of the data are removed .
unfortunately , the institute of medicine committee on dietary reference intakes for vitamin d and calcium did not undertake that task .
they opted instead to wait another 56 y for a definitive rct of vitamin d supplementation .
as kristal and another paper in this issue pointed out , rcts have several problems that , if not carefully addressed , can result in poor - quality studies .
ideally , results from all types of studies clinical , cross - sectional , ecological , laboratory and observational would be considered together and reasons for differences resolved .
findings in the literature for the correlation coefficient for serum 25(oh)d concentrations for a single cohort for different measurement intervals ( table 1 ) are plotted vs. time .
however , because the pearson coefficient is more appropriate for continuous variables , it is used in the graph when both coefficients are given . personal communication from l. rejnmark in addition to published data .
the data for this study are from a recent meta - analysis of prospective cohort studies of all - cause mortality rate .
this study uses the statistics for the 12 studies included in that meta - analysis for hazard ratios per 20-nmol / l - increase in serum 25(oh)d concentration ( fig . 3 in schttker et al .
a complication exists in that the 25(oh)d - mortality rate relationship is nonlinear , but both schttker et al . and | evidence continues to mount that vitamin d reduces the risk and mortality rates of many types of disease .
however , evidence from prospective cohort studies is sometimes weaker than that from case control and ecological studies .
a suggested reason for this discrepancy is that , because serum levels of 25-hydroxyvitamin d [ 25(oh)d ] change over time , a single 25(oh)d concentration measurement taken at study enrollment does not reliably indicate 25(oh)d concentration related to the health outcome . to evaluate this suggestion further ,
this paper plots results from 12 prospective cohort studies of all - cause mortality rate vs. follow - up time .
the regression fit to the hazard ratio per 20-nmol / l increase in serum 25(oh)d concentration vs. time increased from 0.82 ( 95% ci , 0.671.02 ) for 6 y to 0.96 ( 95% ci , 0.901.01 ) for 14 y. the value extrapolated for zero follow - up time was 0.72 ( 95% ci , 0.501.03 ) , giving a hazard ratio reduction 3.5 times higher than the standard result from the meta - analysis [ 0.92 ( 95% ci , 0.890.95 ) ] . using the example of the vitamin d pooling project of rarer cancers , this paper also discusses follow - up time s effect in interpreting prospective cohort studies of cancer outcome .
this paper recommends that meta - analyses of prospective cohort studies account for follow - up time and , if possible , that studies measure serum 25(oh)d concentration every 24 y. |
while brm scales with body mass ( bm ) , several other factors seem to influence brm , such as sociability , environmental and dietary specializations , as well as energetic costs of brain tissue ( mace et al . , 1981 ) .
large brains contain more neurons and neural connections , and thus have greater potential for information processing .
large brains also tend to be more modular , which allows a great amount of connections between neurons ( krubitzer and kaas , 2005 ) . hence , increased brain mass and potential for neural connections
may have facilitated large brained mammals to colonize complex habitats , develop sensory systems and evolved complex societies ( e.g. , budeau and verts , 1986 ) .
for example , the mass - specific metabolic rate of the human brain is nine times higher than that of the body as a whole ( martin , 1981 ) .
the metabolic costs by having a large brain must be paid for by a direct mother metabolic constraint or by a trade - off mechanism between brain mass and energy consumption by other functions ( e.g. , gibbons , 1998 ; pitnick et al . , 2006 ) .
other energy allocations such as relative costs of flight and reproductive strategy in birds may also be reduced to shunt energy to an enlarged brain ( isler and van schaik , 2006 ) .
recent examples also show that brm may become decoupled from bm over short time spans .
2009 ) found that tanganyikan cichlid bm exhibited recent bursts of rapid evolution , a process that is consistent with divergence linked to ecological specialization , while brm showed no bursts of divergence but evolved in gradual manner , consistent with energetic constraints to rapid bm change .
originally , aiello and wheeler ( 1995 ) proposed that a primate is able to meet the high metabolic cost of a large brain without incurring a compensatory increase in relative basal metabolic rate ( bmr ) by decreasing the amount of other metabolically expensive tissues ( i.e. , heart , lung , kidney , liver , and gastrointestinal tract ) .
a similar hypothesis was recently proposed for fish ( kaufman et al . , 2003 ) .
recently , isler and van schaik ( 2009 ) using a large compilation of brain size , bm , and life history data , found evidence that an energetically costly increase in brain size has to be met by either increasing the total energy budget of a species or by compensating changes of energy allocation to other maintenance functions , such as digestion or growth and offspring production , or a combination of these .
martin , 1981 , 1996 ) , for example , found that the energetic investment of the mammalian mother during the development of the fetus and during the postnatal life up to the time of weaning resulted in a weak link between bmr and brain mass .
similarly , jones and maclarnon ( 2004 ) showed that for certain clades of bats , maternal investment plays an important role in the adult brain mass .
while a number of studies have thus focused on these mechanisms of development , little is known about the evolutionary relationship between bmr and brm .
very few studies have tested the generality of the costly brain hypotheses across multiple species with different evolutionary histories , and using phylogenetic approaches .
basal metabolic rate is a fundamental parameter in comparative studies and lineages - specific exponents characterize its allometric scaling ( white et al . , 2009 ) .
recently , isler and van schaik ( 2006 ) controlling both for bm and phylogentic relationships found evidence that bmr correlated with brm in large groups of mammals .
however , brm explained a small % of the variation in metabolic rate at the species and family level ( 2.6 and 10.4% , respectively ) . at higher taxonomic levels , independent contrast ( ic ) revealed a significant correlation only for primates .
it is possible that the use of composite phylogenies lacking resolution and accurate estimates of branch lengths ( bl ) may have obscured the underlying patterns ( e.g. , malia et al . , 2003 ) . other potential confounding
we generated a novel phylogeny of wild rodents using bayesian analysis of cytochrome b sequence data .
we included species where high - quality bmr , brm , and bm data are available .
we then used this analysis to test the hypothesis that bmr and brm are correlated within rodents , after taking into consideration both bm and phylogeny .
cytochrome b for 132 rodent species and six rabbits as outgroups ( wilson and reeder , 2005 ) were downloaded from genbank , and one sequence donated ( table a1 in appendix ) .
cytochrome b was chosen as that marker has proven to be of high utility for species level phylogenetics ( may - collado and agnarsson , 2006 ; agnarsson et al .
, 2010 , 2011 ) sequences were aligned using clustalx 1.83 ( thompson et al . ,
the preferred model for the bayesian analyses was selected with modeltest ( posada and crandall , 2001 ) using the aic criterion ( posada and buckley , 2004 ) .
the best - fitting model was gtr + + i ( yang , 1994 ) .
bayesian analyses were carried out using mrbayes v3.12 ( huelsenbeck and ronchist , 2001 ) with the settings as specified in agnarsson and may - collado ( 2008 ) .
the markov chain monte carlo search was ran with 10,000,000 generations sampling the markov chain every 1,000 generations , and the sample points of the first 7,000,000 generations were removed ( burnin ) , after which the chain had reached stationarity .
data on the log of brm and bm ( g ) , and bmr ( cmo2/h ) were used in this study ( table a1 in appendix ) . for studies comparing traits among species , such as regression analyses ,
it is necessary to account for phylogenetic relationships among the compared species ( felsenstein , 1985 ) .
ignoring phylogenetic relationships can lead to pseudoreplication as species are not independent data points , rather independent evolutionary changes in the traits being compared are the data points , or the ic ( see felsenstein , 1985 ; may - collado et al . , 2007 ) among species and lineages . to describe the evolutionary relationship between bmr and brm we performed various phylogenetic analyses .
( i ) the pdap module in mesquite ( midford et al . , 2008 ) was used to estimate ic ( felsenstein , 1985 ) .
we used bl as estimated by mrbayes testing them for statistic appropriateness using pdap . to correct for bm we regressed bmr and
brm against bm and subsequently regressed the residuals from these regressions ( garland et al . , 1993 ) .
if the residuals are correlated then that is consistent with a relationship among these variables ( bmr and brm ) , that is independent of the bm of , and phylogenetic relationship among , species ( e.g. , may - collado et al . , 2007 ) .
( ii ) to evaluate the correlated evolution among bmr , brm , and bm , we assess the phylogenetic effect on the trends in character relationships between taxa ( i.e. , the observed pattern ) using the best model of evolution that was found for each character . to do this we evaluated the significance of the relationships between the pair of characters using a measure of correlated evolution ( corr ) in a bayesian framework implemented in bayestrait 1.0 ( pagel and meade , 2007 ) , assessing the probability of positively correlated ( corr > 0 ) and negatively correlated evolution ( corr < 0 ) . as the null hypothesis we used a model in which the covariance between characters was set to zero
( i.e. , complete character independence , corr = 0 ) , and the alternative hypothesis was , then , the observed covariance between characters ( pagel , 1999a , 1999b ) . if the null hypothesis was rejected ( i.e. , a significant historical relationship between characters exists ) , then we concluded that the phylogenetic relationship and the models of evolution of the characters influence the observed patterns , and we corroborate the hypothesis of correlated evolution between bmr , brm , and bm .
we used a bayesian approach based on maximum likelihood with 10 test per tree and estimating pagel ( 1999a , b ) escalated phylogenetic parameters ( table a2 in appendix ) .
the sign test was used for statistical comparisons ( zar , 1996 ) with statistica 6.0 ( statsoft , 2001 ) .
the novel phylogeny finds support for the monophyly of each currently recognized taxonomical bat family with the exception that heteromyidae contains geomyidae , and one species of muridae , sigmodon hispidus , groups with cricetidae ( figure a1 in appendix ) .
the phylogeny overall agrees well with recent rodent phylogenies at higher levels ( e.g. , jansa and weksler , 2004 ; montgelard et al . ,
2008 ) and thus represents an reasonable hypothesis for to study the evolution of characters ( e.g. , pagel and harvey , 1988 ) independent contrast revealed associations between bm and bmr ( p < 0.0001 ; r = 0.77 ) , and with brm ( p < 0.0001 ; r = 0.85 ) , and between bmr and brm ( p < 0.0001 ; r = 0.71 ) . when using a single value to represent species mass we also found significant correlation between bmr and brm , after accounting for bm .
when using a single individual weight to represent the species , bmr explained 9.7% of the variation in brm ( p = 0.0003 ; r = 0.097 ; figure 1a ) .
two extreme outliers affected the regression and removing these outliers resulted in much stronger regression ( p < 0.0001 ; r = 0.20 ; figure 1b ) .
the outliers represent 3 species of small rodents ( cricetidae , arvicolinae ) , that inhabit circumpolar northern hemisphere biome ( nowak , 1999 ) .
these lemmings seem to have higher metabolic rate than typical rodents with similar brm , which may be related to living in extreme climates requiring higher metabolism .
this demonstrates that the climate and habitat as well as other potentially confounding factors ( bm , food habits , substrate , a restriction to islands or highlands , use of torpor , and type of reproduction ) make it difficult to demonstrate a significant correlation between bmr and brm , even when it exists ( e.g. , mcnab , 2008 ) .
( a ) independent contrast regression analysis between bmr and brm residuals , corrected by bm . ( b ) ic regression analysis between bmr and brm residuals , corrected by bm and removing outliners ( myopus schisticolor , lemmus sibiricus , and l. lemmus ) .
where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass .
when using species mean bm , bmr explained approximately 3% of variation in brm ( p < 0.05 , r = 0.029 ) .
this correlation , however , disappears when using two bm values for each species , one , the estimated species mean , to calculate residuals of brm , and the second , of the individuals used for the bmr experiments , to calculate residuals of bmr ( p > 0.05 ) .
corr indicated significant correlations between all variables ( p < 0.0001 ; corr
1 ) , with the highest correlation recorded between bm and brm ( p < 0.0001 ; corr = 2.86 ; r = 0.95 ; figure 2 ) , followed by bm and bmr ( p < 0.0001 ; corr = 2.24 ; r = 0.92 ; figure 2 ) , and finally bmr and brm ( p < 0.0001 ; corr = 1.86 ; r = 0.91 ; figure 2 ) .
the series of arrows in the middle of the figure indicates the model of correlated evolution between the studied characters in a bayesian framework ( for more details see materials and methods ) , where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass .
each inset labeled with a lower - case letter contains : on the left - hand side a line graph of a sample of 1,000 markov chain estimations ( x - axis ) of corr ( y - axis ) between pairs of characters , where the continuous black line represents corr = 0 , and the continuous gray line indicates corr = 1 ; and on the right - hand side a histogram of the probability distribution of covariance between pairs of characters , where the black bar indicates the average value and the gray bars indicate the lower 5% percentile ( lp ) and upper 95% percentile ( up ) . in particular , the analyzed characters were ( a ) bmr and brm ( lp = 1.33 , up = 2.46 ) ; ( b ) bm and bmr ( lp = 1.50 , up = 3.12 ) ; and ( c ) bm and brm ( lp = 2.08 , up = 3.77 ) .
brains are the centers of the nervous system of vertebrates , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment ( shultz , 2010 ) .
although brain mass per se does not capture the complexity of brain function , there is general evidence that relative brain size roughly correlates with cognitive ability ( e.g. , barton and harvey , 2000 ) . hence the evolution of brain size is of broad interest , including what factors may favor and constrain the evolution of relatively large , modular and complex brains ( sol , 2009 ) .
basal metabolic rate is influenced by a variety of factors ( bm white and seymour , 2003 ; climate lovegrove , 2000 ; demography kurta and ferkin , 1991 ) .
furthermore , an increase in brm results in increased costs for maintenance and information processing ( niven and laughlin , 2008 ) .
for example , karbowski ( 2007 ) found that in volume - specific cerebral glucose metabolic rate of different brain structures closely scales with brain volume .
these results confirm that information processing in the brain requires large amounts of metabolic energy . here
, isler and van schaik ( 2006 ) found support for this relationship across all mammals combined , and within primates ( ic p = 0.025 , r = 0.20 ) , but not within other orders , such as rodents .
adjustments appear to be clade - specific , the slopes of best - fit lines for brm against bm tend to be higher in analyses of more inclusive taxa ( e.g. , orders and suborders ) and lower in analyses of less inclusive taxa ( families , subfamilies , and genera ; e.g. , finarelli and flynn , 2009 ) .
we hypothesize that the discrepancy between our findings and previous studies is potentially caused by two factors .
first , the use of a composite versus primary - data based phylogenies , and second , differences in accounting for bm .
composite phylogenies often reflect taxonomy , not necessarily phylogeny , and typically lack accurate bl estimates , two aspects that reduce the efficiency of comparative tests .
accounting for bm is a complicated problem , but we argue that using more than a single value for a species , as have prior studies , may introduce confounding variables . thus using an estimate of species bm , such as average species bm to generate brm residuals , and the bm of the individual that happened to be used to evaluate bmr to generate bmr residuals , can likely adds noise that may obscure real patterns . if , for example , the evaluation of bmr happened to have been done on an atypically small or large animal , this would strongly affect the bm - bmr residual , and could readily obscure subtle patterns across two or more variables that are both highly correlated with bm . instead using a single value ,
whether mean bm of a species , or the weight of a single individual seems at least to be a reasonable alternative .
furthermore , species mean bm is a measure independent of the available measures for bmr and brm ( typically single individuals ) , and as such provides a relatively neutral control unlikely to result in systematic error . here
, we used a novel primary - data - based phylogeny with bl estimates and single estimates of species bm ( species average or single individuals ) .
considering the compendium of factors that may contribute to bmr , the up to 20% of variation explained by brm in rodents is high .
clearly , though , further research is necessary to understand the interplay between these variables , and ideally accurate estimates of species means based on multiple individuals would be available for each of these variables ( e.g. , smith and jungers , 1997 ) .
our study is also consistent with two evolutionary paths favoring an increase in brm across rodent species .
path in which an increase in bmr correlates with an increase in brm ( figure 2a ) . alternatively ,
indirect and additive path in which the effect of bm on bmr allows brm to increase ( figures 2a , b ) .
comparatively , the direct scaling effect of bmr on brm is the least important relationship ( figure 2a ) , but the importance of direct scaling increases when considering the indirect path .
dunbar and shultz ( 2007 ) supported the indirect path scenario in primates , where bmr had a limiting effect on brm , while bm had an effect on brm through bmr .
here , we corroborate the hypothesis of isler and van schaik ( 2006 ) that an increase in brain mass is accompanied by an increase in basal metabolic rate , and suggests that this pattern may be general across mammals .
our findings corroborate the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur ( niven and laughlin , 2008 ) .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
aic , akaike information criterion ; bl , branch lengths in phylogenies ; bm , body mass ; bmr , basal metabolic rate ; brm , brain mass ; corr , measure of correlated evolution between characters in a bayesian framework ; gtr , general time reversible models of nucleotide substitution ; ic , phylogenetic independent contrasts ; pdap , phenotypic diversity analysis programs . | brains are the centers of the nervous system of animals , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment .
the evolution of traits that correlate with cognitive ability , such as relative brain size is thus of broad interest .
brain mass relative to body mass ( bm ) varies among mammals , and diverse factors have been proposed to explain this variation .
a recent study provided evidence that energetics play an important role in brain evolution ( isler and van schaik , 2006 ) . using composite phylogenies and data drawn from multiple sources
, these authors showed that basal metabolic rate ( bmr ) correlates with brain mass across mammals .
however , no such relationship was found within rodents .
here we re - examined the relationship between bmr and brain mass within rodentia using a novel species - level phylogeny .
our results are sensitive to parameter evaluation ; in particular how species mass is estimated .
we detect no pattern when applying an approach used by previous studies , where each species bm is represented by two different numbers , one being the individual that happened to be used for bmr estimates of that species .
however , this approach may compromise the analysis . when using a single value of bm for each species ,
whether representing a single individual , or available species mean , our findings provide evidence that brain mass ( independent of bm ) and bmr are correlated .
these findings are thus consistent with the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur . |
surface immobilization
of poly(ethylene glycol ) ( peg ) , or pegylation ,
is widely used to modify nanoparticles ( nps ) with the goal to avoid
undesired , nonspecific biological interactions . the covalent attachment
of peg to nps results in the formation of a relatively inert hydrophilic
corona that reduces uptake by the reticuloendothelial system , thereby
prolonging the circulation time of nps such as therapeutic proteins ,
dendrimers , liposomes , and micelles .
it has been reported
that the length of peg chains plays a major role in determining the
cellular interaction of pegylated nps because it affects the particle
size , peg conformation ( mushroom vs brush ) , and thickness and surface
coverage of the peg corona .
the surface charge of nps is another
important parameter that can affect cellular interactions of nps .
in general , nps with positively charged surfaces ( e.g. , nh2 termini that are protonated under physiological ph )
are known
to nonspecifically interact with cells and cause toxicity via membrane
destabilization , which can be typically diminished by charge neutralization
or carboxylation . in particular , poly(amidoamine ) ( pamam ) dendrimers
have been extensively used to study charge - dependent cellular interactions
because of their chemically well - defined , dendritic structure that
amplifies the effect of surface groups .
although the effects of peg corona and surface charges of nps on
their cellular interactions have been widely studied independently ,
it has not been fully understood to date how they collectively affect
the nano bio interactions .
our group recently synthesized
amphiphilic pegylated dendron - based
copolymers ( pdcs ) consisting of hydrophobic poly(-caprolactone )
( pcl ) , polyester dendron , and multiple hydrophilic peg chains . when the self - assembly characteristics of pdcs
were compared to those of similar linear - block copolymers ( lbcs )
,
pdcs displayed critical micelle concentrations ( cmc ) ( 1010 m ) that were up to
2 orders of magnitude lower than lbcs , indicating their enhanced thermodynamic
stability .
molecular dynamics ( md ) simulations
also revealed that the dendron micelles ( dms ) were almost completely
covered by a dense peg layer , whereas lbc - based micelles displayed
a considerable degree of pcl core exposure at their surface .
this
difference was attributed to the high - density peg layers provided
by the dendritic architecture of pdcs .
interestingly , unlike pamam
dendrimers , after the termini of the peg ( 2000 g / mol ) chains of the
pdcs were modified to primary amines , the resulting dms ( dm2k - nh2 ) did not exhibit any noticeable cellular interactions
or toxicity .
this charge - independent
end - group effect of the dm2k - nh2 was attributed
to a 10-fold higher molecular - weight - to - charge - number of the pdcs
than that of pamam dendrimers and back - folding of the terminal amines
into the ethylene glycol ( ch2ch2o )
backbone .
these results demonstrated that the long peg chain contributes
to the sequestration of charge - dependent end - group effects of the
dms .
our previous findings raised a vital question of whether
the end - group
charge effects of dms could be tuned through changing the peg chain
length . to address this question
, we hypothesized that the lack of
charge - dependent end - group effects of dm2k - nh2 could turn to be charge - dependent by shortening the peg chain lengths .
in this study , we synthesized a series of pdcs with shorter peg chains
( 600 g / mol ) ( pdc600 ) and investigated the relation between
the length of peg chains and the end - group charge effect of dms on
their cellular interactions .
the dm membrane interactions were
further studied in atomistic detail using md simulations .
in addition ,
the effect of polymeric architecture on the encapsulation stability
of the resulting micelles was investigated . by comparing dms and lbc - based
micelles ( lms ) encapsulating a membrane permeable fluorescent dye ,
this study presents a systematic investigation of the effect of surface
charge , peg chain length , and polymeric architecture on the cellular
interactions and stability of polymeric micelles .
boc peg amine , hcl salt
( boc - peg600-nh2 ) ( mw 600 g / mol ) , was purchased
from jenkem technology
usa inc .
regenerated cellulose dialysis membrane ( 3.5 kg / mol mwco ) was
purchased from spectrum laboratories ( rancho dominguez , ca ) .
generation
4 ( g4 ) pamam dendrimers and all other reagents were purchased from
sigma - aldrich co. ( st .
amine - reactive
pcl3.5k - g3-pnp ( 4-nitrophenyl ester ) was synthesized according
to our published protocol followed by
pegylation .
briefly , pcl3.5k - g3-pnp ( 150 mg , 0.026 mmol )
was dissolved in dimethylformamide ( dmf ) ( 4 ml ) for 2 h and added
into nh2-peg600-boc ( 150 mg , 0.249 mmol ) in
dmf ( 3 ml ) in the presence of triethylamine ( tea ) ( 84.17 mg , 0.832
mmol ) .
the reaction was carried out at room temperature for 24 h.
the reaction mixture was then transferred to a dialysis membrane ( 3.5
kg / mol mwco ) , dialyzed against distilled water ( dh2o ) for
48 h with five water changes , and lyophilized using a freezone 4.5
freeze - dryer ( labconco corp . , kanas city , mo ) for 2 days .
the resulting
pcl3.5k - g3-peg600-boc was obtained as powder .
to remove the boc protecting groups in the product , a solution of
pcl3.5k - g3-peg600-boc was dissolved in a 1:1
mixture of dichloromethane and trifluoroacetic acid ( 2 ml of total
volume ) and stirred for 30 min at room temperature .
the solvent was
then completely removed by rotary evaporation , and the purified product
was redissolved in dimethyl sulfoxide ( dmso ) ( 3 ml ) . after dialysis
for 24 h and lyophilization for 2 days ,
briefly , for
acetylation , pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol ) was dissolved in dmso ( 2 ml ) containing tea
( 38 mg , 0.376 mmol ) . acetic anhydride
( 31.95 mg , 0.313 mmol ) was then
added dropwise to the stirring solution . for carboxylation
, pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol )
was dissolved in dmso ( 2 ml ) containing pyridine ( 12.3 mg , 0.156 mmol )
to which a dmso solution ( 1 ml ) of succinic anhydride ( 15.7 mg , 0.156
mmol ) was added dropwise with vigorous stirring .
both reactions were
carried out at room temperature for 24 h. after dialysis for 24 h
and lyophilization for 2 days , pcl3.5k - g3-peg600-ac and pcl3.5k - g3-peg600-cooh were obtained
as powder . to prepare rhodamine - labeled pdc600 , pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol )
was dissolved
in dmso ( 2 ml ) to which an nhs - rhodamine ( 5.16 mg , 0.0098 mmol ) solution
in dmso ( 1 ml ) was subsequently added dropwise .
the reaction was carried
out at room temperature for 24 h. the remaining primary amine groups
were then acetylated by addition of acetic anhydride ( 31.95 mg , 0.313
mmol ) in the presence of tea ( 38 mg , 0.376 mmol ) , followed by stirring
for an additional 24 h at room temperature .
the reaction mixture was
dialyzed against water for 48 h to remove unreacted rhodamine followed
by 2 days of lyophilization , resulting in the final product of pdc600-rhod .
the same method was employed to synthesize pdc2k - rhod and linear pcl3.5k - peg2k - rhod
( lbc2k - rhod ) . in addition
, g4 pamam dendrimers , used
as control in this study ,
were purified using repetitive dialysis and lyophilization as described
earlier , followed by conjugation with
rhodamine , using edc / nhs chemistry , resulting in g4-nh2-rhod . to prepare blank
micelles , each
pdc ( pdc600-nh2 , pdc600-cooh , or
pdc600-ac ) ( 5 mg ) or linear block copolymer ( lbc , pcl3.5k - peg2k ) that was synthesized in our laboratory
as described earlier
was first dissolved
in dmf ( 2 ml ) , vortexed vigorously for 5 min , and stirred for 10 min
until it was completely dissolved .
dh2o ( 1.8 ml ) was then
added dropwise into each of the dmf solutions under stirring .
each
solution was dialyzed against dh2o ( 4 l ) in a dialysis
membrane ( 3.5 kg / mol mwco ) at room temperature for 24 h , followed
by centrifugation at 9300 g for 5 min to remove
large aggregates .
the supernatants containing the resulting micelles
were collected and immediately used for subsequent experiments .
the rhodamine - labeled micelles , dual - dye micelles , and frster
resonance energy transfer ( fret ) micelles were prepared using the
same method described above except for the inclusion of rhodamine - labeled
polymer or hydrophobic dyes in polymer solution . for rhodamine - labeled
micelles ,
pdc - rhod ( 0.5 mg , 10 wt % ) was mixed with each pdc solution
in dmf . for dual dye micelles , pdc600-rhod or lbc2k - rhod ( 0.5 mg , 10 wt % )
was incorporated into each polymer solution
in dmf , along with 3,3-dioctadecyloxacarbocyanine perchlorate
( dio , 25 g , 0.5 wt % ) .
finally , for fret micelles , dio ( 25
g , 0.5 wt % ) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine
perchlorate ( dii , 25 g , 0.5 wt % ) were added into pdc600-ac or pcl3.5k - peg2k in dmf .
particle size of the
various dms ( 1 mg / ml ) was measured using a nicomp
380 zeta potential / particle sizer ( particle sizing systems , santa
barbara , ca ) .
all measurements were performed in triplicate in double
distilled water using unfiltered micelle samples .
the morphology of
each dm was visualized using transmission electron microscopy ( tem ,
jem-1220 , jeol ltd . ,
( 5 l ) of each micellar
suspension ( 0.2 mg / ml ) was placed on a 300 mesh copper grid coated
with carbon ( electron microscopy sciences , hatfield , pa ) .
all samples
were stained with a drop ( 5 l ) of 2% phosphotungstic acid ( ph
7 ) and dried in a desiccator at room temperature for 1 day before
observed using tem as described earlier .
kb cells were seeded
in glass bottom culture dishes at a density of 1 or 4 10 cells / well on 8-well millicell ez slides ( millipore corporation ,
billerica , ma ) or glass - bottomed petri dishes ( mattek corporation ,
ashland , ma ) and incubated for 24 h. cells were then treated with
rhodamine - labeled dm600 ( 8 m ) , rhodamine - labeled
dm2k ( 4 m ) , rhodamine - labeled g4 pamam dendrimer
( 1 m ) , or free rhodamine ( 0.2 m ) all in pbs for 2 or
4 h. the concentrations of all samples were adjusted to normalize
the amounts of rhodamine using fluorescent spectroscopy prior to the
experiment . for
dio - loaded , rhodamine - labeled ( dual - dye ) micelles ,
kb cells were seeded in glass bottom culture dishes using the protocol
described above , and cells were treated with dual - dye dm600 ( 60 g / ml ) or dual - dye lbc(pcl3.5k - peg2k)-based micelles ( 60 g / ml ) in rpmi without supplements for
4 , 8 , 24 , or 48 h. after incubation , the cells were washed twice using
pbs and fixed using a 4% paraformaldehyde solution .
when the nuclear
staining was needed , a mounting agent containing dapi ( vectashield
h-1200 , vector laboratories , inc . , burlingame , ca ) was used after
fixation .
the fixed cells were visualized by a carl zeiss confocal
microscope ( lsm 710 , carl zeiss microimaging gmbh , gena , germany )
using a diode laser ( 405 nm ) for dapi , an argon laser ( 488 nm ) for
dio , and a dpss laser ( 561 nm ) for rhodamine . a 40 objective
( objective c - apochromat
40x/1,20 w corr , carl zeiss
microimaging gmbh , gena , germany ) was used .
all obtained images were
processed using zen software ( carl zeiss microimaging gmbh , gena ,
germany ) .
kb cells were seeded in 12-well
plates at a density of 2 10 cells / well and incubated
for 24 h. cells were then treated with rhodamine - labeled dm600 ( 8 m ) , rhodamine - labeled dm2k ( 4 m ) , rhodamine - labeled
g4 pamam dendrimer ( 1 m ) , or free rhodamine ( 0.2 m ) ,
all in pbs , for 2 h. for dual - dye micelles , cells were treated with
dual - dye dm600 ( 60 g / ml ) or dual - dye lbc ( pcl3.5k - peg2k)-based micelles ( 60 g / ml ) in rpmi
without supplements for 4 , 8 , 24 , or 48 h. the treated cells were
washed twice with pbs and suspended by addition of trypsin / edta .
the
cell suspensions were centrifuged at 228 g for 5 min , and the supernatants were discarded .
the resulting cell
pellets were resuspended in a paraformaldehyde solution ( 1% w / v , 500
l ) and transferred to flow cytometry sample tubes .
the samples
were examined using a lsr fortessa flow cytometer ( becton dickinson ,
franklin lakes , nj ) as described earlier .
kb cells were seeded in 96-well plates at
a density of 1 10 cells / well and grown in rpmi
media for 24 h. cells were then treated with various dms and g4 pamam
dendrimers at various concentrations ranging from 1 to 100 m .
cell viability was
assessed using a celltiter 96 aqueous one solution ( mts ) assay ( promega ,
madison , wi ) according to the manufacturer s protocol .
the
uv absorbance was measured at 490 nm using a labsystems multiskan
plus microplate reader ( labsystems , finland ) .
mean cell viabilities
were determined relative to a negative control ( untreated cells ) and
a positive control ( cells lysed using 0.1% triton - x ) ( n = 5 ) .
fret - dm600-ac micelles
( 60 g / ml ) or fret - lbc ( pcl3.5k - peg2k)-based
micelles ( 60 g / ml ) were incubated in pbs at 37 c for
up to 48 h. fluorescence intensities were measured using a spectrofluormeter
( rf 1501 , shimadzu , japan ) by scanning the emission spectra from 495
to 600 nm at an excitation wavelength of 484 nm .
the md
simulations were performed with namd and
the charmm force field ( charmm27 , c35r revision for ethers and general
force field ) .
nonbonding interactions were
calculated using a cutoff distance of d = 10 ,
and long - range electrostatic interactions were calculated by the pme
method .
the systems were simulated in
the npt ensemble , using the langevin dynamics with a damping constant
of lang = 0.1 p / s , and the time step of 2 fs .
the
micelles were prepared from nagg = 15
individual copolymers ( unimers ) of pdc - nh3 with
two different peg chain lengths of molecular weight 600 and 2000 g / mol .
initially , the unimers were spherically distributed to form the basic
micelle structure and compacted by brief simulations in vacuum .
the
micelles were then simulated alone in ionic solutions for 10 ns at t = 300 k and p = 1 bar .
we also used charmm - gui
( a web - based graphical user interface for charmm ) to prepare a biological membrane consisting of two different types
of lipid molecules .
the dipalmitoylphosphatidylcholine ( dmpc ) and
dipalmitoylphosphatidylglycerol ( dmpg ) lipid molecules were used in
a 3:1 ratio .
similar to the condition used for the micelles , the membrane
( size 230 230 ) was equilibrated for 2 ns ( lang = 1.0 p / s ) using the charmm36 lipid force field .
after
equilibrium , both micelles were placed 5 above the membrane ,
and the whole systems were placed in an ionic solution with a salt
concentration of 150 mm ( physiological salt concentration ) .
first ,
the systems were equilibrated for 5 ns while keeping the micelle and
membrane atoms fixed . then
, all the atoms were released , and the simulations
were continued for 75 ns ( lang= 0.1 p / s ) .
we continued
the micelle simulation without membrane for 30 ns to find out the
effect of the negatively charged membrane on the morphology of positively
charged micelle .
the coulombic potential energy was calculated from
eq 11where u(r ) is coulombic potential energy ,
is the dielectric constant
of water , 0 is the vacuum permittivity , i is the number of positively charged point at the surface
of the micelle , j is the number of negatively charged
point at the upper layer of the lipid bilayer , and rij is the distance between the positive
and negative charge ( qi = qj = 1.6
10 c , = 80 , and 0 = 8.8542 10 c n m ) .
end - group - modified
pdcs were synthesized using a modified version of our previously published
protocol . briefly , alkyne - core - functionalized
polyester g3 dendron ( 870 g / mol ) was conjugated with azido - functionalized
pcl ( 3500 g / mol ) using click chemistry , followed by pegylation of
the dendritic arms using heterobifunctional nh2-peg600-boc .
the end groups of the pdcs were then modified to amine
( pdc600-nh2 ) , carboxylate ( pdc600-cooh ) , and acetamide groups ( pdc600-ac ) , followed by
extensive characterization using h nmr and gpc ( table 1 and figure s1 of the supporting
information )
briefly , the characteristic
peaks of polyester dendron were observed at 4.404.18 and 1.221.15
ppm .
the repeating unit of pcl appeared at 4.06 , 2.30 , 1.62 , and 1.35
ppm , and the repeating unit of peg appeared at 3.62 ppm . also , the
characteristic peaks of terminal groups appeared at 3.13 ppm for pdc600-nh2 , 1.96 ppm for pdc600-ac , and
2.52 and 2.62 ppm for pdc600-cooh .
to employ fluorescence - based
analytical techniques , rhodamine - conjugated pdc600 ( pdc600-rhod ) was also prepared and characterized .
dms were prepared
by self - assembly of the end - group - modified pdcs using the dialysis
method .
figure 1a depicts the structure of a dm assembled from amine - terminated pdcs
as an example .
the particle size distributions , zeta - potentials , and
cmcs of all three end - group - modified dms , along with dm2k - nh2 included as a control , are listed in table 2 .
the hydrodynamic diameters of dm600-nh2 , dm600-ac , and dm600-cooh were
measured to be approximately 47 , 23 , and 47 nm , respectively , which
was consistent with our previous findings where dm2k - nh2 and dm2k - cooh were larger than dm2k - ac .
the zeta - potential measurements
confirmed the surface modification , where dm600-cooh and
dm600-ac were measured at 21 and 0 mv , respectively ,
which was significantly different from + 30 mv of dm600-nh2 .
the cmcs of the three pdcs were consistent with our previously
measured values ( on the order of 10 m ) of pdcs
with 2k peg ( pdc2k ) as well as that of lms ( 2.4
10 m ) .
transmission electron
microscopy ( tem ) images ( figure s2 ) revealed
that all dm600 were spherical with sizes similar to those
of dm2k .
interestingly , the
core size of the both dm600 and dm2k were similar ,
even though the pdc600 has a lower hlb ( 16.56 for pdc2k and 12.37 for pdc600 ) , which suggested an increased
copolymer aggregation number ( nagg ) for
dm600 . collectively , these data demonstrate
that the end - group - modified pdcs were successfully prepared and formed
dms with various surface groups .
number - averaged
molecular weight , mn , estimated by h nmr . measured
by gpc using dn / dc values . calculated
by weight - average molecular
weights divided by number - average molecular weights using the data
obtained by gpc .
not measured
due to the limited
solubility of amine - terminated pdc in thf .
cellular interactions of end - group - modified
dms and g4 pamam dendrimers .
( b ) flow cytometry results of the cell association of end - group - modified
dms and g4 pamam dendrimers after 2 h of incubation .
confocal images
of kb cells treated with ( c ) rhodamine - labeled dm2k - nh2 , ( d ) dm600-nh2 , and ( e ) g4 pamam at
37 c for 2 h. nuclei were stained using dapi ( blue color ) .
note that both amine - terminated dm600-nh2 ( d ) and pamam dendrimers ( e ) exhibit significant
cell interactions as opposed to dm2k - nh2 ( c ) ,
dm600-ac , and dm600-cooh , indicating that the
role of the surface charge and peg chain length on cell interactions
of dms .
the end - group effect of dms
with shorter peg chains on their cell
interactions was first evaluated in terms of cellular uptake and cytotoxicity .
after incubation with kb cells , the in vitro behaviors
of dm600 with nh2 , cooh , and
ac terminal groups , dm2k - nh2 , and g4
pamam dendrimers ( g4-nh2 ) were observed using confocal
microscopy and flow cytometry , as shown in figure 1b e .
pdc600-rhod was incorporated at 10%
( w / w ) of each dm600 for fluorescence - based detection .
no
significant cellular interactions were observed for charge neutral
dm600-ac and negatively charged dm600-cooh .
the positive control g4-nh2 exhibited a strong , charge - dependent
interaction as expected . as we reported earlier , dm2k - nh2 , despite their positively
charged surfaces , did not show any noticeable cellular interactions .
markedly , dm600-nh2 displayed a significant
cellular interaction , which proved our hypothesis that shortening
the peg chain length would enhance the end - group effect of dms , converting
previously observed charge - independent interactions to be charge - dependent .
we verified this result using the mcf-7 cell line as well ( figure s3 ) , which confirmed that the observed
charge - dependent cell interactions of dm600 were not cell
specific .
the charge - dependent cytotoxicity profiles of dm600 were also evaluated using an mts assay ( figure 2 ) . unlike dm2k - nh2 , dm600-ac , and dm600-cooh that all exhibited no apparent
cytotoxicity , dm600-nh2 displayed moderate cytotoxicity
at 100 m .
however , the cytotoxicity of dm600-nh2 appeared to be significantly smaller than that of g4-nh2 , probably due to its 5-fold higher molecular - weight - to - surface - charge
ratio than g4-nh2 .
these cell uptake and cytotoxicity results
indicate that the length of the peg corona modulates the charge - dependent
cellular interactions of dms .
cell viability of kb cells after treated with
end - group - modified
dms and g4 pamam dendrimers at concentrations of 1100 m
for 24 h , as measured using mts assay .
amine - terminated pamam dendrimers
induce significant cytotoxicity , and dm600-nh2 induced a modest level of cytotoxicity only at a high concentration .
all other micelles that do not show cell interactions do not cause
any noticeable cell death .
* p < 0.05 compared
to the negative control . to better understand
these observations , we conducted atomistic molecular dynamics ( md )
simulations of the experimental systems .
figures 3a and 3d show the conformation of dm600-nh3 and dm2k - nh3 in 150 mm nacl solution after 30 ns equilibration .
both micelles were stable , as determined by
the maintenance of their integrity and spherical shape .
we also modeled
a biological membrane in the same solution , using dipalmitoylphosphatidylcholine
( neutral ) and dipalmitoylphosphatidylglycerol ( negatively
charged ) lipid molecules in a 3:1 ratio .
then we placed both dms within
5 of the membrane and simulated the whole system .
over the course of 75 ns simulations , significant
differences in the behavior of the two dms were observed .
after 30
ns of equilibration , individual pdcs were pulled away from the core
of dm600-nh3 and drawn toward the
membrane surface , due to strong coulombic attraction , as shown in
figure 3b .
after 60 ns , dm600-nh3 began to flatten and spread over the membrane
surface , and at 75 ns it formed a pancake - like shape ( figure 3c ) .
coherent binding of the terminal amine groups
of pdcs to the negatively charged membrane .
the short peg chains provide
direct links between the nh3 groups
and the dendron holding them , which means that several nh3 groups attached to the same dendron can act in
a concerted way ( like a polyion ) in the presence of charged membrane
pulling them by coulombic coupling to the membrane .
on the other hand ,
dm2k - nh3 stably settled on the membrane
but did not display any noticeable conformational changes ( figure 3e , f ) .
the coulombic binding energies between dms
and the membrane were calculated with our code ( no ions considered )
and found to be 17% higher for dm600-nh3 than dm2k - nh3 ( 0.224
10 j vs 0.189 10 j ) .
these results were in agreement with our in vitro observations of the dm600-nh3 cell
insertion ( stronger interactions with the membrane ) .
atomistic md simulations
of ( a c ) dm600-nh3 and ( d f )
dm2k - nh3 in an ionic solution ( 150
mm nacl ) with and without
lipid membranes .
the functionalized peg chains are shown in atomistic details
with the amine termini highlighted in red .
( a , d ) show the micelles
in solution ; ( b , c , e , f ) show the micelles placed on lipid membranes
for 30 ns ( b , e ) and 75 ns ( c , f ) .
arrows indicate the stretching
and deformation of dm600-nh3 upon
interaction with the lipid membrane .
next , we analyzed further the mechanism of peg
chain - length - dependent
cellular interactions of dms .
our previous study suggested that the
lack of cellular interactions of dm2k - nh2 was
in part due to the back - folding of the amine termini into the peg
layers , impeding the cellular interactions .
we calculated the number of oxygen atoms in the peg chains present
within a distance of 2.5 from the terminal amine hydrogens
of dm600-nh3 by averaging the results
over 300 frames ( 1.2 ns of equilibration ) to quantitate the number
of intramolecular hydrogen bonds that can be formed .
the average numbers
of oxygen atoms were 165 8.9 and 212 8.6 per frame for
dm600-nh3 and dm2k - nh3 , respectively . a lower number of oxygen atoms
that are available for hydrogen bond formation would indicate less
sequestration of the amine termini by peg chains , thus increasing
the exposure of amine groups at the dm600-nh3 surface .
this phenomenon was also supported by the zeta - potential
measurements of dm600-nh2 and dm2k - nh2 , as summarized in table 2 .
the higher surface exposure of the amine termini on dm600-nh2 resulted in a higher zeta - potential value ( 30
mv ) compared to that ( 21 mv ) of dm2k - nh2 . taken together , these results revealed that charge - dependent end - group
effect of dms could be pronounced or sequestered by the number of
hydrogen bonds formed between the peg backbone and the amine termini .
the increased cellular interactions of dm600-nh2 can be also explained by the intrinsic properties of the
corresponding pdcs .
the charge - number - to - molecular - weight ratio is
approximately 2-fold higher for pdc600 than pdc2k , which accentuates the end - group effect of the dms .
furthermore ,
shorter peg chains lead to a decreased hlb of the copolymer , resulting
in a higher nagg of micelles . a dm with a higher
nagg contains more pdcs , and thus more terminal groups are expected to
be present on its surface .
the lower the number of intramolecular
hydrogen bonds , the higher the charge - number - to - molecular - weight ratio
and the greater nagg , which all contribute
to the increased cellular interactions of dm600-nh2 and a more pronounced end - group effect compared to dm2k - nh2 .
low - generation pamam dendrimers
( generations 25 )
( 26 nm ) have been reported to internalize into cells
via energy - dependent endocytosis . on the other hand , in addition
to energy - dependent mechanisms ,
energy - independent mechanisms such
as nanoscale hole formation have been proposed for larger g7 pamam
( 10 nm ) dendrimers .
the nanoscale
hole formation causes cytoplasm leakage and is usually accompanied
by higher cytotoxicity .
since the end groups of dms are coordinated
by g3 dendrons and the self - assembled structures are much larger than
g7 dendrimers , we sought to determine which dendrimer internalization
mechanism governs cell entry of dm600-nh2 .
the cellular uptake studies were carried out at 4 c to investigate
whether the nonspecific charge - dependent cell interactions of dm600-nh2 were energy - dependent .
figure 4a shows that dm600-nh2 was mostly observed
on the cell surface without significant cell entry , indicating that
their uptake was primarily energy - dependent .
additionally , flow cytometry
analysis ( figure 4b ) revealed that the cell
association of dm600-nh2 at 4 c was only
25% of that at 37 c .
we then concluded that the cellular internalization
mechanism of dm600-nh2 was similar to that of
low - generation pamam dendrimers , which was in good agreement with
their moderate cytotoxicity shown earlier .
however , further investigation
is obviously required to fully understand the uptake mechanism of
dm600-nh2 .
the md simulation results demonstrated that the
nonspecific membrane interactions may likely result in a partial pulling
of individual pdcs from dm600-nh2 , followed
by its large - scale deformations , causing the destabilization of the
micelle structure following the cell membrane interaction .
since this
may deteriorate the therapeutic effect when dms are used as drug carriers ,
we examined the integrity of dm600-nh2 upon
cell internalization . to investigate this
, we prepared a dual - dye
dm system by encapsulating dio into rhodamine - labeled dm600-nh2 , which enabled colocalization studies using confocal
microscopy .
as shown in figure 4c e ,
red fluorescence ( rhodamine ) and green signals ( dio ) were overlapped
inside the kb cells after treatment with dm600-nh2 at 37 c for 4 h. a colocalization analysis of the red and
green signals was then performed using imagej .
the pearson s coefficient of these two colors was
0.939 , which indicated almost complete colocalization of dm600-nh2 with dio . to exclude the possibility
that dio was
released extracellularly and then cointernalized into cells along
with rhodamine - labeled dms , kb cells were also treated with a mixture
of dio and rhodamine - labeled dm600-nh2 . the
poor correlation ( pearson s coefficient : 0.317 ) between the
two signals as shown in figure 4f
h
confirmed that extracellularly released dio was not cointernalized
into cells with dm600-nh2 .
these results demonstrated
that dm600-nh2 maintained its structural integrity
during its nonspecific interaction with the cell membrane and carried
the hydrophobic cargo into the cells .
( a ) kb cells treated with rhodamine - labeled
dm600-nh2 at 4 c for 2 h. ( b ) flow cytometry
histograms representing
the cell association of dm600-nh2 at and 37
c .
the lack of internalization and the significantly reduced
cell association at 4 c indicate that dm600-nh2 internalizes into kb cells in an energy - dependent manner .
( c e ) kb cells treated with dual - dye dm600-nh2 at 37 c for 4 h. ( f h ) kb cells treated with
the mixture of free dio and rhodamine - labeled dm600-nh2 37 c for 4 h. ( c , f : red channel for rhodamine ; d ,
g : green channel for dio ; e , h : merged images ) the correlation ( pearson s )
coefficients between red and green signals are 0.939 and 0.317 in
( e ) and ( h ) , respectively .
the colocalization of the two signals shown
in ( e ) confirms that dm600-nh2 internalizes
into the cells together with the encapsulated dye .
one of the major rationales behind
our dm design was to take advantage
of the dendritic structure to achieve enhanced structural stability
over conventional lbc - based systems owing to its preorganized conical
molecular architecture and ability to accommodate a significantly
denser outer peg layer . to test this hypothesis , we compared dual - dye
dms with lms in terms of their ability to prevent premature release
of hydrophobic cargos .
kb cells were incubated and monitored after
treatment with dual - dye dm600-ac and lms made from pcl3.5k - peg2k - ome . note that we used charge neutral
micelles to inhibit their nonspecific cell entry based on electrostatic
interactions . as shown in figure 5a , b and figure s4 ,
the lack of a rhodamine signal indicated
that neither dm600-ac nor lms were internalized into the
cells even after 48 h of incubation .
interestingly , a negligible uptake
of dio was observed from kb cells treated with dm600-ac ,
whereas a significantly stronger dio signal was observed for cells
treated with lms .
flow cytometry analysis shown in figure 5c further supported that the cell - associated dio
signal was higher for the cells incubated with lms than those with
dm600-ac .
these results are in agreement with a previous
report where hydrophobic probes such as dio that were encapsulated
into lms composed of pcl peg or polylactide peg were
released from the micelles extracellularly . because the both micelles showed a great degree of thermodynamic
stability ( low cmcs ) and maintained structural integrity at the testing
concentration ( 60 g / ml ) as measured by fret ( figure s5 ) , this uncontrolled
release was attributed to the hydrophobic cell membranes extracting
the hydrophobic cargos from the micelle cores .
our results herein
indicate the superior stability of dms over lms for hydrophobic drug
delivery . despite the similar cmc values of dms and lms , the dendritic
architecture , in combination of the dense peg layers of dms , likely
allowed enhanced protection of the encapsulated hydrophobic cargo
molecules compared to lms .
this enhanced stability is particularly
important for the future development of targeted nps to minimize premature
drug release and ultimately improve therapeutic efficacy .
confocal images
of kb cells treated with dual - dye ( a ) dm600-ac and ( b )
lbc ( pcl3.5k - peg2k)-based micelles
at 37 c for 4 h. images a and b are merged images of red ( rhodamine )
and green ( dio ) channels along with differential interference contrast
( dic ) images . ( c )
flow cytometry data of the cellular association
of dm600-ac , lbc - based micelles , and free dio at various
time points .
scale bar : 10 m . a significantly lower extracellular
release of the encapsulated dio was observed in dm600-ac
compared to lbc - based micelles , indicating the enhanced stability
of the encapsulated dye in dms .
here we report that the length of hydrophilic
peg chains can control
the charged end - group effect of dms . with
the short peg chains ( 600
g / mol ) , positively charged dm600-nh2 showed
charge - dependent nonspecific cellular interactions and modest cytotoxicity ,
whereas the dms prepared from longer peg chains ( 2000 g / mol ) , dm2k - nh2 , did not .
this result was further supported
by the md data , where dm600-nh2 exhibited less
hydrogen bonding formation than dm2k - nh2 , thereby
pronouncing the surface charge effect .
surface modification of dm600 to charge neutral or negatively charged groups substantially
decreased the charge - based cell interactions .
in addition , we found
that dm600-nh2 maintained its structural integrity
after binding to kb cell membranes and internalized through an energy - dependent
pathway .
no dms display any noticeable leakage of an encapsulated
dye , whereas the lm allowed a degree of dye leakage , indicating that
the dense , dendritic peg corona enhanced the encapsulation stability
of dms .
our results integrating both in vitro and
md simulation data support the potential to use dms as a drug delivery
platform of which cellular interactions can be facilely modulated
by surface functionalization and the peg chain length .
this study
helps to understand the role of surface charge , peg corona , and polymeric
architecture on tuning the biological properties of nps , providing
a critical design cue for dms as well as other types of polymeric
micelles . | to
systematically investigate the relationship among surface charge ,
peg chain length , and nano bio interactions of dendron - based
micelles ( dms ) , a series of pegylated dms with various end groups
( nh2 , ac , and cooh ) and peg chain
lengths ( 600 and 2000 g / mol ) are prepared and tested in vitro .
the dms with longer peg chains ( dm2k ) do not interact
with cells despite their positively charged surfaces . in sharp contrast ,
the dms with shorter peg chains ( dm600 ) exhibit charge - dependent
cellular interactions , as observed in both in vitro and molecular dynamics ( md ) simulation results .
furthermore , all
dms with different charges display enhanced stability for hydrophobic
dye encapsulation compared to conventional linear - block copolymer - based
micelles , by allowing only a minimal leakage of the dye in
vitro .
our results demonstrate the critical roles of the
peg chain length and polymeric architecture on the terminal charge
effect and the stability of micelles , which provides an important
design cue for polymeric micelles . |
a 55-year - old man who was a known case of scapular bone osteosarcoma , was referred to our clinic with ocular symptoms including acute painful decreased vision , proptosis , conjunctival injection , and chemosis .
he had undergone surgical excision of the original tumor and received systemic chemotherapy 4 months before .
imaging studies and incisional biopsy were performed for the orbital lesion , the histopathological examination confirmed the diagnosis of metastatic osteosarcoma .
the patient was referred to the oncologist for palliative chemotherapy and further intervention ; however , he deceased 2 months later due to sepsis in the context of immunosuppression .
metastatic involvement of the orbit due to osteosarcoma is a rare condition manifesting with orbital mass , pain , diplopia and ocular motility disturbance .
although there is no effective treatment , the combination of modalities such as chemotherapy , radiotherapy , and surgery may delay progression of the disease .
metastatic orbital lesion due to osteosarcoma is a rare condition and manifestations include orbital mass , pain , diplopia , chemosis and ocular motility disturbance depending on the location of the mass and its effect on adjacent structures .
there is no effective treatment , but the combination of treatment modalities including chemotherapy , radiotherapy and surgery for tumor debulking may delay disease progression .
a 55-year - old man was referred to the oculoplastics clinic at farabi eye hospital , tehran , iran with acute onset painful decrease of vision and protrusion of his left eye .
he was a known case of left scapular bone osteosarcoma and had undergone surgical excision of the original tumor and chemotherapy , approximately 4 months before .
visual acuity was counting fingers and 20/20 in the left and right eyes , respectively .
ophthalmic examination revealed proptosis , severe lid swelling , chemosis and conjunctival injection in his left eye [ figure 1 ] .
appearance of the patient at presentation revealed proptosis , severe lid swelling and chemosis of the left eye . in the next step ,
orbital ct - scan revealed a large heterogeneous mass lesion in the left intraconal orbital space with tiny foci of hyperintensity [ figure 2 ] .
for further investigation , mri was done in which an intraconal lesion , hypointense in some planes and hyperintense in others , was noted on t1 images [ figure 3a ] .
the intensity of the lesion was heterogeneous to hyperintense on t2 images [ figure 3b and c ] which also displayed ring enhancement on gadolinium enhanced t1 images [ figure 3d ] .
orbital mri showed an intraconal mass that was heterogeneous to hyperintense on t1 images ( a ) and heterogeneous to hyperintense on t2 images ( b and c ) with gadolinium enhancement at the rim of the lesion on t1 images ( d ) . in some sections
, the mass seems to be hyperintense on t1 images ( a ) and in some others , hypointense ( d ) .
histopathological examination of the tumor revealed malignant spindle cells with osteoid formation and mineralization which was compatible with metastatic osteosarcoma [ figure 4 ] .
histopathological studies revealed malignant spindle cells with osteoid formation and mineralization compatible with metastatic osteosarcoma .
the orbital metastasis being confirmed , the patient underwent systemic evaluation for any other site of metastasis ; however , there was no other organ involvement .
unfortunately , after 2 months of chemotherapy , he deceased in the clinical setting of sepsis probably due to systemic immunosuppression .
osteosarcoma is the most common malignant bone tumor and may involve the long bones of young adults primarily .
it can be seen in association with hereditary retinoblastoma , previous exposure to ionizing radiation or paget disease .
the most common site of involvement is the medulla of the metaphysis in long bones , particularly the distal femur , proximal tibia and proximal humerus .
it has been classified conventionally into osteoblastic ( 50% ) , chondroblastic ( 25% ) and fibroblastic ( < 25% ) variants ; a small percentage of them are categorized into telangiectatic , small cell multifocal , parosteal , periosteal , and extra - osseous variants .
factors affecting prognosis include tumor histopathology , size , raised pre- and post - operative serum alkaline phosphatase levels , involvement of lymph nodes and treatment modality .
however , age , gender , history of trauma or bone fracture and the site of primary osteosarcoma do not influence prognosis .
the most common sites for metastases for tumors originating from long bones is the lung and the second one are bones . by advances in systemic chemotherapy regimens , the long - term survival of patients without metastasis
has now improved to 70% ; however , in patients with metastasis or disease recurrence , survival is < 20% .
metastatic involvement of the orbit is a rare condition ; orbital metastasis can originate from any primary tumor in the body .
the most common solid tumors in adults with orbital metastasis are breast and lung tumors , followed by cancers of the genitourinary tract and gastrointestinal system .
some studies showed that leukemia and neuroblastoma are the most frequent causes of orbital metastasis .
the most common symptoms of metastatic orbital osteosarcoma are pain , diplopia and ocular dysmotility .
the patient was an 8-year - old boy with osteosarcoma in his right tibia with involvement of the right eye 1 month after the diagnosis .
attili et al reported another case with right tibial osteosarcoma , painful protrusion of the right eyeball and metastatic orbital osteosarcoma .
it is interesting that in all previously reported cases of metastatic orbital osteosarcoma , the primary and metastatic sites were both on the right side , but in the present case , both of them originated from the left side . in previous reports , the primary site of osteosarcoma was the right tibia and in our case , it was the left scapula which may explain left orbit preference .
as the current case is only the fifth subject with metastatic orbital osteosarcoma , this finding can be purely coincidental .
however , it is notable that in all cases with metastatic orbital osteosarcoma , the lung was not involved by metastasis .
it is believed that spread of tumor cells to the orbit may occur through botson 's paravertebral venous plexus which has already been hypothesized in other studies .
most metastatic osteosarcomas show hyperintense signal on t1 scans , whereas signal intensity was heterogeneous to high in our case .
we assume that various amounts of necrosis , hemorrhage and mineralization in osteoid tissue may have resulted in this particular presentation on t1-weighted images .
typical ring enhancement was seen on gadolinium enhanced images and a heterogeneous mass was revealed on t2 sections .
combination therapy using different treatment modalities including radiotherapy , chemotherapy and surgery may be effective for palliative measures , maintenance of vision and regression of metastatic tumor . | purpose : to report a case of orbital metastasis from scapular bone osteosarcoma.case report : a 55-year - old man who was a known case of scapular bone osteosarcoma , was referred to our clinic with ocular symptoms including acute painful decreased vision , proptosis , conjunctival injection , and chemosis .
he had undergone surgical excision of the original tumor and received systemic chemotherapy 4 months before .
imaging studies and incisional biopsy were performed for the orbital lesion , the histopathological examination confirmed the diagnosis of metastatic osteosarcoma .
the patient was referred to the oncologist for palliative chemotherapy and further intervention ; however , he deceased 2 months later due to sepsis in the context of immunosuppression.conclusion:metastatic involvement of the orbit due to osteosarcoma is a rare condition manifesting with orbital mass , pain , diplopia and ocular motility disturbance .
although there is no effective treatment , the combination of modalities such as chemotherapy , radiotherapy , and surgery may delay progression of the disease . |
granulocytic sarcoma ( gs ) , also known as chloroma , extramedullary myeloid tumor , myeloid sarcoma , leukocytic sarcoma and myelosarcoma is defined as an extramedullary tumor mass of immature cells of myeloid lineage [ 1 - 3 ] .
was reported by burn in 1811 . in 1853 , king used the term chloroma , because of its greenish color , to refer to gs whose tumoral cells produced myeloperoxidase with exposure to the air , but because this tumor is not always green , granulocytic sarcoma is a more appropriate term . in the head and neck areas ,
gs is often seen in the soft tissues of orbit , nasal cavity and paranasal sinuses , but it can also appear in any location throughout the body including the skin , breasts , gastrointestinal , genitourinary , respiratory tracts , peripheral nerves and lymph nodes [ 7 - 10 ] .
there is a female predilection and most cases occur in childhood . although intensive chemotherapy is the main treatment choice for gs , the related death and relapse rates are the essential factors for prediction of prognosis of gs [ 1 , 2 , 12 ] . recently ,
improving leukemia stem cell biology understanding and considering interaction of the stroma and the host response , may introduce more markers which show the value of result prediction and guide therapy ; for example , cd47 which is highly expressed on leukemic stem cells compared to the normal hematopoietic stem cells and microrna expression patterns are additional markers for predicting worse survival rate .
some degrees of improvement can be achieved after surgery and radiotherapy , but the prognosis of gs is poor and most patients die within months .
it seems that only 2 cases have been reported in the literature with this appearance [ 14 , 15 ] .
a 45-year - old male referred to shahid beheshti maxillofacial pathology department with a two year history of generalized proliferative gingival maxillary lesions with palatal right side swelling and mandibular labially gingival lesions ( figure 1 ) .
the lesions were asymptomatic , without any bleeding or purulent discharge . the right submandibular lymph node was palpable and the patient noticed this swelling after the extraction of the third molar one month prior to his visit to our department .
the gingivally lesions were red and soft with irregular surfaces , and the palatal swelling had a purple - gray appearance with intact overlying mucosa ( figure 2 ) .
laboratory test results were normal , so the patient s diagnosis was stated as inflammatory and reactive hyperplastic lesions .
tissues needed for histopathologic evaluation were obtained from the gingivectomy of gingivally proliferative masses and with a full thickness flap from the palatal swelling .
soft tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin blocks . for standard pathological examination , the sections were prepared with h&e stain .
the slides of both gingival and palatal specimens showed a dense cellular infiltration under the epithelial layer in deeper portions .
the cells were mononuclear which showed pleomorphism with moderately amount of cytoplasm and round to oval nucleus with prominent nucleoli .
therefore , a series of differential diagnosis such as large cell lymphoma , plasmacytoma , poorly differentiated carcinoma and lymphoblastic leukemia was suggested ( figure 3 - 4 ) . for the final diagnosis
the tumor cells were diffusely positive with antibodies against lca , negative with cd20 , cd3 and cd79a and positive with c - kit . therefore , according to the ihc results in combination with morphological features , the final diagnosis was made as granulocytic sarcoma .
bone marrow biopsy and bone marrow aspiration were negative for malignant cells , and the laboratory tests revealed only an increase in the monocyte population .
the treatment protocol was 5 courses of induction chemotherapy with cytarabine , idarubici and doxorubicin and adjuvant whole brain radiotherapy . during the chemotherapy , the patient had neutropenia and thrombocytopenia .
although we tried to manage the complications , the clinical outcome became worse , and the patient died after a heart attack 10 months post diagnosis .
, dock found an association between granulocytic sarcoma and leukemia for the first time [ 16 , 17 ] .
clinically granulocytic sarcoma may occur in three categories : in a patient previously known to have acute myeloid leukemia ( aml ) ; as a sign of blast transformation in a patient with chronic myeloid leukemia ( cml ) , or other chronic myeloproliferative disorders ; in a patient who was previously well . nearly 40 cases of intraoral gs have been reported from 1883 up to now .
involvement of oral tissue without detectable evidence of leukemic cells in peripheral blood and bone marrow was observed in 14 cases [ 6 , 19 - 30 ] ; and only two patients had both jaws involvement [ 13 , 14 ] . the highly invasive and aggressive clinical behavior of gs might be explained by the findings of kabayashi et al . [ 31 , 32 ] . in one study , they found the capability of gs - derived cell line to bind both the bone marrow and the skin fibroblast , which resulted in the formation of extramedulary myeloid tumor .
gingival tissues due to expression of endothelial adhesion molecules , which may increase leukocyte infiltration , are more susceptible to leukemic cell invasion [ 7 , 8 , 16 ] . however , the association of aml with gingival occurrence of gs is extremely rare .
diagnosis of gs in a patient with known myeloid leukemia is not difficult . however , when presented as an isolated entity , like our case , it may be clinically misdiagnosed as an inflammatory lesion such a pyogenic granuloma ( pg ) , peripheral giant cell granuloma ( pgcg ) , gingival or periodontal abscess .
this may lead to surgical procedures such as gingivectomy ; however , the management of gs is different .
positive staining for cd45 is needed to confirm the hematological origin . in the next step ,
one or more myeloid markers such as myeloperoxidase , lysozyme , cd13 , cd14 , cd33 , cd34 , cd68 or c - kit should be positive . in the present case , tumoral cells were stained positively with c - kit . in patients without evidence of leukemia , antileukemic chemotherapy regime upon the initial diagnosis of gs
is associated with a lower probability of aml development , but the overall survival rate of patients with intraoral gs is poor . according to the literature , only four patients survived more than 3 years .
therefore , regular therapies such as treatment with combining growth factors and pharmacologic differentiating agents , and assessing appropriate allograft procedures in the remission period may be of importance .
because gs may be seen before the diagnosis of leukemia , or it could be presented without bone marrow involvement ( the same as our case ) , it is advisable to include gs in the differential diagnosis of severe gingival hyperplasia .
because gs may be seen before the diagnosis of leukemia , or it could be presented without bone marrow involvement ( the same as our case ) , it is advisable to include gs in the differential diagnosis of severe gingival hyperplasia . | granulocytic sarcoma ( gs ) , an unusual extramedullary tumor , is composed of immature granulocytic precursor cells . the intraoral occurrence of this tumor is extremely rare . here , we report a case of gs with palatal swelling , gingival lesions in maxilla and mandible and aleukemic presentation in a 45- year- old male . |
diabetes mellitus ( dm ) is caused by genetic and environmental interactions , along with changing lifestyles and an aging population , increasing incidence of diabetes .
diabetic neuropathy is one of the major complications of diabetes with both type 1 and type 2 . up to 50% of all diabetes
have polyneuropathy which is a major cause of morbidity and associated with increased mortality , and up to 26% of diabetics develop painful diabetic neuropathy with debilitating effects on quality of life [ 13 ] .
the rising incidence of diabetes and its negative impact on quality of life highlights the urgent need to develop biomarkers of early nerve damage .
the gold - standard method to evaluate morphological change in small nerve fibres was the skin biopsy ; however , this technique is limited by cost and invasiveness , provides no information about the function of nerve fibres , and can not be employed as a generalized screening test in all patients .
vitamin b12 ( vit b12 ) is a cofactor for methylmalonylcoa mutase , which converts methylmalonyl coa to succinyl coa .
measurement of total vit b12 suffers from some limitations ; in particular , most of the cobalamin measured is that bound to haptocorrin ( hc ) [ 5 , 6 ] .
methylmalonic acid ( mma ) may contribute to neuronal injury in many human conditions in which it accumulates , including methylmalonyl - coa mutase and vit b12 deficiency [ 7 , 8 ] .
the impaired activity of the enzyme leads to an accumulation of mma and an elevated plasma concentration .
mma is biochemically more stable in urine than in serum and has a 40-fold greater concentration in urine .
urinary methylmalonic acid ( umma ) concentration offers a potentially useful functional marker of vit b12 status .
moreover , the measurement of umma would be a less invasive method for the purpose of screening or epidemiologic studies .
in addition , umma is excreted very efficiently by the kidneys , which concentrates the metabolite in the urine and makes it a sensitive indicator of tissue depletion .
based on the above considerations , we believed that umma was an intermediate metabolite during the course of neuropathy , and there was an important relationship between umma and the formation of neuropathy .
the aim of the present study was to assess umma levels in patients presenting with and without diabetic polyneuropathy ( dpn ) and to determine the role of umma in dpn .
the study population consisted of 176 chinese han patients ( male 97 and female 79 ) with type 2 diabetes mellitus aged 3870 y recruited upon admission to the second hospital of shandong university between june in 2009 and june in 2011 who were eligible for the study .
exclusion criteria were as follows : history of serious heart and lung disease , diabetic kidney disease or other kidney diseases , pernicious anemia , intestinal surgery , and gastrointestinal disease , excluding antibiotics , colchicine , aminosalicylic acid , h2 receptor antagonists and proton pump inhibitors and other effects of gastrointestinal motility drugs , and the patients with the vit b12 or methylcobalamin treatment .
long - term vegetarians ( more than six months ) and those refused concurrent electrophysiological or laboratory testing were excluded too . according to the level of vit b12 , the patients were divided into 3 groups : group a ( vit b12
< 180 ng / l , n = 58 ) , group b ( vit b12 180400 ng / l , n = 68 ) , and group c ( vit b12 > 400
the fasting blood samples ( 22 ml / person ) via venipuncture into edta - coated tubes ( 5 ml ) for a full blood were counted to check glycosylated hemoglobin a1c ( hba1c ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl ) , low - density lipoprotein cholesterol ( ldl ) , serum creatinine ( scr ) , vit b12 , folic acid , ferritin , homocysteine ( hcy ) , hemoglobin ( hgb ) , and mean corpuscular volume ( mcv ) .
holotranscobalamin ( holotc ) level was determined by microparticle enzyme immunoassay on axsym analyzer ( both from abbott , usa ) .
the application of stable isotope dilution method for rapid extraction of the solid phase sample by gas chromatography mass spectrometry was used for the determination of blood methylmalonic acid ( bmma ) and umma .
diabetic polyneuropathy : the diagnosis of bilateral limb sensory and motor nerve conduction velocity ( sncv / mncv ) and amplitude was performed , according to standard techniques protocols , by means of keypoint , dantec equipment , for all individuals .
tests were carried out in a warm room to minimize the effects of temperature on nerve conduction velocity ( cv ) , and the limbs were warmed throughout with an infrared lamp to maintain a skin temperature over 35c .
suprathreshold stimulation was used , the frequency was l hz ( feeling ) and 1 hz ( movement ) , respectively , and course was both for 0.2 ms .
nerve conduction and electromyographic studies were performed in four limbs , which included 2 nerves of upper limbs and 2 nerves of lower limbs , by means of standard techniques .
we measured the amplitude and velocity of motor nerve conduction and sensor nerve conduction in 4 nerves : superficial peroneal , sural , median , and ulnar .
all experimental data were analysed by spss software 16.0 ( spss , chicago , il , usa ) .
the count data were analyzed by test , and logistic regression analysis was used for an independent risk factor of diabetic polyneuropathy .
patients in the two groups with normal vit b12 had decreased serum concentrations of hdl , ferritin , folic acid , holotc , and hgb and increased umma / creatinine and bmma compared to those lack vit b12 . in addition , dm type 2 patients with vit b12 over 400
ng / l had significantly increased ferritin , holotc , and hgb and decreased umma / creatinine compared to those with vit b12 180400 ng / l .
there was no significant difference in ldl , mcv , duration , scr , tg , bmi , and hba1c among the three groups .
multiple regression analysis revealed that umma / creatinine ( p = 0.001 ) , bmma ( p = 0.02 ) , diabetes duration ( p = 0.045 ) , and holotc ( p = 0.003 ) were variables that influenced dpn significantly and independently while the serum concentration of vit b12 did not have an independent significant influence on dpn ( table 2 ) . to value the relationship between umma / creatinine and dpn , we separated data according to whether umma / creatinine was normal or elevated .
it was observed that nerve sensory conduction velocity and nerve amplitude in the group of umma / creatinine > 3.5 mmol / mol decreased significantly .
all individual nerve conduction parameters except median nerve sensory and motor conduction velocity and ulnar nerve motor conduction velocity were statistically significant between the two groups ( see table 3 ) .
some significant correlations were revealed between the umma / creatinine and other electrophysiological markers of neuropathy severity ( table 4 ) . superficial peroneal nerve sensory and conduction velocity ( r = 0.496 , p = 0.0005 ; r = 0.327 , p = 0.003 , resp . ) and ulnar nerve sensory active nerve potential amplitude ( r = 0.315 , p = 0.005 ) were inversely correlated with the umma / creatinine .
sural nerve compound motor active potential amplitude ( r = 0.196 , p = 0.02 ) , sural nerve motor conduction velocity ( r = 0.205 , p = 0.01 ) , superficial peroneal nerve compound motor active potential amplitude ( r = 0.197 , p = 0.015 ) , and ulnar nerve sensory conduction velocity ( r = 0.187 , p = 0.02 ) showed modest inverse correlations with umma / creatinine .
our data showed polyneuropathy was present in 24.7% of our diabetic patients using electromyogram ( emg ) , which was higher than the previous report , maybe because of sample selection or the diagnostic criteria of neuropathy .
we demonstrated a significant association between polyneuropathy and duration of diabetes in our patients and no association with age , while , in the knuiman and young studies , neuropathy was correlated with age and duration of diabetes [ 12 , 13 ] .
this may reflect the age distribution ( all were below 70 years of age in the study ) .
the bmma level in the patients with vit b12 deficiency was significantly higher than in the people with normal vit b12 , while there was no significant difference between the two groups with normal vit b12 , which meant bmma was not sensitive for early vitamin b12 deficiency .
it is not considered the gold standard for the determination of vit b12 deficiency , because its concentrations are also elevated in renal failure , thyroid disease , small - bowel bacterial overgrowth , and conditions of hemoconcentration . in the study ,
there was great difference among the three groups , and it was closely related to dpn .
we thought that holotc is a sensitive marker for vit b12 deficiency , as previously reported .
holotc measurement has come of age ; however , it has not acquired wide clinical acceptance to date , maybe due to the test 's cost and limited availability . the present results demonstrated that when part of vit b12 was in normal range , umma had obviously exceeded the normal level , and there was great difference among the three groups , so we thought that umma was sensitive for the early deficiency of cobalamin , consistent with the previous reports [ 18 , 19 ] . the fasting urinary mma concentration corrected for creatinine was highly correlated with diabetic polyneuropathy .
the mechanism was unclear , and the probable mechanism was that methylmalonic acid affected energy metabolism and the citric acid cycle and then led to neurotoxicity and the occurrence of dpn .
in addition , umma was reported related to the enhanced inflammation in acute myocardial infarction .
the umma measurements are a noninvasive method that requires only a 1 ml urine specimen , and for months if frozen , it may offer a new method of analysis for identifying true tissue cobalamin deficiency .
the nerve conduction velocity and amplitude of four limbs decreased obviously as umma / creatinine was above normal .
there was no significant difference between the decreased conduction velocity of median and ulnar nerves and the increased umma / creatinine .
the upper limb sensory nerves appeared to be preferably affected by axonal damage as demonstrated by the finding that the sensory active nerve potential amplitude of the median and ulnar nerves was reduced in a remarkably high percentage of study patients , versus the conduction velocity of the two nerves .
the abnormality in the superficial peroneal and sural nerves in the lower limb was more severe than in the median and ulnar nerve .
the presence of conduction block and abnormal amplitude in our patients also raises the possibility that it was a manifestation of a more widespread demyelinating polyneuropathy , according to .
the correlation analysis showed that the nerve conduction block of lower limbs was more serious in the group of the increasement of umma / creatinine .
although we identified patients prospectively , they were not randomly selected from a population with type 2 diabetes with or without dpn .
being an invasive operation , emg could not reflect early neuropathy screening , so some early neuropathy patients might be missed .
we excluded the patients with type 1 diabetes because of their expected metformin restriction and the distinct pathophysiological mechanisms , and further study would be conducted in depth . in conclusion , umma and holotc
the relationships among cobalamin deficiency , decline holotc , elevated umma levels , and the polyneuropathy are controversial , and further work is needed to prove a direct causal relationship and the mechanism , but both cobalamin deficiency and elevation of its serum metabolites are associated with the presence of a sensorimotor peripheral neuropathy .
umma may exacerbate polyneuropathy as a result of other unknown mechanisms ; a clear understanding of its role necessarily awaits further research on the pathogenesis of dpn . despite these limitations
, we believe that umma is a potential iatrogenic contributor to the severity of the polyneuropathy in the population described .
it can be recommended that umma , alone and in combination with holotc , has better diagnostic efficiency in diagnosing vit b12 deficiency for the diabetic patients and is expected to become more sensitive predicting and monitoring indicators of diabetic polyneuropathy . | the rising incidence of diabetes and its negative impact on quality of life highlights the urgent need to develop biomarkers of early nerve damage .
measurement of total vitamin b12 has some limitations .
we want to determine the levels of urinary methylmalonic acid and its relationships with serum vitamin b12 and polyneuropathy .
the 176 chinese patients with type 2 diabetes mellitus were divided into 3 groups according to the levels of vitamin b12 .
a gas chromatography mass spectrometric technique was used to determine blood methylmalonic acid and urinary methylmalonic acid .
the diagnosis of distal diabetic polyneuropathy was based on the determination of bilateral limb sensory and motor nerve conduction velocity and amplitude with electromyogram .
multiple regression analysis revealed that urinary methylmalonic acid / creatinine , blood methylmalonic acid , and so forth were variables that influenced diabetic polyneuropathy significantly .
nerve sensory conduction velocity and nerve amplitude in the group of urinary methylmalonic acid / creatinine > 3.5 mmol / mol decreased significantly .
superficial peroneal nerve sensory and motor conduction velocity and ulnar nerve compound motor active potential amplitude were inversely correlated with urinary methylmalonic acid / creatinine .
urinary methylmalonic acid correlates with serum vitamin b12 levels in person with diabetes and is a sensitive marker of early polyneuropathy . |
the radiographic comparative analysis , in which antemortem and postmortem records are compared , is frequently used for personnel identification either in human remains or in living persons .
particular attention has been paid to the skull where several structures have the potential to identify an individual , including teeth , frontal sinuses ( fs ) , mastoid processes , sella turcica , vascular groove patterns , nasal septum .
the fs are paired , irregular - shaped air - containing chambers , lined by mucoperiosteum , and are located between the outer and inner tables of the frontal bone , posterior to the supercilliary arches , and at the roof of the nose .
they point upwards beyond the middle part of the supercilious and backwards to the medial part of the orbital roof .
the sinuses are divided into several recesses , which communicate with each other through incomplete bony septa .
the fs become radiographically evident at the age of 5 or 6 years and develop fully by the age of 20 years .
the posterioanterior ( pa ) skull projection is designed to provide a clear view of the frontal and ethmoid sinuses without loss of definition by superimposition of portions of the sphenoid bone . the fs alone has the clearest silhouette in this projection and presents the least chance for error in interpretation .
schuller suggested that the fs are slightly bigger in males than in females , and the presence of metopic suture is associated with the absence of fs .
some factors can modify the normal anatomy of the fs such as fractures , neoplasias , severe infections , growth hormone level , and mucoceles . in the forensic field , fs is a very convenient part of the human skeleton for personal identification .
the fs has very strong walls and is preserved intact in human remains as its internal bony structure and arched nature protect it from damage and decomposition .
it has been suggested that fs radiographic size and patterns have a potential to be used as aids for personal identification , age , and sex estimation .
the nasal septum ( ns ) is the bone and cartilage in the nose that separates the nasal cavity into the two nostrils .
the cartilage is called the quadrangular cartilage and the bones comprising the septum include the maxillary crest , vomer and the perpendicular plate of the ethmoid .
a deviated ns is an abnormal condition in which the top of the cartilaginous ridge leans to the left / right or takes sigmoidal / reverse sigmoidal shape .
these patterns are stable throughout the adult life and are clearly visible on pa skull views .
this study was undertaken to examine the combined variations in patterns of fs and ns as observed on a pa cephalogram and to use the data in personal identification .
the study was conducted on patients coming for orthodontic treatment , in surendera dental college & research institute , sriganganagar , rajasthan , from march to august 2014 .
the 149 randomly selected patients comprised 74 males and 75 females aged between 20 to 45 years ( mean age 26.01 0.23 years ) .
the exclusion criteria included non - visualization of the fs or ns on radiograph , history of trauma or surgery of the skull or nose , any pathology affecting the nasal cavity or fs , frontal rhinosinusitis , endocrinal or metabolic disorder affecting the growth , and development of fs or nose and syndromic patients .
all the subjects were briefed about the purpose of study and a written informed consent was obtained .
a clinical examination was carried out for each patient and the selected patients were subjected to pa cephalometry using
kodak 8000c digital panoramic and cephalometric system with exposure parameters of 74 kvp , 10 ma , and 13 seconds .
the selected radiographs were analyzed for fs and ns patterns by single oral and maxillofacial radiologist .
the fs was classified according to symmetry as symmetrical , right or left dominant asymmetry , unilateral ( right or left ) and bilateral aplasia .
[ figure 1 ] the greatest horizontal dimension was measured from the central septum on both sides .
the difference in the right and left side dimensions was divided by the greatest dimension and multiplied by 100 .
the number and highest point of lobulation including both sides and center were also recorded .
the measurements were taken in mm using 6.12.24.0 kodak dental imaging software according to the sequence : various frontal sinus patterns ( a ) symmetrical ( b ) left dominated asymmetry ( c ) right dominated asymmetry ( d ) unilateral aplasia ( e ) bilateral aplasia , r = right side
draw a line directly between both the orbital cavities , at the nasofrontal suture.the diameter of the fs at the widest points that was the distance between two projected lines that delineate the maximum lateral limits of the right and left fs.the height was measured by drawing a parallel line to the nasofrontal line at the highest superior point of the frontal sinuses.measure the distance between the both .
the diameter of the fs at the widest points that was the distance between two projected lines that delineate the maximum lateral limits of the right and left fs .
the height was measured by drawing a parallel line to the nasofrontal line at the highest superior point of the frontal sinuses .
the measurements were accomplished according to the parameters as : only air - filled cavities were considered ; two equally high points , measure the one closer to the intersinus septum ; when the highest point was located at a large open curve lobulation , measure the point at the middle of the lobulation ; when the highest point was located at a plateau lobulation measure the middle of the plateau .
the ns patterns were recorded according to the features of deviation as seen on radiograph as : straight ( s ) , simple deviation to the left ( l ) or right ( r ) , sigmoid type ( rl ) , reverse sigmoid type ( lr ) , and others ( epsilon- and reverse epsilon - type ; rare types ) [ figure 2 ] .
various nasal septum patterns ( a ) straight ( b ) right deviated ( c ) left deviated ( d ) sigmoid ( e ) reverse sigmoid , r = right side the data obtained were tabulated and input to statistical package of social sciences ( spss ) software version 20 ( spss inc . ,
the correlation between various parameters of fs and ns was obtained using pearson 's correlation coefficient and two tailed t - test .
the data obtained were tabulated and input to statistical package of social sciences ( spss ) software version 20 ( spss inc . , chicago , il , usa ) .
the correlation between various parameters of fs and ns was obtained using pearson 's correlation coefficient and two tailed t - test .
the fs symmetry was observed in 78.5% of the individuals ( 58 males and 59 females ) while asymmetry was observed in 7.3% of the subjects ( 6 males and 5 females ) .
the fs bilateral aplasia was seen in 5.3% ( 4 males and 4 females ) and unilateral aplasia was observed in 8.7% of the subjects . the highest point of the fs lobulations was noted on the left side ( 53.9% ) followed by the right ( 28.4% ) and center ( 17.7% ) [ table 1 ] .
distribution and correlation between frontal sinus patterns with both the genders and highest point of sinus lobulation the total number of right and left lobulations was higher in males while center lobes were observed slightly more often in females .
the maximum numbers of lobes were observed in the fs symmetrical pattern , followed by left asymmetrical and right asymmetrical patterns .
a highly significant correlation was observed between distribution of lobes and different patterns of fs . also , a significant correlation was noted for distribution of lobes and both sexes ( p = 0.001 ) [ table 2 ] .
distribution and correlation of frontal sinus lobes with gender and patterns of frontal sinus the observed order of ns patterns decreased from straight ns ( 40.9% ) , to reverse sigmoid ( 28.2% ) , sigmoid ( 20.1% ) , right deviated ( 8.05% ) , and left deviated ( 2.7% ) .
a significant correlation was found between various patterns of ns and fs except in right dominated asymmetrical fs ( p > 0.05 ) [ table 3 ] .
correlation of different patterns of frontal sinus and nasal septum the dimensions ( width , height , and area ) of fs were assessed in both the sexes . the mean width ( 46.93 6.43 mm ) and height ( 28.54 3.67 mm ) of fs
also , the mean ratio of width / height of fs was observed more in males .
a highly significant correlation was observed in both the sexes and ratio of width / height of fs .
the mean area of fs was more in males than females [ table 4 ] .
the uniqueness of fs was initially observed by zukerkandl in 1875 due to its asymmetrical morphology . in 1927 ,
culbert and law described the first human identification through morphological analysis of fs that was accepted in a united states court of law .
the study was conducted on subjects of 20 years and above , as the growth of fs completes in both sexes by that age .
, and david et al . the present study utilized pa cephalogram as fs and ns patterns could be easily assessed with minimal distortion from a single standardized radiograph , if previous records are available . during the fetal period ,
it is possible that earlier ossification of the cartilage will interfere with their further development , manifesting as a hypoplastic or aplastic sinus . in the present study ,
, reported non - existence of fs in chinese han population to be 16.6% , white and pharoah had mentioned fs absence in 4% .
similarly , ponde et al . , had found aplasia of 24.7% in their study .
a highly significant correlation was found between the aplasia of fs and both sexes in the present study whereas no significant difference was found by verma et al . in our study , the symmetry of fs was found in 78.5% of subjects , whereas taniguchi et al . , obtained 43.1% symmetry in the japanese population and in a study by david and saxena symmetry of fs was reported in 58% of the subjects .
the discrepancy between the frequency in our population and other populations may be due to the number of patients examined , the patient sample , the difference in examining techniques and equipment .
in addition , individual health and environmental factors control the fs configuration within each population , contributing to its variability . in accordance with previous studies ,
the mean width , height and area of fs in males was significantly larger than females .
morphological differences in the cranium between both sexes are determined mainly by genetic factors which might be the reason for larger fs in males . according to prossinger and bookstein
, the estimated maximum growth rates of fs for males occur 3.02 years later than females .
these results are in accordance with taniguchi et al . , and david and saxena studies .
genetic factors , trauma , compression of nose during childbirth , some genetic connective tissue disorders such as marfan syndrome , ehlers - danlos syndrome , homocystinuria cause deviated ns . in the present study , all other causes were ruled out except genetic . in the present study , the highest point of fs
was located maximally at the left cavity ( 53.9% ) , whereas rubira - bullen et al .
gulisano et al . , observed that left fs lobes tend to be larger than the right ones , a fact that could be observed by ponde et al .
there was a significant correlation found between various patterns of ns and fs except in right dominated asymmetrical fs ( p > 0.05 ) .
hence it has been suggested that the combined use of different fs and ns patterns contributes to a more precise identification than using either pattern alone .
first , only individuals age 20 years and above were considered due to the varying fs size in childhood development .
second , area was measured by a mathematical method , thus a more precise measuring tool should be used with less margin of error .
a significant correlation was found between patterns of ns and fs except in right dominated asymmetrical fs pattern , hence combined use of fs and ns patterns could strongly be used in forensics for identification by exclusion . | background : skull radiographic examination is a potentially useful procedure for the personal identification in cases where fragments of skull persist with no likelihood of identification based on dental arch.aims:the study was to determine the uniqueness and reliability of combined frontal sinus ( fs ) and nasal septum ( ns ) patterns as observed on posterioanterior ( pa ) cephalograms for personal identification.materials and methods : the randomly selected 149 digital pa cephalograms taken on kodak 8000c digital panoramic and cephalometric system were evaluated for patterns of fs and ns . also the distribution of lobulations , area , and ratio of height / width of fs was calculated .
the data obtained was statistical analyzed using pearson 's coefficient correlation.results:fs symmetry was observed in 78.5% and asymmetry in 7.3% subjects .
bilateral aplasia was noticed in 5.3% and unilateral aplasia in 8.7% of subjects .
the total lobulation of fs was noted more in males on both sides while center lobes were observed slightly more in females .
the straight ns was maximally seen followed by reverse sigmoid .
the mean ratio of width / height of fs was observed more in males and highly significant correlation was observed with both sexes .
the mean area of fs was noted more in males .
there was significant correlation found between patterns of ns and fs except in right dominated asymmetrical fs.conclusion:the combined use of fs and ns patterns could be used as method for identification by exclusion in forensics . |
regional cerebral blood flow ( rcbf ) studies have been employed to investigate the pathophysiology of migraine headache [ 13 ] .
the introduction of brain single - photon emission computed tomography ( spect ) using technetium tc 99m- ( tc- ) labelled hexamethylpropyleneamine oxime ( hmpao ) or ethyl cysteinate dimer ( ecd ) has facilitated assessment of rcbf .
a number of rcbf studies using tc 99 m hmpao or ecd spect have been reported in patients with migraine during prodromes or headache phases [ 38 ] .
lomerizine , 1-(bis(4-fluorophenyl)methyl)-4-(2,3,4-trimethoxybenzyl ) piperazine dihydrochloride , is a calcium channel blocker with antimigraine properties .
we first report a unique case in which prophylactic treatment with lomerizine recovered brain hypoperfusion on tc ecd spect during interictal period , in addition to dramatic amelioration of migraine attacks .
a 70-year - old man developed visual disturbance frequently at walking exercise for the recent 3 months . visual disturbance consisted of scintillating scotoma in both eyes , which continued 520 minutes .
he had prior history of migraine with aura ( ma ) and without aura ( mo ) from 30 years of age .
after 60 years of age , migraine attacks were decreased to a few times per one year .
fasting blood sugar level was 144 mg / dl , and hemoglobin a1c was 6.6% ( normal 4.35.8 ) .
brain spect scanning with tc - ecd was performed at the interictal time of migraine .
spect was scanned at 10 minutes after intravenous bolus injection of 1.5 ml ( 600 mbq ) , tc - ecd spect examination was performed using a rotating -camera ( prism 3000 ; picker corp .
one of spect analyses used the revised version of 3-dimensional stereotaxic region of interest template ( 3dsrt ) by takeuchi et al . .
global cbf was calculated from all data of 636 rois in whole brain , including both cerebral hemispheres and cerebellum .
spect images were divided as regional cbf into 24 symmetrical ( right and left ) regions per patient : the callosomarginal , the precentral , the central region , the parietal region , the angular region , the temporal region , the posterior region , the pericallosal region , the lenticular nucleus , the thalamus , the hippocampus region and the cerebellar hemisphere .
brain tc - ecd spect before lomerizine treatment revealed hypoperfusion in the frontal , temporal , parietal , and occipital lobes ( table 1 ) .
lomerizine ( 10 mg / day , po ) was administered for 3 months .
there were no migraine attacks and visual disturbance whenever he walked and exercised for long time .
we showed that prophylactic treatment with lomerizine ameliorated brain hypoperfusion during the interictal period in a patient with ma , together with complete prevention of migraine attacks and visual auras .
lomerizine , an antimigraine calcium channel blocker , is prescribed widely in japanese migraineurs [ 1013 ] .
lomerizine belongs to the same class of diphenylpiperazine - type calcium antagonists as flunarizine , and this drug is prescribed for migraine prophylaxis in japan .
previous clinical trials of lomerizine suggested that this drug reduced the frequency of migraine attacks over 12 weeks [ 1012 ] .
little is known about how these drugs influence rcbf on brain spect during the headache attack or the interictal phase in migraineurs .
prophylactic effects of magnesium citrate supplementation ( 600 mg / day , po ) were assessed by means of clinical evaluation , visual evoked potential , and statistical parametric mapping of brain spect before and after 3 months treatment .
magnesium treatment significantly increased cbf in the inferolateral frontal , inferolateral temporal , and insular regions .
inhibitory effects of lomerizine on the cortical hypoperfusion and expression of c - fos - like immunoreactivity induced by spreading depression in anaesthetized rats were mediated via the effects of ca - entry blockade , which may include an increase in cbf and the prevention of excessive ca influx into brain cells .
these results provide the possibility that lomerizine may potentiate cbf and inhibit cortical spreading depression in migraine .
lomerizine had a greater effect on cbf than on blood pressure and heart rates in anaesthetized rats and beagle dogs .
this drug is reported to inhibit voltage - dependent ca channels and 5-hydroxytryptamine ( 5-ht)2a receptors , leading to suppression of 5-ht - induced contraction in rat basilar artery .
recent study has disclosed that lomerizine recovered visual function in an experimental animal model of optic nerve injury .
therefore , these experimental profiles supported that lomerizine could be clinically effective in cerebral circulatory disturbances , such as migraine status .
further spect studies with numerous migraineurs are needed to elucidate the precise prophylactic mechanism of lomerizine .
after lomerizine administration had improved ma or visual aura in our patient , brain spect revealed restoration of decreased cbfs during the interictal period .
clinicoradiological features of our patients indicated that antimigraine mechanism of lomerizine could contribute to alleviation of interictal cerebral hypoperfusion . | previous studies of brain single - photon emission tomography ( spect ) showed changes of regional cerebral blood flow ( rcbf ) in migraineurs during prodromes or headache attacks .
little is known about how successful medication of migraine prevention can reflect rcbf in migraineurs .
we highlighted alternation of brain spect findings in a migraineur with aura before and after prophylactic treatment with lomerizine , a calcium channel blocker .
a 70-year - old man with migraine developed visual disturbance frequently at walking exercise for the recent 3 months .
after this visual attack , a mild - degree of throbbing headache occured occasionally .
brain spect using 99mtc - ethyl cysteinate dimer was performed at interictal time of migraine .
brain spect before lomerizine treatment revealed hypoperfusion in the frontal , parietal , and occipital regions .
he was diagnosed with recurrence of migraine with aura ( ma ) .
lomerizine ( 10 mg / day , po ) was administered for 3 months .
ma and visual aura without headache were dramatically improved .
migraine attacks and visual disturbance were not induced at exercise . at 3 months after lomerizine medication ,
brain spect showed remarkable increase of rcbf .
these spect changes of our patient indicated that antimigraine mechanism of lomerizine could contribute to restoration of cerebral hypoperfusion . |
histological studies have shown that the inferior alveolar nerve typically courses through the mandible as one major trunk with branches extending to apices of the teeth . however , there are multiple smaller branches of the inferior alveolar nerve ( ian ) running roughly parallel to the major trunk
such bifid canals are seen on panoramic and cone beam computed tomography ( cbct ) images .
patients with bifid canals are at greater risk of inadequate anesthesia or difficulties with jaw surgery . even trifid mandibular canal has been reported .
the major importance of location and configuration of mandibular canal variations is in the lower jaw surgeries , such as extraction of an impacted third molar , dental implant treatment , and sagital split ramus osteotomy .
there are many causes of failure of ian blocks , including poor technique , anatomical variations , the presence of an acute infection , inability to enter the needle to the appropriate site or a reduced pain threshold .
this case study focuses on one example of an anatomical variation : bifid mandibular canal ( bmc ) .
when we reviewed the literature regarding bmc , we encountered only a few case reports that had two mental foramens .
the following case report describes a patient with bilateral bmcs with two mental foramens by using cbct .
a 47-year - old man was referred to his clinicians with persistent dull pain , 1 year after retrograde root canal treatment of tooth#24 .
bifid mandibular canal with different patterns were located in each side of the jaw . while two mandibular canals with two distinct mandibular nerves ended to two different mental foramens in the left side [ figure 1 ] , in the right side the conjunction of two different mandibular canals ended to an isolated mental foramen [ figure 2 ] . cbct images of right mandibular canal , a : panoramic view , b : coronal view , c : sagittal - oblique view , d : crosssectional view . in the right side
, the mandibular canal ended to two mandibular foramens and only one mental foramen cbct images of left mandibular canal , a , b : cross - sectional image , c : panoramic view , d : lateral sagittal , e : coronal view .
in the left side , two mandibular canals including two mandibular nerves opened to two mental foramens
the occurrence of bifid mandibular canal has been reported to be 0.35% by sanchis et al . , 0.8% by grover , 0.9% by nortje et al . , and 0.95% by langlais et al .
reported that mandibular canal of specimen cadavers is not visible in 36.1% of panoramic radiographs .
also , lindh et al . reported that the mandibular canal of specimen cadavers is clearly visible in almost 25% of panoramic radiographs .
thus it was considered that there was a limitation in the observation of the mandibular canal by means of panoramic radiography . nowadays by the accurate new diagnostic equipments such as cbct , this statistics may increase .
reported four cases of bmc by using cbct images that only two of them can be detected by panoramic radiography .
cbct has enabled dentists to visualize the anatomy of the mandibular canal and ramified canals better than past . according to these finding and our observation
, we suggest that cbct is more useful than conventional radiography such as panoramic images in detection of bmc .
the first included bmcs extending to the area surrounding the third molar or to the tooth itself ; the second included bmcs arising from the same foramen but forming two separate canals which rejoined to form a single canal anteriorly ; the third type included a combination of the first two types ; and the last type included two radiographically separate canals with separate origins that eventually fused into a single canal anteriorly .
nevertheless , the fact that , as opposed to the upper canals , the duplicate canals on the left side extended into different mental foramen allows us to consider this case as a new division .
it is believed that one of the reasons that previous studies could not identify this type was using conventional radiographies and subsequently failing in detection narrow second mental foramen .
the most common and first complication of bifid canal may appear in performing mandibular nerve block .
the responsible nerve maybe long buccul or accessory ian . conventionally , anesthesia of the ipsilateral lip , chin , and teeth is indicative of an effective ianb .
if a patient experiences only soft tissue anesthesia around the injection site , but not of the ipsilateral lip or chin , then a problem with local anesthesia technique is likely to be the cause of the failure .
however , if there is soft tissue anesthesia of the lips and chin but not the teeth , one should consider anatomical variation .
when dentists encounter this situation , if their problem is only inadequate anesthesia they should use another technique for local anesthesia .
but if the surgical process must be done in this region , the anatomical variation of ian must be evaluated with diagnostic images .
the best diagnostic imaging modality is cbct that has considerable benefits , such as low dose , appropriate cost , and well visual quality .
the most common and first complication of bifid canal may appear in performing mandibular nerve block .
the responsible nerve maybe long buccul or accessory ian . conventionally , anesthesia of the ipsilateral lip , chin , and teeth is indicative of an effective ianb .
if a patient experiences only soft tissue anesthesia around the injection site , but not of the ipsilateral lip or chin , then a problem with local anesthesia technique is likely to be the cause of the failure .
however , if there is soft tissue anesthesia of the lips and chin but not the teeth , one should consider anatomical variation .
when dentists encounter this situation , if their problem is only inadequate anesthesia they should use another technique for local anesthesia .
but if the surgical process must be done in this region , the anatomical variation of ian must be evaluated with diagnostic images .
the best diagnostic imaging modality is cbct that has considerable benefits , such as low dose , appropriate cost , and well visual quality .
bifid mandibular canal may exist in each patient . to manage this anatomic variation , dentists should choose appropriate images and local anesthesia technique . | one of the normal interesting variations that we may encounter in the mandible is bifid mandibular canal .
this condition can lead to difficulties when performing mandibular anesthesia or during extraction of lower third molar , placement of implants , and surgery in the mandible .
therefore diagnosis of this variation is sometimes very important and necessary . |
loss of genome stability is an enabling hallmark of tumor progression , in which elevated rates of mutation and numerical / structural chromosomal aberrations combine to drive unrestrained cellular proliferation and resistance to cell death ( hanahan and weinberg , 2011 ) .
mechanisms that drive mutagenesis and copy - number changes in cancer represent the molecular underpinnings of tumor initiation and progression , and many of these mechanisms have been well described ( gordon et al . , 2012 ; helleday et al . ,
2014 ) . however , the causes of genomic instability are not yet comprehensively understood , and identifying new mechanisms remains a critically important focus in cancer cell biology . a new mechanism promoting genomic instability in cancer cells , in which the nuclear envelope ruptures and exposes chromosomal dna to the cytoplasmic environment , has been described ( vargas et al . , 2012 ) .
depending on the cellular context , the phenomenon of nuclear rupture inflicts various degrees of dna damage to cells .
rupture of primary nuclei , although transient due to quick repair of the nuclear envelope , promotes a low level of dna damage that is localized to small chromosomal regions adjacent to rupture sites ( denais et al .
by contrast , rupture of micronuclei , which are commonly generated by abnormal chromosome segregation , is permanent and leads to catastrophic chromosomal damage ( hatch et al .
nuclear envelope rupture is significantly more common in cancer cells than normal cells , although the basis for this difference remains unresolved ( vargas et al . , 2012 ) .
understanding how defects in this foundational nuclear fortification contribute to genome instability represents a burgeoning field in cancer research . here
we summarize the known causes and consequences of nuclear rupture and speculate on the cell biological factors that may regulate this phenomenon .
selective pressure to shield dna from damage in the face of increasing cellular complexity helped drive the evolution of the nuclear envelope ( cavalier - smith , 2010 ) .
this radical innovation in cellular organization marked the beginning of eukaryotic life and enabled rampant evolutionary diversification . reflecting its role of chaperoning access to critical genetic information ,
the metazoan nuclear envelope is complex and dynamic , engaging in multiple interactions with components of the cytoplasm and the cytoskeleton at any given time ( chow et al . , 2012 ; hatch and hetzer , 2014 ) .
the nuclear envelope is composed of three distinct components : the lamina , the inner and outer nuclear membranes , and nuclear pore complexes ( hetzer , 2010 ) .
the lamina plays a central role in maintaining the structural integrity of the nuclear envelope and is composed of lamins a / c , b1 , and b2 ( dechat et al . ,
lies the inner nuclear membrane , which runs contiguous with both the outer nuclear membrane and the endoplasmic reticulum ( figure 1a ) .
finally , dotted throughout the nuclear envelope and extending through both the inner and outer membranes and lamina are nuclear pore complexes ( wente and rout , 2010 ) .
nuclear pore complexes regulate the active transport of molecules greater than 40 kda in size in and out of the nucleus ( wente and rout , 2010 ) .
the various components of the nuclear envelope work in concert to promote cross - talk between the nucleus and the remainder of the cell while simultaneously protecting the genetic contents within from the potentially damaging environment of the cytoplasm . mechanisms of nuclear envelope rupture .
( b ) transient rupture of the nuclear envelope occurs at sites exhibiting structural deficiencies in the underlying lamina . although quickly repaired , these rupture events promote the localized mixing of nucleoplasmic and cytosolic contents .
( c ) chromosome bridges that form as a consequence of defective mitosis result in elongated
defective lamin deposition around these chromosome bridges creates a localized region of the nuclear envelope that is particularly susceptible to rupture .
( d ) micronuclei ( insets ) are defective in nuclear lamina assembly and undergo complete and irreversible nuclear envelope rupture .
( e ) under conditions of hyperactivated rho gtpases , increased actomyosin contractility may puncture the nuclear envelope and promote rupture .
in physiologically normal cells , breakdown of the nuclear envelope occurs only during mitosis to enable condensed chromosomes to freely segregate to daughter cells ( smoyer and jaspersen , 2014 ) .
this highly regulated process of mitotic nuclear envelope disassembly is governed in large part by activation of the mitotic kinase cdk1 , which phosphorylates key components of the lamina and nuclear pore complexes to promote their disassembly ( heald and mckeon , 1990 ; peter et al . , 1990 ; guttinger et al . ,
, the nuclear envelope rapidly reassembles around the decondensing chromosomes to once again shield them from the surrounding cytoplasm .
by contrast , nuclear envelope breakdown during interphase is anomalous and strongly indicative of an underlying cellular defect ( hatch and hetzer , 2014 ) .
transient nuclear envelope rupture has been best studied with regard to laminopathies , in which expression of mutant lamin proteins leads to gaps in the nuclear lamina structure and predisposes the nuclear envelope to intermittent nonlethal loss of integrity during interphase ( de vos et al . , 2011 ;
viruses also promote nuclear envelope rupture upon infection , perhaps as a means to gain access to the nucleus ( de noronha et al . , 2001 ; cohen et al . , 2011
it has been demonstrated that cancer cells are predisposed to nuclear envelope rupture ( vargas et al . , 2012 ) .
this is perhaps unsurprising , considering that the nuclear envelope of cancer cells has long been known to be commonly structurally impaired ( bernhard and granboulan , 1963 ; bell and lammerding , 2016 ) . indeed , as far back as 1860 , abnormalities in nuclear size and shape were used to identify cells that had become cancerous ( beale , 1860 ) .
a key unresolved question regarding nuclear envelope rupture is why the phenomenon commonly occurs in cancer cells yet is exceedingly rare in normal cells .
one obvious explanation lies in the fact that cancer cells commonly exhibit aberrant expression of lamin proteins , which impairs the structural integrity of the nuclear lamina ( chow et al .
, 2012 ; ho and lammerding , 2012 ; bell and lammerding , 2016 ; vargas et al . , 2012 ) .
confirming this notion , studies have revealed that depletion of b - type lamins increases the frequency of nuclear envelope rupture , whereas , conversely , overexpression of b - type lamins mitigates rupture rates in cancer cells ( vargas et al . , 2012 ; hatch et al . ,
an alternative , non mutually exclusive explanation for the elevated rate of nuclear envelope rupture in cancer cells is that many cellular defects commonly associated with cancer have been shown to promote nuclear rupture .
for example , abnormalities in chromosome segregation during mitosis that produce chromosome bridges and/or micronuclei are frequent in transformed cells yet rare in nontransformed cells ( ganem and pellman , 2012 ; gordon et al . , 2012 ) .
chromosome bridges typically arise from dicentric chromosomes , which originate from inappropriately repaired double - strand breaks or from fusions of critically shortened telomere regions , which are then pulled to opposing poles during anaphase and persist well into the next cell cycle . by physically connecting the two nuclei ,
a direct or indirect consequence of this defect is that nuclear lamins fail to efficiently assembly around chromatin bridges , and this predisposes the envelope around the bridge to rupture ( maciejowski et al . , 2015 ;
similarly , micronuclei , which form when anaphase lagging chromosomes reassemble nuclear envelopes independently of the spatially separated primary nucleus during telophase , are also highly prone to rupture ( hatch et al .
research suggests strongly that compositional defects , including insufficient lamin deposition , are also responsible for this effect ( hatch et al . , 2013 ) .
it is tempting to speculate that other defects arising in mitosis may similarly disrupt normal nuclear lamina assembly and promote rupture .
for example , asynchronously segregating chromosomes may delay and/or disrupt the efficient rebuilding of the nuclear lamina .
similarly , mitotic or cytokinetic failures that produce tetraploid cells with abnormally large nuclei may also impede efficient nuclear lamina assembly and predispose such large cells to rupture .
in addition to defects in lamina structure , it is now clear that mechanical forces from the actin cytoskeleton can also promote nuclear rupture .
two studies recently demonstrated that cells forced to migrate through narrow microfabricated constriction channels are prone to rupture due to increased internal pressure in the nucleus ( denais et al .
this rupture occurred at the leading edge of the nucleus and was preceded by the appearance of a nuclear protrusion lacking nuclear pore complexes and lamina .
of importance , rupture was driven , at least in part , by forces from actomyosin contraction , as inhibition of myosin ii activity significantly reduced the occurrence of rupture ( denais et al . , 2016 ) .
this raises the possibility that deregulation of actomysosin contractility may play a significant role in promoting rupture even in nonmigrating cells . in this sense ,
stimulation of actin contractility due to hyperactivation or overexpression of rho gtpases , which is common in many cancer cells , may offer an additional explanation for increased rupture events ( mark petronczki , personal communication ; vega and ridley , 2008 ) .
one intriguing possibility is that abnormal actin fibers may directly puncture fragile nuclear envelopes that are already weakened by a structurally defective lamina ( figure 1e ) .
alterations in the microtubule cytoskeleton and/or increases in centrosome number may also play a role in promoting nuclear envelope rupture . during mitosis ,
centrosomes and microtubules play important roles in facilitating nuclear envelope breakdown , and depletion of centrosomes has been shown to delay nuclear envelope disassembly ( smoyer and jaspersen , 2014 ) .
thus it is plausible that extra centrosomes , which are a hallmark of cancer cells , facilitate nuclear envelope rupture .
indeed , a convergence of multiple abnormalities in cancer cells , such as abnormal nuclear lamina structure , extra centrosomes , and/or hyperactive rho , may be required to promote rupture .
having established that nuclear envelope rupture is a defect commonly observed in human cancer cells , we are left to ask what functional consequences result from such a phenomenon .
indeed , the dramatic disruption of cellular architecture after rupture is not a benign event ; nuclear rupture is now known to induce significant , and sometimes catastrophic , genome instability ( chow et al . , 2012 ; crasta et al . , 2012 ;
recent studies have shown that dna damage appears minutes after nuclear envelope rupture and at a chromosomal location immediately adjacent to the rupture site ( denais et al . , 2016 ; raab et al . , 2016 ) .
however , the mechanism underlying the generation of this damage has yet to be fully resolved .
one possibility is that the chromosomal dna becomes exposed to harmful cytoplasmic factors from which it otherwise would have been shielded .
for example , evidence suggests that a cytoplasmic exonuclease , trex1 , may cleave chromosomal dna outside the nucleus ( maciejowski et al . , 2015 ) .
cytoplasmic dna may also be subject to the mechanical stress generated by the actin and microtubule cytoskeletons , which could cause shearing , particularly since the chromosomal dna is not condensed and may be more susceptible to damage .
it is also possible that regions of single - stranded dna , either from actively transcribing regions or regions undergoing replication , are more prone to acquiring damage in the cytoplasm .
in addition , it has been proposed that the temporary loss of compartmentalization , including the integration of organelles and other cytosolic elements into the nucleus , may indirectly promote dna damage ( bernhard and granboulan , 1963 ; de noronha et al . , 2001 ;
reactive oxygen species generated by mitochondria that have infiltrated the nucleus represent one potential source of such damage ( sieprath et al . , 2012 ; vargas et al . , 2012 ) .
whereas transient rupture of the primary nucleus represents a source of dna damage , expeditious repair and resealing of the nuclear envelope driven by endosomal sorting complexes required for transport iii ( escrt iii ) machinery tempers its catastrophic potential ( denais et al .
this is not the case in the context of micronuclei , where nuclear envelope rupture is permanent , leaving the chromosomal contents therein completely exposed to the surrounding environment ( figure 1d ; hatch et al . , 2013 ) .
it has been directly demonstrated that such chromosomes undergo a catastrophic shattering and rearrangement event called chromothripsis in which thousands of clustered chromosomal rearrangements occur in a single event ( stephens et al .
, micronuclear rupture promotes chromosome damage only in s - phase cells , as no major damage results after rupture of micronuclei in g0 or g1 cells ( crasta et al .
, 2012 ; hatch et al . , 2013 ; zhang et al . , 2015 ) .
one simple explanation for this result is that rupture may lead to the rapid dilution of dna replication factors from actively replicating chromatin , thus generating stalled or collapsed replication forks that are prone to cytoplasmic nucleases ( leibowitz et al . , 2015 ) .
similar to micronuclei , rupture of the nuclear envelope surrounding chromosome bridges also promotes massive dna damage ( maciejowski et al . , 2015 ) .
thus nuclear rupture may represent a major source of the complex genetic variability found in chromosomally unstable cancers .
of importance , it is interesting to consider whether exploiting the phenomenon of nuclear envelope rupture may have therapeutic value , especially given that the phenomenon predominantly occurs in cancer cells .
supporting this proposition , inhibition of nuclear envelope repair in cancer cells is lethal in conjunction with inhibition of dna repair mechanisms ( raab et al . , 2016 ) .
only escrt iii is known to be involved in nuclear envelope resealing after rupture , and its contribution is relatively minor compared with its integral role in nuclear envelope reformation after mitosis ( olmos et al . , 2015 ; vietri et al . , 2015 ;
, 2016 ) . therefore identifying additional factors that are required for nuclear envelope repair after rupture but are dispensable for nuclear envelope reformation after mitosis may represent more ideal therapeutic targets .
other vulnerabilities that arise in cancer cells as a result of the need to accommodate this fragile - nucleus phenotype may similarly represent additional novel therapeutic opportunities .
cancer cells have been characterized by their abnormal nuclear structure for well more than a century , despite the functional consequences of this defect being largely unknown ( beale , 1860 ) .
thus the recent finding that this widely appreciated abnormality is indicative of elevated rates of nuclear rupture and resultant dna damage is a discovery that reaches across decades to integrate early observations with contemporary molecular understanding .
the demonstration that the weakened cancer cell nucleus , with its propensity for rupture , serves as a previously unappreciated source of mutagenic stimuli and genome instability has brought the field of nuclear envelope dynamics into sharp focus . elucidating why cancer cells are more prone to rupture than their nontransformed counterparts and uncovering the causes and consequences of nuclear envelope rupture in the context of tumor progression are inquiries rife with biological significance .
indeed , the exploitation of altered nuclear envelope dynamics in tumor cells presents an entirely untested therapeutic arena to explore . investigating nuclear envelope rupture as an important driver of genome instability and
tumor progression is likely to be a rewarding line of scientific inquiry for years to come , with significant implications in both basic and translational cancer biology . | the nuclear envelope , composed of two lipid bilayers and numerous accessory proteins , has evolved to house the genetic material of all eukaryotic cells . in so doing , the nuclear envelope provides a physical barrier between chromosomes and the cytoplasm . once believed to be highly stable , recent studies demonstrate that the nuclear envelope is prone to rupture .
these rupture events expose chromosomal dna to the cytoplasmic environment and have the capacity to promote dna damage .
thus nuclear rupture may be an unappreciated mechanism of mutagenesis . |
the physical examination revealed no specific findings except for the scar from the transplant operation .
his serum creatinine on presentation was 1.7 mg / dl and urine analysis showed adequate red blood cells .
there were no abnormal findings in the bladder during cystoscopy and he underwent an abdominopelvic computed tomography ( ct ) scan and retrograde pyelography , which revealed a 1.01.0 cm sized mass at the lower pole of the left renal pelvis ( fig .
1 ) . the abdominopelvic ct scan , as performed , did not reveal metastasis to other organs or lymph nodes but there was a left renal pelvis mass .
the bone scan did not reveal bone metastasis , and the chest x - ray did not reveal any particular findings . a left nephroureterectomy with bladder cuff excision was performed and the pathological analysis showed a high - grade papillary transitional cell carcinoma ( tcc ) of the pelvis .
six months later , a regular follow - up cystoscopy confirmed four tiny papillary masses on the left lateral wall .
pathological tests showed a high - grade tcc , and the tmn staging was t1nomo ; mytomycin - c instillation therapy was performed .
however , the bladder cancer recurred , and transurethral resection of the bladder tumor was performed six times in the next 5 years .
an invasion into the bladder muscle layer was found , and the tnm staging was t2n0m0 .
however , the patient refused additional surgery because of his anxiety over losing a functional organ .
he came in for dialysis owing to decreased renal function . on the sixth month after the sixth transurethral resection of the bladder , a regular follow - up abdominopelvic ct revealed an enlarged lymph node near the right iliac vessel and multiple paraaortic and aortocaval lymphadenopathy with aggravated bladder wall thickening .
in addition , the patient complained of left thigh pain ; thus , magnetic resonance imaging ( mri ) of the thigh was performed .
the mri showed a 3.16.5 cm sized oval - shaped t1 slightly high and t2 heterogenous high - signal intensity lesion in the mid portion of the sartorius muscle ( fig .
ultrasonography - guided biopsy of the nodular mass lesion in the left sartorius muscle was performed ( fig .
the pathological analysis of the biopsy specimen revealed poorly differentiated metastatic urothelial carcinoma ( fig .
4 ) . chest ct and abdominopelvic ct showed lung , bone , and lymph node metastasis .
radiation therapy to the left metastatic skeletal muscle lesion and chemotherapy to the metastatic bladder cancer were performed .
although skeletal muscle represents approximately 50% of the total body mass and receives a large portion of total cardiac output , hematogenous metastatic disease to the skeletal muscle is extremely rare .
several factors have been postulated to contribute to the resistance of skeletal muscle to metastatic disease .
these factors include muscle motion and mechanical tumor destruction , inhospitable muscle ph , and the muscle 's ability to remove tumor - produced lactic acid that induces tumor neovascularity in other tissues . despite these defensive factors , metastases to skeletal muscle
have been reported from pancreatic , renal , colonic , pulmonary , gastric , and ovarian primary malignancies . the malignant tumor to muscle metastasis per se isevidence of systemic spread , and
metastatic disease to skeletal muscle tends to be found in people with advanced - stage neoplasms .
the largest muscles , such as the erector spinae , psoas , and gluteals , are the most common sites of metastatic involvement .
one large autopsy study of 5,298 people who died of malignancies found that 6% ( 298 ) involved the anterior chest wall or abdominal wall musculature and that 13% ( 666 ) involved the diaphragm .
many of these lesions were microscopic and unlikely to be detected by any imaging technique .
in this case , the patient complained of muscular pain , which is compatible with studies showing that most lesions are painful .
skeletal muscle metastasis may be an incidental finding on ct of the chest or abdomen because most of the lesions identified were neither painful nor palpable .
the general ct findings of skeletal muscle metastasis show an increase in ring - shaped enhancement with central hypoattenuation , and mri findings , although not specific , show low to intermediate signal intensity in the t1-weighted image and uniform high - signal intensity in the t2-weighted image . in this case ,
mri showed a 3.16.5 cm sized oval - shaped t1 slightly high and t2 heterogenous high - signal intensity lesion in the sartorius muscle and strong enhancement after contrast injection , which suggested intramuscular metastasis .
reported that if painful muscle metastasis is present , en bloc excision has good effects .
also , pop et al reported that although not statistically significant , there was a slight difference in survival rates among patients who underwent radiotherapy or chemotherapy compared with those who underwent different surgeries .
however , klune et al . reported that surgical treatment was selectively possible only in patients meeting various conditions and recommended radiotherapy or chemotherapy . regarding bladder tumor metastasis to skeletal muscle , nabi et al
. reported that the mean survival rate after receiving chemotherapy was 8 months , but because such cases are rare , specific details are not known .
reported that in the case of lung cancer metastasis to skeletal muscle , the survival rate is considerably reduced compared with the absence of metastasis , and in the case of three patients , the survival rate was not good at 3 , 6 , and 30 months . | hematogenous metastasis to skeletal muscle from urothelial carcinoma is extremely rare and metastatic disease to skeletal muscle tends to be found in people with advanced - stage neoplasm .
we report in this paper a case of left sartorius muscle metastasis from urothelial cell carcinoma .
a left nephroureterectomy with bladder cuff excision was performed and revealed a high - grade papillary transitional cell carcinoma ( tcc ) of the pelvis . and 6 month later , recurrent bladder cancer was found regular cystoscopy and then treated with transurethral resection of the bladder .
after 6 times resection of bladder , an invasion into the bladder muscle layer was found .
we recommended additional radical cystectomy to prevent the disease from advancing .
however , the patient refused additional surgery .
6 month later , the patient complained of left thigh pain , so ultrasonography - guided biopsy of the nodular mass lesion in the left sartorius muscle was performed .
the pathological analysis of the biopsy specimen revealed poorly differentiated metastatic urothelial carcinoma . |
inverted nipple may cause various cosmetic , psychological , breast feeding and hygienic problems . providing a symmetrical nipple shape and position with permanent projection
nonsurgical treatments include breast shells , breast pumps and syringe techniques . however , the beneficial and permanent effects of these treatments are controversial . in the teimourian technique which is the one of the most commonly used surgical technique ,
all the lactiferous ducts are cut and the dermal flaps are used as supportive bulk under the nipple for correcting moderate and severe nipple inversion . in the present study , we aimed to make a new modification of the teimourian method .
after the release of the nipples , the flaps were tunnelised beneath the nipple and the tip of each flap was fixed to the nipple base in reverse s - shaped design .
the modified design of the traditional triangular dermal flap procedure provides much more suspension effect as well as filling dead space under the nipple .
it represents simple and reliable surgical technique to avoid the recurrence of inversion and to provide satisfying shape and projection of the nipple with minimal scar .
three nipples of two female patients treated with the reverse s - shaped fixation method are presented . according to the criteria of han and hong , the nipples were classified as grades 2 and 3 [ figure 1 ] .
( b ) preoperative view of the unilateral case the operation was performed under local anaesthesia .
two opposite linear incisions at 3 and 9 oclock on the two sides of the everted nipple were made within the range of the areola .
afterwards , the areolar skin was dissected and the epidermis was separated from the dermis over the planned flap area .
the base width of the flaps was determined as equal to radius of the nipple .
on the two sides of the triangles with no epithelium , the incisions were deepened into the subcutaneous fatty tissue .
on the two sides of the triangles with no epithelium , two conic shape areolar dermal flaps were created the retracting ducts and fibrous bands beneath the nipple were released with blunt dissection .
if the nipple was still not free after these dissections , all the retracting ducts were transected so as not to restrict the nipple .
the conic shape triangular flaps on both sides of the inverted nipple were turned down and advanced through the tunnel beneath the nipple as the filling tissue for the dead space [ figure 3 ] .
the tip of each flap was sutured to the nipple base where any incision was not made at 6 and 12 oclock , also next to the base itself in reverse s shape [ figure 4 ] . because the length of the flap is shorter than the diameter of the areolar external circle , the fixations of the flaps provided a strongly suspending effect and the flaps was completely able to fill out the dead space beneath the nipple .
any complication was not observed and the patients recovered uneventfully during the follow - up period .
the conic - shape triangular flaps on both sides of the inverted nipple were turned down and advanced through the tunnel beneath the nipple as the filling tissue for the dead space the tip of each flap was sutured to the nipple base where any incision was not made at 6 and 12 oclock , also next to the base itself in reverse s shape early postoperative view of the patient
three nipples of two female patients treated with the reverse s - shaped fixation method are presented . according to the criteria of han and hong , the nipples were classified as grades 2 and 3 [ figure 1 ] .
( b ) preoperative view of the unilateral case the operation was performed under local anaesthesia .
two opposite linear incisions at 3 and 9 oclock on the two sides of the everted nipple were made within the range of the areola .
afterwards , the areolar skin was dissected and the epidermis was separated from the dermis over the planned flap area .
the base width of the flaps was determined as equal to radius of the nipple .
on the two sides of the triangles with no epithelium , the incisions were deepened into the subcutaneous fatty tissue . two conic shape areolar dermal flaps
on the two sides of the triangles with no epithelium , two conic shape areolar dermal flaps were created the retracting ducts and fibrous bands beneath the nipple were released with blunt dissection .
if the nipple was still not free after these dissections , all the retracting ducts were transected so as not to restrict the nipple .
the conic shape triangular flaps on both sides of the inverted nipple were turned down and advanced through the tunnel beneath the nipple as the filling tissue for the dead space [ figure 3 ] .
the tip of each flap was sutured to the nipple base where any incision was not made at 6 and 12 oclock , also next to the base itself in reverse s shape [ figure 4 ] . because the length of the flap is shorter than the diameter of the areolar external circle , the fixations of the flaps provided a strongly suspending effect and the flaps was completely able to fill out the dead space beneath the nipple .
any complication was not observed and the patients recovered uneventfully during the follow - up period .
the conic - shape triangular flaps on both sides of the inverted nipple were turned down and advanced through the tunnel beneath the nipple as the filling tissue for the dead space the tip of each flap was sutured to the nipple base where any incision was not made at 6 and 12 oclock , also next to the base itself in reverse s shape early postoperative view of the patient
during the postoperative follow - up period , surgical complications such as traction pain , infection , haematoma , permanent numbness , or skin necrosis were not observed .
follow - up examinations were performed at 3 to 9 months ( average , 6 months ) and revealed no evidence of recurring inversion .
inverted nipple can simply be defined as nipples that lie on a lower plane than the areola due to shortened lactiferous ducts embedded in short fibrous bands .
the grading system by han and hong is commonly used to determine the grade of deformation . according to the grading system , in grade 2 cases , the nipples can be pulled out but can not maintain projection and have strongly tendency to go back again .
blunt dissection for surgical release should be performed until the inversion does not recur without traction .
the lactiferous ducts are mildly retracted and do not need to be cut for the release of fibrosis in the majority of the patients . a temporary traction suture with a suitable dressing is generally preferred after surgery for 2 weeks . in grade 3
cases , the nipple can hardly be pulled out manually due to the severe fibrosis . for correcting grade 3 cases ,
two or three deepithelialised dermal flaps should be used to make up for soft tissue deficiency after the release of fibrotic bands and a traction suture is also applied postoperatively .
there are numerous surgical and nonsurgical techniques introduced in the literature and among these , the incision - release and local flap methods are generally used in the treatment of grades 2 and 3 nipple inversion cases .
many of the flap descriptions over the last two decades are designed on the teimourian technique . in these techniques , two or three
triangular flaps tunnelled and approximated below the nipple are used to fill the dead space and achieve eversion .
the presented method can also be suggested as a modification of the teimourian method and the technique presented by kim et al . in this study , we introduced a new nipple suspension technique using nipple - based dermal flaps for correcting moderately and severely inverted nipples .
the most significant difference of the recommended method is in its geometrical design as compared to similar surgical methods using dermal triangular flaps . in our procedure , because two triangular dermal nipple - based areolar flaps are fixed on the opposite side of the nipple base , their geometrically design was called reverse s shape . as a consequence of this design ,
the reverse s - shaped fixation of triangular flaps provides more traction effect to keep the nipples in protruded position after they are released .
the other advantages and differences of our technique is as follows :
linear incision is used to create the triangular flaps on the areola , thus the scar is more uneventful and minimal;the operation time is relatively shorter because the flaps are raised without deepithelialisation;the triangular dermal flaps provide enough bulk beneath the inverted nipple;the size of the triangular flap is slightly shorter than the diameter of the areola , the flaps cause an efficiently suspending effect under the released nipple ; andthe lactiferous ducts can be preserved in grade 2 cases or if the women particularly hope to breast feeding .
linear incision is used to create the triangular flaps on the areola , thus the scar is more uneventful and minimal ; the operation time is relatively shorter because the flaps are raised without deepithelialisation ; the triangular dermal flaps provide enough bulk beneath the inverted nipple ; the size of the triangular flap is slightly shorter than the diameter of the areola , the flaps cause an efficiently suspending effect under the released nipple ; and the lactiferous ducts can be preserved in grade 2 cases or if the women particularly hope to breast feeding . the technique introduced is not to be able to preserve the lactiferous tubules .
however it is well - known that these functional units beneath the nipple areolar complex may already be absent in grade 2 and especially grade 3 inverted nipple cases .
optimal surgical procedure in the treatment of the moderate or severe nipple inversion cases should provide a satisfying shape , sufficient projection and preserve ductal function when required . in conclusion , as an alternative nipple suspending method , the reverse s - shaped fixation method of triangular flaps is recommended to be used in any kind of nipple inversion with satisfying results and minimal scar . | the primary aim in the treatment of inverted nipple is to achieve a satisfactory and permanent projection of the nipple .
the drawbacks of reported techniques include sensory disturbance of the nipple , marked scarring of the nipple and areola , destruction of breast function and incomplete correction . in the present study ,
the authors introduced a new modification of using two opposite nipple - based areolar dermal flap in the treatment of grades 2 and 3 inverted nipple cases .
nipple - based areolar flaps designed at 3 oclock and 9 oclock were raised by two linear incisions and the tip of each flap was sutured on the base of the nipple close to the base itself in a reverse s shape at 6 and 12 oclock .
successful outcome was obtained due to a strongly suspending effect of the design of the flaps .
the mean follow - up period was 6 months .
the patients were satisfied with the result and the scars were minimal .
the authors recommend the use of the technique that is a simple , reliable and with minimal scars for correcting grades 2 and 3 inverted nipples . |
a 75 year old woman presented to the emergency room with a seven months history of increased fatigability , loss of appetite , weight loss and progressive abdominal distension . on examination
the patient was markedly pale , asthenic , dehydrated and febrile and had altered mental status .
her general physical examination revealed anemia and pedal edema without any lymphadenopathy . on inspection abdomen was distended , without any visible vessel , scar or sinus .
a periumblical bluish violet swelling 5 cm in diameter was also noted ( figure 1 ) .
on palpation a firm liver edge 4 cm below the right costal margin with a hard periumblical swelling was also found .
the cardiovascular system was normal except for a systolic flow murmur at the apex and base .
investigations revealed a low hemoglobin of 3% , microcytic hypochromic type with a reticulocyte count of 5% and no abnormal cells on peripheral blood smear .
kidney function tests , electrolytes , blood sugar , urine examination and septic screen ( blood , urine and asitic fluid cultures ) were normal .
ascites and pleural taps revealed a lymphocytic exudate without malignant cells and adenosine deaminase in a suspect range .
ultrasound of the abdomen revealed ascites with an enlarged liver , normal pancreas , gall bladder , adnexae and no para - aortic lymphadenopathy .
fine needle aspiration cytology ( fnac ) of the umbilical nodule done showed metastatic deposits of well - differentiated adenocarcinoma ( figure 2 ) .
the patient succumbed to massive rectal bleeding and despite all measures could not be resuscitated .
a visible periumblical bluish violet swelling suggestive of a sister mary joseph nodule metastatic deposits of a well differentiated adenocarcinoma documented by fnac
umbilical tumors are relatively rare and can be classified as benign or malignant.1 malignant tumors can be primary or metastatic tumors .
sister mary joseph nodule is used to describe a malignant umbilical tumor usually associated with advanced metastasizing intra - abdominal cancer and generally indicating a poor prognosis .
this sign was first identified by sister mary joseph ( 1856 - 1939 ) who as a surgical assistant to dr william james mayo drew attention to the presence of a hard umbilical nodule in a patient being prepared for surgery in 1928 .
sister mary joseph nodule in the 11 edition of his textbook demonstrations of physical signs in clinical surgery in 1949 .
the nodule usually presents as a firm , indurate often vascular swelling which may be fissured or ulcerated and may have serous , mucinous , purulent or bloody discharge .
the nodule has been described as white , bluish violet and brownish red and is occasionally pruritic .
it is usually irregular in shape , generally painless when palpated except if the overlying skin has ulcerated .
it is typically less than 5 cm in diameter but occasionally enlarges enough to form a protruding tumor .
patients with sister mary joseph nodule present with a number of clinical symptoms consistent with intra - abdominal cancer including epigastric pain , abdominal distension , weight loss , nausea , ascites and bleeding per rectum.2 the sign of sister mary joseph nodule has been extensively described in literature .
the evaluation of an umbilical mass should be directed by suspicion of its being a metastatic deposit keeping in mind its potential to be either a primary malignant umbilical lesion or a benign disease . in a patient with a known malignancy an umbilical mass represents a spread or seeding of the primary tumor and
thus can influence therapeutic decision making.3 the most common origins of sister mary joseph nodule are gastrointestinal ( 52% ) , gynecologic ( 28% ) , stomach ( 23% ) and ovarian ( 16% ) carcinomas .
about 15 - 29% of all cases have an unknown origin ( as depicted by the presented case ) and 3% originate from the thoracic cavity .
primary tumors in many other sites like gall bladder , uterus , liver , endometrium , small intestine , fallopian tube , appendix , cervix , penis , prostrate , urinary bladder , breast , lung and kidneys have also been reported to cause sister mary joseph nodules .
histology of the metastatic umbilical tumor usually reveals adenocarcinoma but rare reports of umbilical metastasis from sarcomas , mesotheliomas and melanomas have also been seen.24 ct scan ( abdomen and chest ) and fine needle aspiration cytology ( fnac ) of the tumor are invaluable in the diagnosis of sister mary joseph nodule and help to exclude a primary benign umbilical neoplasm . in 14 - 33% of cases , umbilical metastases
lead to the diagnosis of previously occult neoplasms.5 in 40% of patients with a known neoplasm the nodule was an early sign of relapse.6 spread of metastatic carcinoma to the umbilical region has been hypothesized to occur by either contiguous spread of peritoneal cancer , hematogenous spread through arterial and venous systems or lymphatic spread ( mainly pancreatic carcinoma ) with extension along ligaments of embryonic origin ( round ligament of liver , urachus , vitello intestinal duct reminant and the obliterated vitelline artery).67 the presence of a sister mary joseph nodule verified histological signifies advanced metastatic carcinoma and a poor prognosis with a survival time of 10 months ( range 2 - 17 months ) and inoperability .
treatment of established sister mary joseph nodule is palliative as wide excision , surgery and radiotherapy have all proved ineffective .
nevertheless a sister mary joseph nodule is a time tested , honored clinical sign emphasizing the importance of a careful physical examination of the abdomen .
ihd examined , planned the work up , executed the investigative profile and wrote the manuscript . | sister mary joseph nodule or sister mary joseph sign refers to a palpable nodule bulging into the umbilicus as a result of metastasis of a malignant cancer in the pelvis or abdomen .
a rare case of sister mary joseph nodule , manifesting as ascites , cachexia and bleeding per rectum , is presented without any primary tumor despite extensive search for the same . |
this is a prospective , observational study approved by the institutional review board , and the study was conducted according to the tenets of the declaration of helsinki .
axial length ( al ) and anterior chamber depth ( acd ) were measured using an iolmaster ( carl zeiss meditec , jena , germany ) .
central corneal thickness ( cct ) was measured using a specular microscope sp-2000p ( topcon , tokyo , japan ) .
individuals were excluded if they met any of the following criteria : ( 1 ) preoperative iop > 20 mmhg ; ( 2 ) trabecular meshwork invisible in any quadrant on static gonioscopy ; ( 3 ) refractive error exceeding 6 diopters ( d ) spherical equivalent or 3 d of astigmatism ; ( 4 ) history of ocular disease or trauma ; ( 5 ) previous ocular surgery except uncomplicated cataract surgery of the contralateral eye ; or ( 6 ) glaucomatous optic disc changes such as excavation , thinning or notching of the neuroretinal rim , or disc hemorrhages .
individuals were excluded from the analysis if they had surgical complications during or after phacoemulsification , such as posterior capsular rupture , intraocular lens ( iol ) placement in the ciliary sulcus , or postoperative iop spike .
unexpected cataract surgery of the contralateral eye within 1 month was also considered a reason for exclusion .
patients were treated with topical 0.5% levofloxacin and 0.1% diclofenac sodium for four times a day starting 3 days before surgery .
surgery was performed by a single surgeon at a single surgical center from may 2014 to september 2014 . after making a 2.2-mm incision in the temporal clear cornea
a single - piece acrylic foldable posterior chamber iol was implanted in the capsular bag in all cases .
then , the ophthalmic viscoelastic agent was removed and the incision site was closed by stromal hydration with no suture .
a single observer measured iop using a single tono - pen avia ( reichert inc . ,
iop readings were always obtained after calibration of the tono - pen and in the right eye first .
the iop value for each posture was the mean of two consecutive measurements that were within 2 mmhg and that had < 5% error as indicated on the tono - pen or the median of three measurements if the first two differed by 3 mmhg or more .
the patients underwent iop measurements in different body postures from 7 to 11 a.m. on the day of surgery . to assign a randomized sequence of iop measurements in different body postures ( sitting , supine , or ld ) ,
every patient was asked to pick up one card , where a different body posture was written on the back , one after another until all the three cards were opened .
first , each patient was asked to sit on a chair under dim light conditions and undergo iop measurement with a goldmann applanation tonometer ( gat ) .
then , the patient was asked to assume and maintain each of the body postures in the randomized sequence predetermined in an aforementioned manner .
all participants rested their head on a soft pillow to keep the head parallel to the bed and they were instructed to neither turn their heads nor bury their eyes into the pillow .
a pilot study revealed that the standard deviation ( sd ) of the iop reduction after cataract surgery of the operated eye was 2.8 mmhg in ld position ( 2.4 mmhg for supine position and 1.8 mmhg for sitting position ) .
a sample size calculation , using g*power software ( version 3.0.10 ; universitt kiel dusseldorf , germany ) with = 0.05 , determined that 28 patients would be required to detect an iop difference of > 1.6 mmhg in the operated eye at an sd of 2.8 mmhg with a power of 80% .
accounting for the loss of follow - up to be about 20% , we estimated a final sample of 35 patients in this study .
wilcoxon signed - rank test was used to compare iop changes after cataract surgery in different body postures .
the amount of iop changes after surgery with different body postures was compared using the friedman test .
we performed spearman 's correlation analysis to evaluate the relationship between iop change and ocular biometric parameters , with the change of iop as a dependent parameter and the ocular biometric parameters as independent parameters .
p values were considered statistically significant at values < 0.05 unless the bonferroni correction method for multiple comparisons was used , in which case a p < 0.017 was considered statistically significant .
a pilot study revealed that the standard deviation ( sd ) of the iop reduction after cataract surgery of the operated eye was 2.8 mmhg in ld position ( 2.4 mmhg for supine position and 1.8 mmhg for sitting position ) .
a sample size calculation , using g*power software ( version 3.0.10 ; universitt kiel dusseldorf , germany ) with = 0.05 , determined that 28 patients would be required to detect an iop difference of > 1.6 mmhg in the operated eye at an sd of 2.8 mmhg with a power of 80% .
accounting for the loss of follow - up to be about 20% , we estimated a final sample of 35 patients in this study .
wilcoxon signed - rank test was used to compare iop changes after cataract surgery in different body postures .
the amount of iop changes after surgery with different body postures was compared using the friedman test .
we performed spearman 's correlation analysis to evaluate the relationship between iop change and ocular biometric parameters , with the change of iop as a dependent parameter and the ocular biometric parameters as independent parameters .
p values were considered statistically significant at values < 0.05 unless the bonferroni correction method for multiple comparisons was used , in which case a p < 0.017 was considered statistically significant .
of the 35 patients recruited in this study , 5 patients were excluded due to loss to follow - up and one patient was excluded due to unanticipated phacoemulsification of the contralateral eye within 1 month .
sixteen patients were male and 13 were female , with a mean age of 64.2 9.1 years ( range : 5083 ) .
baseline demographics of the patients who underwent phacoemulsification table 2 lists the iop measurements obtained in the different body postures and shows the statistical differences of iop changes before and after cataract surgery . for the operated eye ,
all iops measured in different body postures showed significant reduction after phacoemulsification ( p < 0.01 ) , except the iop measured by tono - pen in the sitting position ( p = 0.266 ) .
however , the amount of postoperative iop change measured by tono - pen in the sitting position was significantly different between the operated and the nonoperated eyes ( 0.6 2.5 mmhg vs. 0.5 2.2 mmhg , p = 0.045 ) .
neither significant difference was found for the nonoperated eye in the sitting position ( p = 0.253 ) nor in the recumbent positions .
the amount of iop reduction in the operated eye was significantly different among the various body postures ( p < 0.001 , friedman test ) . although these differences were not always significant in each pair of comparisons with bonferroni correction , in which case a p < 0.017 was considered statistically significant , the amount of iop reduction was significantly larger in ld position than that in sitting position ( sitting vs. supine , p = 0.048 ; sitting vs. ld , p = 0.001 ; supine vs. ld , p = 0.028 ) .
the mean postoperative iop reduction in the operated eye was 3.0 3.5 mmhg in the ld position , 1.7 2.5 mmhg in the supine position , and 0.6 2.5 mmhg in the sitting position .
intraocular pressure in different body postures and biometric parameters before and after phacoemulsification when compared between the preoperative and postoperative measurements , the posture - induced iop elevations obtained by changing from the sitting to the supine or ld position were significantly reduced after the surgery [ table 3 ] . before the surgery , iop was higher ( + 2.0 1.8 mmhg ) in the supine position than in the sitting position . however , after the surgery , this posture - induced iop elevation was reduced to 0.8 2.0 mmhg ( p = 0.048 ) . such perioperative reduction in iop rise
was also noted for changing body posture from the supine to the ld position ( p = 0.028 ) .
posture - induced intraocular pressure changes before and after phacoemulsification table 4 shows the spearman 's correlation analysis performed to evaluate the association between ocular biometric parameters and iop reduction .
higher preoperative iop was correlated with larger iop reduction after phacoemulsification in every body posture .
furthermore , the iop difference in the ld position showed the strongest correlation coefficient ( spearman 's rho = 0.730 ) [ fig . 1 ] .
however , preoperative acd , al , cct , and body mass index did not correlate with the iop differences in the supine and ld positions .
spearman correlation analysis between preoperative ocular parameters and intraocular pressure changes before and after phacoemulsification correlation between preoperative intraocular pressure and mean decrease of intraocular pressure after phacoemulsification of the operated eye in different body postures .
note that the higher preoperative intraocular pressure was correlated with larger intraocular pressure reduction in every position .
a number of previous studies reported mean iop reductions ranging from 1.5 to 8.3 mmhg after phacoemulsification in eyes with or without glaucoma .
recent studies also demonstrated that phacoemulsification decreased diurnal iop fluctuations in patients with pseudoexfoliation syndrome with open or occludable angles and decreased nocturnal iop fluctuation in patients with primary angle closure .
however , such iop reductions may not always occur after phacoemulsification , and cataract surgery may even raise iop postoperatively . in the present study , we found that iop was reduced ( from 13.8 1.9 mmhg to 12.6 2.1 mmhg ) after phacoemulsification when measured by gat in the sitting position ( p = 0.007 ) .
although the tono - pen iop readings in the sitting position did not show a significant iop reduction after cataract surgery ( p = 0.266 ) , the amount of postoperative iop change obtained with tono - pen differed significantly between the operated and the nonoperated eyes ( 0.6 2.5 mmhg vs. 0.5 2.2 mmhg , p = 0.045 ) . in addition to postoperative iop reduction in the sitting position , we also found that phacoemulsification reduced iop in the recumbent postures . to the best of our knowledge ,
this is the first study to look at the iop changes in the supine and ld postures after phacoemulsification in nonglaucomatous eyes . in this study ,
the amount of postoperative iop reductions was greater in the recumbent positions than in the sitting position ( 1.7 2.5 mmhg vs. 0.6 2.5 mmhg , p = 0.048 ; 3.0 3.5 mmhg vs. 0.6 2.5 mmhg , p = 0.001 ) and was greater in the ld position than in the supine position ( 3.0 3.5 mmhg vs. 1.7 2.5 mmhg , p = 0.028 ) .
moreover , the posture - induced iop elevation was also reduced after cataract surgery . by changing from the sitting to the supine position
, iop increased by 2.0 1.8 mmhg before the surgery , whereas this elevation was reduced to 0.8 2.0 mmhg at 1 month after cataract surgery ( p = 0.048 ) .
similar postoperative mitigation of posture - induced iop elevation was also found between the supine and ld positions .
thus , these findings suggest that uncomplicated cataract surgery may also be beneficial for iop management during the night time . the mechanisms underlying the iop reduction after cataract surgery are not yet fully understood .
possible explanations include ( 1 ) increased aqueous outflow by widening of the anterior chamber angle , increased postoperative acd , and/or an increase in prostaglandin production after surgery and ( 2 ) reduction of aqueous production resulting from traction on the ciliary body due to fibrosis and contraction of the posterior lens capsule .
some researchers have proposed that extraction of the cataractous lens allows the anterior lens capsule to assume a more posterior location , thereby exerting zonular traction on the ciliary body and scleral spur .
the increased posterior traction on the scleral spur may expand the trabecular meshwork and schlemm 's canal , leading to enhanced aqueous outflow .
as to the greater reduction of iop in the recumbent postures found in the present study , we are not able to fully explain the mechanisms . however , we speculate that the increase in outflow facility after cataract surgery may allow for greater accommodation of increased iop , helping to prevent its rise after lying down or in the decubitus position .
usually , iop is elevated when the body posture is altered from the sitting to a recumbent ( supine or ld or prone ) position .
such posture - induced iop alterations have been reported to be higher in glaucoma patients compared to normal controls , and these iop changes have been associated with progression of glaucoma . moreover
, sawada and yamamoto also reported that successful trabeculectomy reduced the posture - induced iop changes and that measuring these changes could be a method for assessing the bleb 's filtering function .
therefore , information on the iop not only in the sitting position but also in the recumbent positions may improve our understanding of the effects of cataract surgery on iop and glaucoma .
a recent study investigated the effect of cataract surgery on the circadian iop pattern in primary angle closure glaucoma patients using the contact lens sensor known as triggerfish ( sensimed , lausanne , switzerland ) .
this study demonstrated that the mean range of iop fluctuations during the nocturnal period was significantly decreased after cataract surgery although 24-h iop fluctuation was not significantly changed .
the nocturnal iop reduction after surgery may be explained by our findings of the greater iop - reducing effects in recumbent postures .
however , our results need to be verified in open - angle glaucoma patients . there are some limitations to the present study . first , the sample size of the study population was small .
thus , the present findings can not be directly applicable to glaucoma patients who have a coexisting diagnosis of cataract .
third , one may criticize the discrepant results between the perioperative iop readings obtained by two different tonometers in the sitting position .
however , the amount of iop change between the operated and nonoperated eyes was significantly different when iop was measured using tono - pen in the sitting position .
fourth , the short duration ( 10 min ) of maintaining each position may also be a limitation .
hence , the current finding may not be extrapolated into the effect of longer duration supine or ld position on iop .
finally , the postural variations in iop were checked only once at 1 month after cataract surgery .
multiple checkups of iop variations in different postures over a longer - term postoperative period are needed .
we have shown that phacoemulsification significantly reduced iop of the nonglaucomatous eye in the supine as well as in the ld body postures .
in addition , our findings suggest that phacoemulsification may help to reduce posture - induced iop elevations during the night time . however , further studies are required to investigate the effect of phacoemulsification on posture - induced iop variations in eyes with glaucoma .
| purpose : to investigate the effect of phacoemulsification on intraocular pressure ( iop ) in different recumbent body postures including supine and lateral decubitus ( ld ) positions.materials and methods : this prospective , observational study included patients who had no glaucoma and who had planned to undergo phacoemulsification and intraocular lens implantation in one eye . before and 1 month after cataract surgery , iop was measured in both eyes using the tono - pen avia .
we measured iop in the sitting , supine , and ld ( with the operated eye placed on the lower side ) positions .
iop was measured 10 min after assuming each position in a randomized sequence .
the wilcoxon signed - rank test was used to compare the iop changes before and 1 month after phacoemulsification in all postures.results:twenty-nine patients participated in this study .
postoperative iop was lower than the preoperative iop when measured by goldmann applanation tonometry in the sitting position ( 13.8 1.9 mmhg vs. 12.6 2.1 mmhg , p = 0.007 ) .
the postoperative iop was lower than the preoperative iop for the supine and ld positions .
the average iop reduction of the operated eye was 0.6 mmhg , 1.7 mmhg , and 3.0 mmhg in the sitting , supine , and ld positions , respectively ( sitting vs. supine , p = 0.048 ; sitting vs. ld , p = 0.001 ; supine vs. ld , p = 0.028 ) . in the nonoperated eye ,
iop did not change significantly after surgery ( all p > 0.05).conclusions : cataract surgery lowered iop in the sitting position as well as in the supine and ld positions .
such postoperative iop reductions were greater in the recumbent positions than in the sitting position . |
the association of hepatitis b virus ( hbv ) and hematologic malignancies , including hodgkin lymphoma and non - hodgkin lymphoma ( nhl ) has been previously studied ( 1 ) .
hepatitis b virus causes a wide range of liver diseases , including subclinical infection , acute , chronic and fulminant hepatitis .
chronic hbv may lead to cirrhosis or hepatocellular carcinoma ( hcc ) ( 2 ) .
hepatitis b virus is a dna virus , which belongs to the hepadnaviridae family ( 3 ) .
the genome of hbv is a partially double stranded circular dna with molecular weight of 3.2 kbp .
the viral genome encodes four open reading frames ( orfs : s , c , p and x ) .
the orf s encodes hbsag , which consists of pre - s1 , pre - s2 and s regions .
the orf x encodes the x protein ( hbx ) and is a transactivator that influences the transcription of hbv genes by regulating the activity of transcriptional promoters and orf p , which encodes dna polymerase and reverse transcriptase ( 4 ) .
genotypes a and d are prevalent in europe , united states , and africa , whereas genotypes b and c are prevalent in asia .
genotype e is the dominant genotype in west africa whereas f and h have been isolated in the america ( 5 ) .
based on amino acid substitutions in positions 122 and 160 of the s region of hbv dna , four major subtypes : adw , adr , ayw and ayr , and nine minor subtypes have been described ( 6 ) . hepatitis b virus is a hepatotropic virus but recently hbv dna has been detected in peripheral blood mononuclear cell ( pbmc ) of patients with acute and chronic infection ( 7 - 9 ) .
some studies have suggested that the hbv dna could replicate in pbmc and proved this suggestion by detection of hbv mrnas and expression of hbsag and hbcag in pbmcs ( 10 - 15 ) .
these observations show that the lymphoid system could be an important reservoir for hepatitis b virus ( 16 ) .
the hbv dna integration in pbmcs has been demonstrated previously ( 7 , 17 - 19 ) .
hodgkin lymphoma and non - hodgkin lymphoma are lymphoid malignant tumors , and are prevalent worldwide .
recent studies have revealed the association between hbv infection and hodgkin and non - hodgkin lymphoma ( 20 - 22 ) .
the aim of this study was to determine the prevalence of hbv dna and s region mutations among block samples of patients with hodgkin and non - hodgkin lymphoma in ahvaz city , iran .
paraffin embedded tissues of 41 block samples including 12 ( 29/26% ) hodgkin and 29 ( 70.73% ) non - hodgkin patients were collected from imam khomeini hospital located in ahvaz , iran , during 2002 to 2011 .
the following steps were then carried out for the detection of hbv dna in the patient samples .
statistical analysis was done with the chi square test using the spss software , version 20 .
the dna extraction of all the tissue samples was carried out using the high pure pcr template kit ( roche , germany ) , according to the manufacturer s instructions .
the nested pcr for detection of hbv dna was performed for all the tissue samples .
the primers for partial sequencing of the s region are presented in table 1 ( 23 ) .
for the first round , 5 l of the extracted dna from each sample was added to a pcr reaction mixture containing 0.5 l dntp ( 10 mm ) , 2.5 l pcr buffer ( 10x ) , 0.15 l 5u taq dna polymerase ( roch , germany ) , 50 pmol/l of the fhbs1 and rhbs1 primers , 0.5 l dntp ( 10 mm ) and 15.85 l distilled water . the samples were placed in the thermal cycler ( techne company , uk ) and the first round amplification was carried out with initial denaturation at 94c for five minutes , followed by denaturation at 94c for 30 seconds , annealing at 56c for 30 seconds and extension at 72c for 30 seconds , for a total of 30 cycles . for the second round , 5 l of the pcr product
was added to 25 l of reaction mixture containing the same components mentioned in the first run including dntp , pcr buffer and taq dna polymerase with 50 pmol/l of each of fhbs2 and rhbs2 primers .
amplification was carried out in the thermal cycler with the same program as the first round .
next , 8 l of the nested pcr product ( 417 bp ) was analyzed by 2% agarose gel electrophoresis .
amongst the ten samples positive for hbv dna , six samples were sequenced ( applied bi benier company , south korea ) .
the sequences of all the six samples were submitted to ncbi and registered in gen bank ( table 2 and figure 1 ) .
hepatitis b virus sequences were aligned with the hbv reference sequence from gene bank using the hbv data - base and online blast .
a phylogenetic tree was constructed using the mega 5 software . to evaluate the hbs subtypes and s region mutations of the positive samples , ( figure 1 )
the amino acid substitutions in positions 122 and 160 of the s region of hbv dna were compared with the hbv reference sequence .
interestingly , the presence of an unpredictable amino acid substitution ( l88p ) was observed , which indicated a new mutation in the
s region of hbv dna in patients with hodgkin and non - hodgkin lymphoma , ( figure 2 ) .
the consensus amino acid sequence of the reference d genotype is shown on the top .
the partial sequences of all the six hbs genes in position 88 compared with the consensus sequence amino acid indicated a proline substitution with leucine . so far this type of mutation has not been reported in any hbv d genotype .
the dots show conserved amino acid and the amino acid code indicates differences with the consensus amino acid .
statistical analysis was done with the chi square test using the spss software , version 20 .
the dna extraction of all the tissue samples was carried out using the high pure pcr template kit ( roche , germany ) , according to the manufacturer s instructions .
the nested pcr for detection of hbv dna was performed for all the tissue samples .
the primers for partial sequencing of the s region are presented in table 1 ( 23 ) .
for the first round , 5 l of the extracted dna from each sample was added to a pcr reaction mixture containing 0.5 l dntp ( 10 mm ) , 2.5 l pcr buffer ( 10x ) , 0.15 l 5u taq dna polymerase ( roch , germany ) , 50 pmol/l of the fhbs1 and rhbs1 primers , 0.5 l dntp ( 10 mm ) and 15.85 l distilled water . the samples were placed in the thermal cycler ( techne company , uk ) and the first round amplification was carried out with initial denaturation at 94c for five minutes , followed by denaturation at 94c for 30 seconds , annealing at 56c for 30 seconds and extension at 72c for 30 seconds , for a total of 30 cycles . for the second round , 5 l of the pcr product was added to 25 l of reaction mixture containing the same components mentioned in the first run including dntp , pcr buffer and taq dna polymerase with 50 pmol/l of each of fhbs2 and rhbs2 primers .
amplification was carried out in the thermal cycler with the same program as the first round .
next , 8 l of the nested pcr product ( 417 bp ) was analyzed by 2% agarose gel electrophoresis .
amongst the ten samples positive for hbv dna , six samples were sequenced ( applied bi benier company , south korea ) .
the sequences of all the six samples were submitted to ncbi and registered in gen bank ( table 2 and figure 1 ) .
hepatitis b virus sequences were aligned with the hbv reference sequence from gene bank using the hbv data - base and online blast .
a phylogenetic tree was constructed using the mega 5 software . to evaluate the hbs subtypes and s region mutations of the positive samples , ( figure 1 )
the amino acid substitutions in positions 122 and 160 of the s region of hbv dna were compared with the hbv reference sequence .
interestingly , the presence of an unpredictable amino acid substitution ( l88p ) was observed , which indicated a new mutation in the
s region of hbv dna in patients with hodgkin and non - hodgkin lymphoma , ( figure 2 ) .
the consensus amino acid sequence of the reference d genotype is shown on the top .
the partial sequences of all the six hbs genes in position 88 compared with the consensus sequence amino acid indicated a proline substitution with leucine .
so far this type of mutation has not been reported in any hbv d genotype .
the dots show conserved amino acid and the amino acid code indicates differences with the consensus amino acid .
out of 12 hodgkin samples , seven ( 58.33% ) and five ( 41.66% ) were males and females , respectively .
the age of the patients with hodgkin lymphoma was between 5 and 34 with a mean age of 20 7 years .
of the 29 non - hodgkin samples , 17 ( 58.62% ) and 12 ( 41.37% ) were males and females , respectively , table 3 . out of the 41 samples , three out of twelve ( 25% ) hodgkin samples and seven of twenty - nine ( 24.13% )
all the positive hbv patients were within the normal range for alanine aminotransferase ( 20 - 32 iu / dl ) and aspartate aminotransferase ( 18- 27 iu / dl ) .
the youngest hodgkin patient was a female with 17 years of age and the oldest was a male with 29 years of age .
the youngest non - hodgkin patient with hbv infection was a male with two years of age and the oldest patient was a female with 75 years of age .
the sequences of six positive samples ( two hodgkin and four non - hodgkin lymphoma ) were registered in genbank with the following accession numbers , kj398939.1 to kj398944.1 .
the results of the alignment showed that the d genotype was the predominant genotype among the patients with hepatitis b infection .
mutation analysis indicated amino acid substitutions in positions 88 , 122 and 160 of the s region of hbv dna .
all of the six sequences were recognized as the awy subtype because of substitution in positions 122 and 160 .
the sequences of six positive samples ( two hodgkin and four non - hodgkin lymphoma ) were registered in genbank with the following accession numbers , kj398939.1 to kj398944.1 .
the results of the alignment showed that the d genotype was the predominant genotype among the patients with hepatitis b infection .
mutation analysis indicated amino acid substitutions in positions 88 , 122 and 160 of the s region of hbv dna .
several epidemiological studies have revealed the persistence of hepatitis b virus ( hbv ) infection amongst patients with hodgkin ( hl ) and non - hodgkin lymphoma ( nhl ) . in our study , 25% of hodgkin and
24.13% of non - hodgkin patients had positive hbv dna , which indicates the high rate of hbv infection among these patients .
the youngest hodgkin patient with hbv infection was a female with 17 years of age and the oldest was a male with 29 years of age .
the youngest non - hodgkin patient with hbv infection was a male with two years of age and the oldest was a female with 75 years of age .
the frequency of hbv infection among the male and female patients was not significantly different ( p > 0.05 ) . in our study
fernndez et al . in spain reported the ad and ay hbs subtypes amongst 40 chronic hbv patients ( 24 ) .
de souza et al . from brazil reported hbsag subtypes adw2 , adw4 , and ayw amongst patients with mental problems ( 25 ) . in our study
genes , showed mutation at position 88 ( l88p ) . so far this type of mutation has not been reported in any hbv d genotype . in the study conducted by okamoto et al .
a point mutation was found in the s gene of hepatitis b virus for two blood donors carrying a surface antigen with subtype adyr or adwr ( 26 ) .
the patient s history revealed that they were not tested for hbv and hepatitis c virus and it is not clear whether these patients had chronic hbv or occult hbv infection .
however , there are some reports about the association of occult hbv infection and non - hodgkin lymphoma ( 27 , 28 )
. the presence of unpredictable amino acid proline at position 88 of hbs gene indicates a new mutation in the
s region of hbv dna in patients with hodgkin and non - hodgkin lymphoma . in our survey
the d1 genotype was dominant in all the hbv patients , which is similar to the report of marcucci et al .
the role of other viruses including hepatitis c virus in hodgkin and non - hodgkin lymphoma was not studied however this requires further investigations ( 30 , 31 ) .
there have been reports about the reactivation of hepatitis b and hepatitis c virus ( hcv ) infections during nhl treatment ; in these studies it was revealed that the patients who were under chemotherapy showed hbv and hcv reactivation , yet other patients who received lamivudine before chemotherapy did nt show any reactivation for hbv or hcv infection ( 32 - 35 ) . in conclusion , a high prevalence of hbv infection ( 25% and 24.13% , respectively ) was found in patients with hodgkin and non - hodgkin lymphoma , indicating the importance of hbv and hcv screening by sensitive tests such as pcr before chemotherapy to prevent hbv or hcv reactivation in patients with hl and nhl . | background : hepatitis b virus ( hbv ) is responsible for chronic , acute , and fulminant hepatitis , which are prevalent worldwide .
chronic hbv may lead to cirrhosis and hepatocellular carcinoma .
several epidemiological studies have indicated that hepatitis b virus is involved in b - cell hodgkin and non - hodgkin lymphoma ( nhl).objectives : the aim of this study was to evaluate the association between hepatitis b infection and hodgkin and non - hodgkin lymphoma.materials and methods : paraffin embedded of 41 block samples including 12 ( 29.26% ) hodgkin and 29 ( 70.73% ) non - hodgkin patients were collected .
next , dna extraction was carried out for all the samples followed by hbv dna detection by the nested polymerase chain reaction ( pcr ) .
the positive hbv dna samples were sequenced , and hbv genotypes and hbv subtypes were determined.results:three out of 12 ( 25% ) hodgkin samples and seven out of 29 ( 24.13% ) non - hodgkin showed positive hbv dna results .
the results of sequencing revealed that the d genotype was predominant among the positive hbv patients .
interestingly an unpredictable amino acid proline was detected in position 88 of the hbs gene , which indicates a new mutation in the
s region of hbv dna in patients with hodgkin and non - hodgkin lymphoma.conclusions:a high rate of 25% and 24.13% of hbv dna was detected among patients with hodgkin and non - hodgkin lymphoma , respectively . |
while ecstasy is a drug used for fun today , it has serious side effects and sometimes it can be fatal .
these effects are due to idiosyncratic reactions as a result of various stimulations in adrenergic receptors .
short after it is swallowed , it causes sweating , tachycardia , tiredness , muscle spasms , and jaw locking ; and in long - term , it may cause delirium , convulsions , cerebral hemorrhage , cardiac arrhythmias , hyperthermia , liquid and electrolyte imbalance , disseminated intravascular coagulation ( dic ) , rhabdomyolysis , acute kidney failure , and acute liver failure . in this case report , we present a case of thrombotic thrombocytopenic purpura ( ttp ) associated with the use of ecstasy .
this case is a 36-year - old male who complains of fever , speech difficulty , change in conscience , and red rashes on legs .
in addition , he was pale , icteric and had petechial rashes on his legs .
his blood pressure was 150/100 mmhg , and heart rate 92 beats / min in normal rhythm .
his tests revealed that he had anemia and thrombocytopenia , increased lactate dehydrogenase ( ldh ) , indirect bilirubin and creatinine kinase , a two - fold increase in transaminases , and reticulocytes were 7.4% .
his urinalysis revealed that he had proteinuria , calculated to be 320 mg/24-h . in his peripheral smear , there were 810 schistocytes , 56% fractioned neutrophils , 22% clubbings , 14% lymphocytes , 4% monocytes , 2% myelocytes and 2% metamyelocytes in every region in the small magnification [ figure 1 ] , so he was diagnosed as microangiopathic hemolytic anemia . together with thrombocytopenia , fever , neurologic symptoms and proteinuria , the patient was labeled as ttp .
his cranial tomography and cranial magnetic resonance imaging ( mri ) did not reveal any pathology .
the patient did not use any medication , did not have any autoimmune condition , and did not have any infection findings , and his abdominal and thorax tomography taken for any possible malignancy did not reveal any pathology .
his viral serology tests and anti - nuclear antibodies , anti - neutrophil cytoplasmic antibodies , extractable nuclear antigens screen , rheumatoid factor , anti - cardiolipin igm , and igg were negative .
iga , igg , igm , c3 , c4 were found to be normal . as the patient had been using 3,4 methylenedioxymethamphetamine ( mdma ) ( ecstasy ) intensively for approximately 10 years
he was given 1 g / day methylprednisolone and vincristine 2 mg / day ( single dose ) for 3 days .
when his platelets got above 100.000 mm , plasmapheresis was started to be performed once every 2 days .
the patient 's ldh decreased , and level of his platelets increased so he was discharged . on follow - up visits
he had left flank pain , decreased urine output and was hypertensive ( 200/120 mmhg ) , so he was put on amlodipine 10 mg / day and ramipril 5 mg / day .
follow - up laboratory workup showed creatinine to be 2.4 mg / dl , creatine kinase : 559 u / l , platelet : 130.000 mm , ldh : 351 u / l and 24-h urine protein 7.6 g / day .
kidney biopsy was performed and showed bowman capsules to have peak lesions in some glomeruli , along with adhesions and visceral epithelial proliferation . in these areas ,
focal minimal atrophy with few foamy cell group tubules , and a mild wall thickening in arterioles and arteries were observed . with these findings
, the patient was diagnosed with focal segmental glomerulosclerosis ( fsgs ) [ figure 2 ] .
it was thought that the patient developed fsgs secondary to ecstasy , and his proteinuria increased as a result of accelerated hypertension .
the patient was given 1 g / day pulse methylprednisolone and cyclophosphamide for 3 days .
then , the patient 's treatment continued with 1 mg / kg / day methylprednisolone , and his hypertension was controlled .
plasmapheresis was performed on the patient for 1 more week , and when his ldh and platelets level turned to normal , his apheresis procedures were ended .
laboratory workup after 3 months showed levels of platelets , creatinine , creatine kinase and ldh to be normal . in his peripheral smear , schistocytes in every region in the small magnification focal minimal atrophy in a few foamy cell group tubules and a mild wall thickening in arterioles and arteries were observed
thrombocytopenic purpura is a rare clinical syndrome with the cardinal features of hemolytic anemia , thrombocytopenia , fever , renal impairment , and neurological dysfunction , although all five features being observed in only 40% of the patients .
ttp is usually idiopathic , but it can be seen as a secondary condition after infections , medications , autoimmune diseases , malignancies , pregnancy , and bone marrow transplantation . while ttp was observed to develop after the use of cocaine and heroin , very rare cases were reported of ttp after the use of ecstasy .
ttp was first defined in 1924 by moschcowitz , and it occurs through immune and nonimmune mechanisms .
recently , a specific metalloproteinase ( adamts13 ) that breaks up the von willebrand factor was defined .
while enzyme activity deficiency can be congenital , it has also been shown that it can be caused by auto - antibodies . with the absence of signs of any infection in our case , and the presence of hemolytic anemia , normal creatinine , proteinuria , fever , and change in consciousness
, we diagnosed the patient as ttp rather than hemolytic uremic syndrome . with the use of amphetamine
procoagulant factors are released from platelets , and microthrombuses are formed in arteriol capillary level , resulting in secondary microangiopathic hemolytic anemia .
we hypothesize that there could be an association between the consumption of mdma in our case and the induction of ttp based on certain similarities between the molecular structure of mdma and thienopyridines that are known to cause ttp .
nephropathy secondary to ecstasy use was first defined in 2007 by bohatyrewicz et al . as a membranous glomerulonephritis that developed secondary to ecstasy use .
then hyponatremia occurs due to the antidiuretic hormone secreted as a response and drinking too much water .
the rennin angiotensin aldosterone system is activated , and hence , water and salt are retained in the body .
it exhibits sympathomimetic effect by stimulating adrenergic receptors , by blocking catecholamine reuptake and by inhibiting monoamine oxidase activity .
hypertension occurs as a result of all these mechanisms . while chronic kidney failure can develop due to hypertension within weeks or months after ecstasy use , acute kidney failure and nephropathy can also develop as a result of accelerated hypertension . in our case , who developed nephropathy secondary to ecstasy use ( fsgs ) , distinctive increase in proteinuria as a result of accelerated hypertension was observed .
in addition , it should be kept in mind that dic , rhabdomyolysis , and ecstasy can have a direct toxic effect on nephropathy . as a result , although reported very rarely , it should be kept in mind that ttp accelerated hypertension and nephropathy can develop secondary to ecstasy use . | ecstasy is a drug , which causes serious side effects and sometimes it can be lethal .
these effects are due to idiosyncratic reactions as a result of various stimulations in adrenergic receptors . here
we present a case of a 36-year - old male patient who was diagnosed with thrombotic thrombocytopenic purpura associated with the use of ecstasy .
plasmapheresis along with methylprednisolone treatment restores patient condition to normal . |
increased industrialization is posing a threat to human health in the form of environmental pollution . environmental remediation of organic pollutants by photocatalytic degradation using wide bandgap semiconductors has attracted considerable attention in recent times.14 further , these materials , in a nanostructured form , exhibit enhanced properties and therefore have potential application such as the fabrication of nanodevices . various semiconductors , including zinc oxide ( zno ) and titanium dioxide ( tio2 ) , are being used in photocatalytic application because they have a low - cost , high - photosensitivity , nontoxic , and environmentally friendly nature.5,6 zno is established to be a more efficient photocatalyst than tio2 because of its high surface reactivity , owing to its large number of active surface defect states .
it has also been shown that zno has high reaction and mineralization rates7 because of its more efficient hydroxyl ion production.8 when zno is irradiated with ultraviolet ( uv ) light , electrons are excited from the valence band to the conduction band and electron hole pairs are created . these electron
hole pairs activate the surrounding chemical species and then promote the chemical reactions.9,10 as photocatalytic activity of metal oxide nanostructures depends on the surface area and surface defects , one - dimensional nanostructures such as nanowires / nanorods are ideal candidates for the application in photocatalysis , because they offer a larger surface - to - volume ratio than nanoparticulate thin films.11 photocatalysis using suspended nanoparticles is an efficient method for removing pollutants in a liquid
. however , the successive removal of the nanoparticles from the liquid can be cumbersome and includes an additional process step .
also , freely suspended nanoparticles can enter the human body through skin pores and may lead to health - related problems.12 therefore , it is advantageous if the nanoparticulate photocatalyst is attached to a support .
the importance of catalyst support has been discussed by pozzo et al,13 who observed an inverse relationship between the adhesion of a catalyst to a support and its photocatalytic activity .
thus , zno nanorods , which can be strongly attached to any type of substrates through proper surface treatment before seeding,14 might be an attractive choice for photocatalytic applications .
various one - dimensional zno nanostructures , such as nanobelts , nanowires , nanorods , and nanotubes,1518 have been reported in literature .
several methods have been employed for synthesizing zno nanorods , such as thermal evaporation , chemical vapor deposition , physical vapor deposition , and electrochemical processes.1922 however , these methods require vacuum conditions , a high temperature , sophisticated equipment , and other rigid experimental conditions , which may increase the cost . comparatively , the solution approach is more attractive for its simplicity , commercial feasibility , and good potential for large - scale production .
wet chemical synthesis has been proven to be a versatile approach for the growth of zno nanorod arrays , due to its convenience , simplicity in fabrication , environmentally friendly reactions , low temperature , large - scale production , and high crystalline quality . in this work ,
zno nanorod arrays on an si substrate were grown using a wet chemical route for the photodegradation of organic dyes .
the presented work may open up the possibility for the successful utilization of zno nanostructures as a future photocatalyst .
well - aligned zno nanorod arrays were synthesized on the zno particles deposited on an si ( 100 ) substrate using high purity analytical grade chemicals ( sigma - aldrich , st louis , mo , usa ) . to obtain the final product ,
initially , zno nanoparticles were synthesized by acetate route and were used as a seed layer on an si substrate .
zno nanoparticles acted as the nucleation center for the formation of zno nanorods on the si substrate .
zno nanocrystals to be used as the seed layer were synthesized by the acetate route .
prior to coating , si substrate was cleaned ultrasonically for 10 minutes , first in acetone and then deionized water , to remove any impurity and oxide layer . in a typical synthesis procedure , 20 mm zinc acetate dihydrate ( zn[ch3coo]2 2h2o ) and 10 mm sodium hydroxide ( naoh ) solution in ethanol was mixed in a beaker and kept at 70c for 2 hours under stirring , to produce a white suspension of zno seeds .
subsequently , the suspension was used to produce a seed layer on an si substrate by a drop casting method .
the coated si substrate was dried at 200c in an oven for 1 hour to attain better adhesion of the seed particles to the substrate . a solution containing 0.2 m aqueous solution of zinc nitrate hexahydrate ( zn[no]3 6h2o ) and 0.01 m hexamethylenetetramine ( c6h12n4 )
lastly , the si substrate now with zno nanorods was thoroughly washed with deionized water to eliminate residual salts and dried in air at 80c for 2 hours before characterization .
various characterizations were carried out on the as - obtained product , such as x - ray diffraction ( xrd ) using a rigaku ultima iv x - ray diffractometer ( rigaku corporation , tokyo , japan ) with cuk radiations ( =1.5406 ) operated at a voltage of 40 kv and current of 30 ma .
xrd spectrum was recorded at a scanning rate of 0.5/minute in the range of 20280. morphology was studied using a field emission scanning electron microscope ( fesem ) , ( jeol , tokyo , japan ) at an operating voltage of 15 kv .
energy dispersive x - ray spectroscopy ( edx ) was used for ascertaining the elemental composition of zno nanorods .
an fesem ( jem-2100f ; jeol ) operated at 200 kv was used for obtaining transmission electron microscopy ( tem ) micrographs , selected area electron diffraction pattern , and high - resolution transmission electron microscopy ( hrtem ) images . to prepare samples for tem examination ,
zno nanorods were scraped off an si substrate and then dispersed in ethanol solution , followed by an ultrasonic treatment for 10 minutes .
a minute drop of zno nanorod suspension was cast on to a carbon - coated copper grid and subsequently dried in air before transferring it to the microscope .
room temperature raman spectrum was recorded using a raman spectrometer ( thermo fisher scientific , waltham , ma , usa ) with a 532 nm solid - state primary laser as an excitation source in the backscattering configuration .
methylene blue ( mb ) was used as the test contaminant for the photolysis experiments .
a 10 m aqueous solution of mb was prepared , into which the zno nanorod arrays substrate was dipped .
a high - pressure mercury lamp ( 125 w ) was used as a light source .
the beaker containing mb and zno nanorod arrays was then placed under the uv light , with the nanorod surface facing the light source for photodegradation of mb . optical absorption spectrum in the uv - visible region
was recorded after every fixed time interval , to follow the degradation of the mb .
zno nanocrystals to be used as the seed layer were synthesized by the acetate route . prior to coating ,
si substrate was cleaned ultrasonically for 10 minutes , first in acetone and then deionized water , to remove any impurity and oxide layer . in a typical synthesis procedure , 20 mm zinc acetate dihydrate ( zn[ch3coo]2 2h2o ) and 10 mm sodium hydroxide ( naoh ) solution in ethanol was mixed in a beaker and kept at 70c for 2 hours under stirring , to produce a white suspension of zno seeds .
subsequently , the suspension was used to produce a seed layer on an si substrate by a drop casting method .
the coated si substrate was dried at 200c in an oven for 1 hour to attain better adhesion of the seed particles to the substrate .
a solution containing 0.2 m aqueous solution of zinc nitrate hexahydrate ( zn[no]3 6h2o ) and 0.01 m hexamethylenetetramine ( c6h12n4 ) was prepared in a beaker . the seeded si substrate described in the previous section
lastly , the si substrate now with zno nanorods was thoroughly washed with deionized water to eliminate residual salts and dried in air at 80c for 2 hours before characterization .
various characterizations were carried out on the as - obtained product , such as x - ray diffraction ( xrd ) using a rigaku ultima iv x - ray diffractometer ( rigaku corporation , tokyo , japan ) with cuk radiations ( =1.5406 ) operated at a voltage of 40 kv and current of 30 ma .
xrd spectrum was recorded at a scanning rate of 0.5/minute in the range of 20280. morphology was studied using a field emission scanning electron microscope ( fesem ) , ( jeol , tokyo , japan ) at an operating voltage of 15 kv .
energy dispersive x - ray spectroscopy ( edx ) was used for ascertaining the elemental composition of zno nanorods .
an fesem ( jem-2100f ; jeol ) operated at 200 kv was used for obtaining transmission electron microscopy ( tem ) micrographs , selected area electron diffraction pattern , and high - resolution transmission electron microscopy ( hrtem ) images . to prepare samples for tem examination ,
zno nanorods were scraped off an si substrate and then dispersed in ethanol solution , followed by an ultrasonic treatment for 10 minutes .
a minute drop of zno nanorod suspension was cast on to a carbon - coated copper grid and subsequently dried in air before transferring it to the microscope .
room temperature raman spectrum was recorded using a raman spectrometer ( thermo fisher scientific , waltham , ma , usa ) with a 532 nm solid - state primary laser as an excitation source in the backscattering configuration .
methylene blue ( mb ) was used as the test contaminant for the photolysis experiments .
a 10 m aqueous solution of mb was prepared , into which the zno nanorod arrays substrate was dipped .
a high - pressure mercury lamp ( 125 w ) was used as a light source .
the beaker containing mb and zno nanorod arrays was then placed under the uv light , with the nanorod surface facing the light source for photodegradation of mb . optical absorption spectrum in the uv - visible region
was recorded after every fixed time interval , to follow the degradation of the mb .
the xrd pattern of the zno nanorod arrays grown on the si substrate is depicted in figure 1 .
it is clear from figure 1 that the xrd peaks positions are very well matched with the standard pdf values ( jcpds , 361451 ) .
it is also seen from the figure that the relative intensities of these peaks are different from those of zno nanopowder,23 as shown in the inset of figure 1 . on the basis of the xrd results
, we can conclude that the prepared nanorod arrays are highly crystalline , having wurtzite structure with preferable c - axis orientation .
the intensity of the ( 002 ) peak was observed to be very strong in comparison with other peaks .
trace amounts of other diffraction peaks , such as ( 101 ) , ( 102 ) , and ( 103 ) , do not mean that some zno nanorods were grown in those directions , but c - axis zno nanorods were so randomly oriented that these diffraction planes happen to meet bragg s law.24 the morphological studies of vertically aligned zno nanorod arrays were carried out using fesem , and the micrograph is shown in figure 2a .
the fesem image ( figure 2a ) of the sample shows that the product consists of nanorods with an average diameter of ~8090 nm and length of about ~350400 nm , respectively , and each individual zno nanorod has well - developed hexagon facets . the elemental composition of the sample determined by edx analysis is presented in figure 2c .
figure 2c shows the presence of only zn and o peaks , which confirms that the nanorods are primarily made of zno .
the hrtem image and its corresponding selected area electron diffraction pattern ( inset ) are shown in figure 2b .
it is also confirmed from figure 2b that the synthesized zno nanorods are single crystalline with ( 001 ) growth direction .
the interplanar spacing was found to be 0.262 nm , corresponding to the ( 002 ) plane of the hexagonal structure of zno , corroborating the xrd results .
raman spectroscopy measurements were performed at room temperature to examine the structure , crystallization , and defects of the zno nanorod arrays .
zno has a wurtzite structure and belongs to the c46v space group with two formula units per primitive cell.25 group theory analysis , at the center of the brillouin zone , suggests that the a1+e1 + 2e2 modes are raman active.26 representative raman spectrum of the zno nanorod arrays is shown in figure 3 .
only the e2 high modes at 439 cm and al ( lo ) at 578 cm were observed , which further confirms that the zno nanorod arrays are highly c - axis oriented because , in the present experiment , the incident light was normal to the substrate : ie , the incident light is parallel to the c - axis of the nanorods and the raman signal was recorded in the backscattering geometry . in this configuration , only a1 ( lo ) and e2 modes are allowed and the other modes can not be observed from the vertically aligned nanorods but can be observed from the tilted and randomly oriented nanorods , according to the raman selection rules.27,28 photocatalytic properties of the zno nanorod arrays were examined by decomposition of mb . the characteristic absorption of mb at ~660 nm was chosen to monitor the photocatalytic degradation process .
figure 4 illustrates the time - dependent absorption spectra of mb aqueous solutions during the uv light irradiation in the presence of zno nanorod arrays . as a control ,
the absorbance peak of the mb solution was monitored under two different conditions : 1 ) with a photocatalyst in the dark and 2 ) without photocatalysts under uv light illumination .
the change in the absorbance peak of mb under these conditions is found to be negligible , indicating that there is no loss of mb without an irradiated photocatalyst . for zno nanorod arrays placed in mb solution , the maximum absorption of the mb solution
was found to decrease with illumination time and disappeared almost completely after irradiation for about 270 minutes .
this indicates that zno nanorod arrays show uv light photocatalytic properties in the degradation of mb .
figure 5 shows the relative concentration ( c / c0 ) of mb as a function of time , where c is the concentration of mb at the irradiation time ( t ) and c0 is the concentration of the dye before irradiation .
after uv light irradiation for 270 minutes , more than 95% of mb is degraded in the presence of zno nanorods .
this indicates that zno nanorods are an effective photocatalyst for the degradation of organic dyes .
figure 6 shows the percentage degradation of mb as a function of irradiation time with zno nanorod arrays photocatalyst
. the higher photocatalytic performance of ~80% was achieved within 60 minutes of photoirradiation for zno nanorod arrays , whereas after 270 minutes of illumination with uv light , mb was almost completely removed over zno nanorod arrays . by comparing the photocatalytic performance of the zno nanorod arrays obtained in this study with other reported nanostructures , zno nanorod arrays showed better photocatalytic properties .
for example , nipane et al29 showed that zno nanorods degraded 50% of mb under uv light irradiation for 120 minutes of irradiation time , whereas about 90% of mb was degraded with zno nanocauliflowers . in the present work ,
zno nanorod arrays degraded ~92% of mb for 120 minutes , and for 270 minutes of uv light irradiation , mb was almost completely degraded .
therefore , the zno nanorod arrays obtained in the present work are more superior photocatalytics than others .
the photocatalytic activity of a catalytic material is influenced by several factors , such as carrier recombination , size of the particles , surface area , surface acidity , and presence of a higher number of hydroxyl groups .
the enhancement of photocatalytic activity was most likely due to the relative increase of active morphological surface resulting from increased surface to volume ratio as well as the low recombination rate of the electron
photocatalytic degradation was generally operated by the action of hydroxyl radicals formed during the reaction.30 the photocatalytic mechanism is proposed as follows : when the zno nanorod arrays are illuminated with light , electrons are excited from the valence band of zno to the conduction band , leaving a hole in the valence band .
the hydroxyl groups present on the surface of the zno react with the photogenerated hole to produce hydroxyl radicals . similarly ,
this peroxide takes one proton to yield a superoxide ( ho2 ) followed by the formation of hydrogen peroxide ( h2o2 ) .
a hydroxyl radical was also produced by the attack of a photogenerated electron to the hydrogen peroxide .
these reactive radicals and intermediate species react with dye and degrade them into nontoxic organic compounds .
we have successfully grown well - aligned zno nanorod arrays on an si substrate with a cheap , low - temperature , and environmentally friendly wet chemical solution route .
xrd , hrtem , and raman analyses revealed the hexagonal wurtzite structure , single phase nature , and high crystalline quality of zno nanorod arrays .
fesem and tem micrographs showed that the nanorods were typically of ~8090 nm in diameter and ~350400 nm in length , respectively .
raman measurements confirmed the characteristic peak of zno : ie , e2 ( high ) mode for zno nanorod arrays .
the photocatalytic properties of zno nanorod arrays toward mb under uv light irradiation were found to be attractive .
the results of photodegradation show that zno nanorod arrays are capable of degrading ~80% of mb within 60 minutes of irradiation , whereas ~92% of degradation was achieved for 120 minutes . upon prolongation of irradiation time to 270 minutes , mb degrades almost completely .
due to the excellent photocatalytic performance and easier preparation method , the prepared zno nanorod arrays are believed to have potential application in the containment of environmental pollution . | well - aligned and single - crystalline zinc oxide ( zno ) nanorod arrays were grown on silicon ( si ) substrate using a wet chemical route for the photodegradation of organic dyes .
structural analysis using x - ray diffraction , high - resolution transmission electron microscopy , and selected area electron diffraction confirmed the formation of zno nanorods grown preferentially oriented in the ( 001 ) direction and with a single phase nature with a wurtzite structure .
field emission scanning electron microscopy and transmission electron microscopy micrographs showed that the length and diameter of the well - aligned rods were about ~350400 nm and ~8090 nm , respectively .
raman scattering spectra of zno nanorod arrays revealed the characteristic e2 ( high ) mode that is related to the vibration of oxygen atoms in the wurtzite zno .
the photodegradation of methylene blue ( mb ) using zno nanorod arrays was performed under ultraviolet light irradiation .
the results of photodegradation showed that zno nanorod arrays were capable of degrading ~80% of mb within 60 minutes of irradiation , whereas ~92% of degradation was achieved in 120 minutes .
complete degradation of mb was observed after 270 minutes of irradiation time .
owing to enhanced photocatalytic degradation efficiency and low - temperature growth method , prepared zno nanorod arrays may open up the possibility for the successful utilization of zno nanorod arrays as a future photocatalyst for environmental remediation . |
partial duplication of intracranial arteries , so called fenestration , is known to have an angiographical incidence of 0.3 to 0.9% , and is frequently associated with aneurysms22 ) .
fenestrated arteries are often observed in the posterior circulation , but are known to be relatively unusual in the anterior circulation19 ) .
aneurysms arising from the fenestrated a1 segment of anterior cerebral artery ( aca ) are even more rare , and are considered to be unique because they may be associated with other vascular anomalies . however , the characteristics of fenestrated a1 aneurysms has not been well determined until now . in the present report
, we demonstrate a case of ruptured saccular aneurysm arising from the proximal end of a fenestrated a1 artery . by literature review
, we classified fenestrated a1 aneurysm into three subtypes based on the anatomical location of the aneurysm and found the close relationship between the occurrence of fenestrated a1 aneurysm and the presence of several vascular anomalies including azygos aca and hypoplastic a1 .
a 59-year - old woman was admitted with complaints of a sudden onset of dysarthria and left side weakness .
subarachnoid hemorrhage was also identified in the anterior falx and sulci of medial frontal lobes ( fig .
cerebral angiography demonstrated a saccular aneurysm which arised from the proximal end of the right fenestrated a1 segment .
there was another saccular aneurysm at the right middle cerebral artery ( mca ) bifurcation which was considered as an unruptured one because of the long distance from co - existing intracerebral hematoma ( fig .
a ruptured saccular aneurysm with broad neck was identified on the proximal end of fenestrated a1 segment intraoperatively .
an unruptured mca bifurcation aneurysm was also treated by surgical clipping and the intracerebral hematoma was removed ( fig .
the postoperative course was uneventful , and the patient was discharged without any neurologic deficits . in the literature review , we found 18 cases of aneurysms arising from the fenestrated a1 segment .
characteristics of 19 cases ( including present case)1 - 4,6,7,9 - 13,16 - 20,22 ) are summarized in table 1 .
there were 9 males and 9 females ( 1 case , not recorded ) with a mean age of 49.7 years ( range 33 - 78 years ) .
the sizes of aneurysms were recorded in 7 out of 19 cases , and the mean aneurysm size was 5.05 mm ( range 2.9 - 7.0 mm ) .
twelve aneurysms were developed at the right side while seven were located at left side .
three among the aneurysms were treated by endovascular interventions , while the other aneurysms except for those three were treated by aneurysm clipping .
we could classify the aneurysms into three types based on the arising point of the aneurysm ( fig .
3 ) . of the 17 cases in which exact location of the aneurysm was described on the original article ( including the present case ) , 14 ( 82% ) aneurysms developed on the proximal end of the fenestrated a1 segment ,
which was classified as type i. two cases ( 12% ) were classified as type ii which represent the aneurysm located on the midportion of the fenestrated a1 .
one case ( 6% ) which demonstrated multiple aneurysms including one at the proximal end and another at the posterior aspect of the fenestrated artery was classified as type iii .
of the nine reports in which the presence of azygos aca has been investigated azygos aca was noted in 3 cases ( 33% ) .
hypoplastic a1 or aplasia of a1 was demonstrated in 4 cases ( 31% ) out of 13 cases in which the status of a1 hypoplasia or aplasia were described . in one case ,
finally , multiple aneurysms were found in 3 cases ( 16% ) including the present case .
a 59-year - old woman was admitted with complaints of a sudden onset of dysarthria and left side weakness .
subarachnoid hemorrhage was also identified in the anterior falx and sulci of medial frontal lobes ( fig .
cerebral angiography demonstrated a saccular aneurysm which arised from the proximal end of the right fenestrated a1 segment .
there was another saccular aneurysm at the right middle cerebral artery ( mca ) bifurcation which was considered as an unruptured one because of the long distance from co - existing intracerebral hematoma ( fig .
a ruptured saccular aneurysm with broad neck was identified on the proximal end of fenestrated a1 segment intraoperatively .
an unruptured mca bifurcation aneurysm was also treated by surgical clipping and the intracerebral hematoma was removed ( fig .
the postoperative course was uneventful , and the patient was discharged without any neurologic deficits .
in the literature review , we found 18 cases of aneurysms arising from the fenestrated a1 segment .
characteristics of 19 cases ( including present case)1 - 4,6,7,9 - 13,16 - 20,22 ) are summarized in table 1 .
there were 9 males and 9 females ( 1 case , not recorded ) with a mean age of 49.7 years ( range 33 - 78 years ) .
the sizes of aneurysms were recorded in 7 out of 19 cases , and the mean aneurysm size was 5.05 mm ( range 2.9 - 7.0 mm ) .
twelve aneurysms were developed at the right side while seven were located at left side .
three among the aneurysms were treated by endovascular interventions , while the other aneurysms except for those three were treated by aneurysm clipping .
we could classify the aneurysms into three types based on the arising point of the aneurysm ( fig .
3 ) . of the 17 cases in which exact location of the aneurysm was described on the original article ( including the present case ) , 14 ( 82% ) aneurysms developed on the proximal end of the fenestrated a1 segment ,
which was classified as type i. two cases ( 12% ) were classified as type ii which represent the aneurysm located on the midportion of the fenestrated a1 .
one case ( 6% ) which demonstrated multiple aneurysms including one at the proximal end and another at the posterior aspect of the fenestrated artery was classified as type iii . of the nine reports in which the presence of azygos aca has been investigated azygos aca
hypoplastic a1 or aplasia of a1 was demonstrated in 4 cases ( 31% ) out of 13 cases in which the status of a1 hypoplasia or aplasia were described . in one case ,
finally , multiple aneurysms were found in 3 cases ( 16% ) including the present case .
there were only few articles which have proposed several theories for the development of fenestrated a1 segment .
fenestration of the a1 segment might occur due to the absence of fusion of the plexiform anastomosis which was present in the distal primitive aca during the 18- to 43-mm stage of the embryo1,15 ) .
this failure would possibly increase blood flow in one of the a1 segments because of the contralateral a1 hypoplasia , resulting in increased hemodynamic stress on the ipsilateral a1 segment .
such increased blood flow and hemodynamic stress would prevent normal fusion of the plexiform anastomosis in the distal primitive aca , resulting in formation of the a1 fenestration , and could also cause aneurysm formation on the fenestrated a1 segment4 ) .
however , the pathophysiologic mechanism for the development of fenestrated a1 segment is still not clearly understood .
based on literature review , we classified fenestrated a1 aneurysm into three subtypes based on the location of aneurysms associated with a1 fenestrations , and to our knowledge , this is the first article to make a classification based on the location . in majority of the cases ,
the aneurysm had developed on the proximal end of the fenestration ( 82% ) , while 12% was developed on the trunk of duplicated a1 . in our case ,
the aneurysm was located at the proximal end of the fenestrated a1 , and could be determined as type i. additionally most of fenestrated a1 aneurysms ( 79% ) were ruptured at the time of presentation .
considering that cerebral saccular aneurysms usually arise from the arterial bifurcations where blood flow creates the greatest hemodynamic stress , the development of fenestrated a1 aneurysm also may be strongly associated with the hemodynamic force at the bifurcating point .
abnormal fusion of paired a2 from the medial branch of the primitive olfactory artery at the 16-mm stage of the embryo , and persistence of the median artery of the corpus callosum at the 20- to 24-mm stage may involve in the regression or lack of development of the acas4,8,15 ) .
the azygos aca is known to be closely associated with the occurrence of saccular aneurysms1,2 ) .
considering these theories and reviewing several previous studies , we found that the incidence of azygos aca in patients with fenestrated a1 aneurysm is much higher than the overall incidence of azygos aca in normal population , which suggests that azygos aca may be associated with the development of fenestrated a1 aneurysm .
the hypoplasia of the proximal aca on one side is shown in 7 - 10% of general population , and is regarded as a normal variant3 ) . however , by literature review , we found that the incidence of a1 hypoplasia or aplasia in patients with fenestrated a1 aneurysm ( 31% ) was significantly higher than that reported in patients without aneurysms .
the presence of hypoplastic a1 may lead to the increase of blood flow at the opposite side a1 , in which additional hemodynamic stress may be provided .
these could be one of the important predisposing factors for development of a fenestrated a1 aneurysm on the contralateral side of hypoplastic a1 . in our case , left a1 aplasia was demonstrated in cerebral angiography .
in addition , there was an azygos aca supplying blood flow to both cerebral hemispheres .
therefore , the right a1 segment was likely to receive strong hemodynamic stress , which may have affected the development of fenestrated a1 segment aneurysm and subsequent rupture . in the literature review ,
multiple aneurysms were shown in 16% of patients with fenestrated a1 aneurysms , which was comparable to approximately 15 - 20% of incidence of multiplicity in all patients with aneurysms5,14,21 ) .
therefore , fenestrated a1 aneurysm may not be associated with the occurrence of multiple aneurysms .
although fenestration of the proximal anterior cerebral artery is a very rare vascular anomaly , it is likely to accompany saccular type aneurysms in the vicinity of the vascular anomaly .
the fenestrated a1 aneurysm is also often co - existed with the azygos aca or contralateral hypoplastic a1 . as the aneurysms originated from fenestrated proximal aca are prone to rupture , early treatment may benefit patients harboring those aneurysms . | the aneurysm arising from fenestrated proximal anterior cerebral artery ( aca ) is considered to be unique .
the authors report a case of a 59-year - old woman who presented with a subarachnoid hemorrhage ( sah ) secondary to a ruptured aneurysm originating from the fenestrated a1 segment of right aca .
the patient had another unruptured aneurysm which was located at the right middle cerebral artery bifurcation .
she was successfully treated with surgical clipping for both aneurysms . from the previously existing literatures
, we found 18 more cases ( 1983 - 2011 ) of aneurysms associated with fenestrated a1 segment .
all cases represented saccular type of aneurysms , and 79% of the patients had sah .
there were three subtypes of the fenestrated a1 aneurysms depending on the anatomical location , relative to the fenestrated segment .
the most common type was the aneurysms located on the proximal end of fenestrated artery ( 82% ) .
azygos aca and hypoplastic a1 were frequently accompanied by the aneurysm ( 33% and 31% , respectively ) , and multiple aneurysms were shown in three cases ( 16% ) . considering that fenestrated a1 segment is likely to develop an aneurysm , which has high risk of rupture , early management may benefit patients with aneurysms accompanied by fenestrated proximal aca . |
left ventricular noncompaction has been classified as a primary cardiomyopathy with a genetic origin.1 morphologically , this condition is characterized by a thickened , two - layered myocardium with numerous and prominent trabeculations and deep , intertrabecular recesses.13 left ventricular noncompaction may be an isolated finding or it may be associated with a variety of other congenital heart defects.1 left ventricular noncompaction is associated with numerous sarcomere mutations and this fact created the view that there is a spectrum spanning hypertrophic cardiomyopathy ( especially apical hcm ) , dilated cardiomyopathy and left ventricular noncompaction.4 current diagnostic criteria for left ventricular noncompaction require the visualization of two distinct myocardial layers.5 the work of stllberger departs from this stipulation and he coined the term
left ventricular hypertrabeculation.5 this entity , which can be viewed as a less severe form of noncompaction , is characterized by the presence of more than three trabeculations located apically to the papillary muscles.5
we present a case report of a three month old male infant who presented with sudden infant death syndrome .
the macroscopical characteristics of this case was reported before.6 this three month old non - caucasian died suddenly and unexpectedly at his day care centre .
he did not have any known medical problems and no surgical procedures were ever performed .
no known allergies were present and no known family history of sudden , unexpected death were present . unfortunately the family members were lost to follow up , before electrocardiographic and echocardiographic screening could be performed to assess their risk for sudden unexpected death
. postmortem examination of the heart revealed numerous apical trabeculations of the left ventricle ( see fig .
no abnormalities were detected in any other organs during the postmortem examination and no thrombi were detected in the arterial system or the left ventricle .
figure 1 clearly demonstrates more than three trabeculations in the left ventricle in a location apical to the papillary muscles . according to stllberger7,8 this case which presented as a sudden infant death syndrome
histological assessment of sections underlying the left ventricular trabeculae revealed prominent areas of subendocardial fibrosis ( see figs . 2 , 3 and 4 ) .
figure 5 is a histological section of the apex of the left ventricle of a three month old male infant who also presented as a sudden infant death syndrome , but without left ventricular hypertrabeculation to serve as a control .
endocardial fibrosis with prominent elastin deposition have been described in cases of left ventricular noncompaction.5,9,10 currently , it is thought that ischaemia may play a major role in the pathogenesis of left ventricular noncompaction : mri and thallium-201 scintigraphy has shown subendocardial and transmural perfusion defects which corresponds to areas of noncompacted myocardium.5,1114 reduced coronary flow reserve , indicating microvascular dysfunction has also been shown to be present in isolated ventricular noncompaction by pet.12 currently , a chicken and egg dilemma exists regarding the pathogenesis of isolated ventricular noncompaction : either an impairment in the development of the myocardial microcirculation impairs the normal compaction process of the myocardium or vice versa . in this case report
current literature notes the presence of fibrosis in cases of ventricular noncompaction , this is the first case describing fibrosis in ventricular hypertrabeculation the less severe form of noncompaction .
secondly , the presence of such striking subendocardial fibrosis implies that the involved areas in the left ventricle must experience ischaemia already in utero .
lastly , we propose that these fibrotic areas may act as the foci of ventricular arrhythmia as the underlying cause of death .
l du - toit - prinsloo , wfp van heerden and g saayman did the post - mortem analysis and histological assessment . | left ventricular noncompaction has been classified as a primary cardiomyopathy with a genetic origin .
this condition is morphologically characterized by a thickened , two - layered myocardium with numerous prominent trabeculations and deep , intertrabecular recesses .
recently , it has become clear that these pathological characteristics extend across a continuum with left ventricular hypertrabeculation at one end of the spectrum.the histological findings include areas of interstitial fibrosis.we present a case of left ventricular hypertrabeculation which presented as sudden infant death syndrome .
histologically areas of subendocardial fibrosis was prominent and we propose that this entity may be a hidden cause of arrhythmic death in some infants presenting as sudden infant death syndrome .
, with areas of subendocardial fibrosis as possible arrhythmogenic foci . |
coronary artery bypass grafting ( cabg ) with cardiac vale repair is an uncommon surgery in infants .
cabg is technically demanding in infants due to the small size not only of the coronary arteries but also the potential graft arteries .
the short and long - term outcome of surgery is not known and thus has largely been avoided .
we report the case histories of two infants in whom cabg was undertaken successfully as a life - saving measure .
case 1 : this infant needed an arterial switch operation after which the right coronary artery ( rca ) was stenosed resulting in low cardiac output .
after the right internal mammary artery ( rima ) was used to anastamose the rca , the hemodynamic status improved drastically .
case 2 : this infant underwent surgical correction for anomalous left coronary artery from pulmonary artery ( alcapa ) .
she was found to have an occluded left coronary artery and mitral regurgitation ( mr ) . after she underwent left internal mammary artery ( lima ) to left anterior descending ( lad ) anastamosis and mitral valve repair , the clinical condition improved dramatically .
the long term results are not known and there are very few reports for the same .
though such an operation is best avoided , it can be used as a desperate life saving measure .
coronary artery bypass graft ( cabg ) surgery is technically difficult in infancy due to the small size not only of the coronary arteries but also the potential graft arteries .
the short and long - term outcome of surgery can be unpredictable , and thus has largely been avoided .
we report the case histories of two infants in whom coronary artery grafting was undertaken successfully as a life - saving measure .
a neonate ( three day old , 2.2 kilogram ) investigated for cyanosis at birth was found to have transposition of great arteries ( tga ) , intact ventricular septum and restrictive patent foramen ovale .
balloon atrial septostomy ( four day old ) was done , saturations improved and the baby was taken for urgent arterial switch operation ( aso ) on the sixth day of life ( 1 - 10 - 06 ) .
intraoperatively tga was confirmed , and aso was performed under cardiopulmonary bypass ( cpb ) with both antegrade and retrograde cardioplegia .
the aorta and pulmonary arteries were divided after accurate measurement to prevent any tension along the anastamotic lines .
the coronary buttons were transferred to the root of the divided pulmonary artery ( pa ) after which the divided distal aorta was brought below the pa confluence ( lecompte manoeuvre ) for anastamosis with the pa ( neoaorta ) . the pa defect resulting from dissection of coronary buttons
the heart came off cpb easily after which the chest was closed and baby shifted to intensive care .
the postoperative hemodynamics were excellent with normal electrocardiogram ( ecg ) , normal troponin levels and echocardiography ( echo ) showing good ventricular function .
follow up ( december 2006 ) , he was investigated for found to have respiratory symptoms .
captopril , digoxin , frusemide and spironolactone were started for cardiac failure . during cardiac catheterization ( december 2006 )
however there was faint visualization of the rca , filling from left coronary artery ( lca ) collaterals .
though the need for surgical intervention was obvious , no surgery could be done immediately , as the parents refused to give consent . during subsequent
follow up , the cardiac failure worsened , the dosages of the medications had to be increased . at this stage ,
parental consent was obtained and we decided to perform surgical revascularization . on 15 july 2007 , myocardial radionucleide scan ( 99m- technetium- tetrofosmin myocardial perfusion scintigraphy ) showed perfusion deficits in the inferior and basal segments suggesting impaired coronary perfusion .
after detailed deliberations and discussions it was decided to perform an off pump coronary artery grafting for the rca using right internal mammary artery ( rima ) .
considering the difficulty of weaning a poor ventricle off cpb , it was decided to perform an off pump cabg .
intraoperatively , the rima was found to be barely 1.5 mm size while the rca was only 1 mm size .
the rca was stabilized between stay sutures and opened for grafting to rima using 8 - 0 prolene sutures .
postoperative echo done one month postoperatively showed good flow across the rima to rca anastamosis . at two years
follow up the baby has optimum weight ( 7.8 kilogram ) , is active , has no ecg changes and is not on any medication .
a 3 month baby ( 2.6 kilogram ) with dysponea was found to have severe lv dysfunction with mild mitral regurgitation ( mr ) in echo .
cardiac catheterization ( 5 - 11 - 05 ) showed late filling of left coronary artery ( lca ) from left pulmonary artery ( lpa ) .
surgical correction of anamolous left coronary artery from pulmonary artery ( alcapa ) was done on cardiopulmonary bypass ( cpb ) ( 10/12/05 ) .
lca origin was cut off with a liberal button of main pulmonary artery ( mpa ) and dissected to allow a tensionless transfer to the aorta .
postoperatively she continued to have poor myocardial function and needed high dose infusion of dopamine , dobutamine and adrenaline .
intraoperatively ( 23/12/05 ) , the ventricles were barely contracting , much dilated and the la appeared distended . the left main coronary artery ( lmca )
there was a slight narrowing of the lmca just distal to its implantation to the aorta .
even after full mobilization , the left subclavian artery ( sca ) was barely 1 cm long and rudimentary .
since , the left internal mammary artery ( lima ) was 1.5 mm size and had good pulsations , lima was harvested .
the posterior annulus was reinforced and shortened by two pairs of continuous sutures coming to the centre of the pml from opposite commissures . since the expected mv annulus diameter for this baby was 11 mm both pairs of the sutures
the lad was identified and lima anastamosed to the lad using 7 - 0 prolene sutures .
soon sinus rhythm resumed and cpb was discontinued effortlessly ; however dobutamine was infused at 4 micrograms /kilogram /minute .
the postoperative course was uneventful and she was eventually discharged from the hospital on digoxin and frusemide . at three years
follow up , she has gained weight ( 9.6 kilogram ) , echo shows good left ventricular function and no mr .
these findings have been confirmed by cardiac catheterization ( 21 - 07 - 08 ) , ( figure 1 ) .
coronary revascularization along with cardiac valve repair is also an unusual surgery in infants.1 however coronary revascularization is indicated in babies who need aso or surgical correction for coronary abnormalities .
the coronary arteries are very small and delicate and re - implantation of these arteries or constructing an anastamosis for these arteries becomes technically demanding .
these situations may come up immediately postoperatively or in the later years during follow up.34 during the initial surgical correction itself , care was taken to avoid any tension in the newly implanted coronary arteries . in the first case , the arteries were checked after repositioning ; and they were found to be free from any tension . after the re implantation of the left coronary artery button to the aorta ( case 2 ) there was no tug on the artery . however with fibrosis in the postoperative period , these arteries can get pulled or deformed causing narrowing .
also , cardiac dilatation due to congestive cardiac failure also may have led to stretching of the artery causing stenosis .
bergond et al have stated that provided that the left main coronary artery bifurcation was not involved in the stenotic process , surgical arterioplasty of the main coronary trunks led to good functional and anatomic midterm results.4 in both infants from our study , the main coronary trunks were not visualised during angiography .
also , dissection of the main coronary arteries at the aortic root is very difficult due to dense adhesions from previous surgery . in case 1 ,
intraoperatively , the rca was identified in the anterior atrioventricular groove and grafted . in case 2
, there was a slight narrowing of the lmca just distal to its implantation to the aorta .
in this situation it was decided to graft the lima to lad so that it could supply the entire left coronary system .
these infants have benefitted as their cardiac function improved ( clinically and by echo ) , ecg changes reverted , they have put on weight and are free from medication .
subclavian artery has been reportedly used as graft in an infant for reconstruction of lmca.5 in both cases the sca was found to be unsuitable for grafting as it was short and rudimentary .
dissection of a coronary artery in a redo open heart case can be a daunting task even in adults .
cpb further increases the risk of bleeding and low cardiac output . in the first baby ( post aso ) , it was technically difficult to cannulate the aorta ( for cpb ) as the pa lies anterior to the aorta . also weaning off a sick lv from cpb
we had to wait two years before consent was given for cardiac catheterization , that too in only one patient .
there are reports are of cabg in bigger children and in adults with congenital cardio - vascular lesions.236 small , delicate arteries , non availability of good calibre conduits and co existing cardiac failure makes surgical revascularization extremely challenging.345 however in these two infants revascularization was done as a desperate life saving measure . though long term results are not known , this surgery did improve the hemodynamic parameters and increased the life span of these infants . with increasing number of surgical corrections for coronary anomalies in infants , | backgroundcoronary artery bypass grafting ( cabg ) with cardiac vale repair is an uncommon surgery in infants .
cabg is technically demanding in infants due to the small size not only of the coronary arteries but also the potential graft arteries .
the short and long - term outcome of surgery is not known and thus has largely been avoided.resultswe report the case histories of two infants in whom cabg was undertaken successfully as a life - saving measure .
case 1 : this infant needed an arterial switch operation after which the right coronary artery ( rca ) was stenosed resulting in low cardiac output .
after the right internal mammary artery ( rima ) was used to anastamose the rca , the hemodynamic status improved drastically .
case 2 : this infant underwent surgical correction for anomalous left coronary artery from pulmonary artery ( alcapa ) .
postoperatively , she was in low cardiac output .
she was found to have an occluded left coronary artery and mitral regurgitation ( mr ) . after she underwent left internal mammary artery ( lima ) to left anterior descending ( lad ) anastamosis and mitral valve repair , the clinical condition improved dramatically.conclusioncabg is an uncommon operation in infants .
this surgery is technically difficult .
the long term results are not known and there are very few reports for the same .
though such an operation is best avoided , it can be used as a desperate life saving measure . |
vinyl chloride ( vc ) is both a known carcinogen and a regulated chemical , and its production capacity has almost doubled over the last 20 years , currently 27 million tons / year worldwide . according to recent reports
it is still a cause for concern .
vc has been found as a degradation product of chloroethylene solvents ( perchloroethylene and trichloroethylene ) and in landfill gas and groundwater at concentrations up to 200 mg / m(3 ) and 10 mg / l , respectively .
worldwide occupational exposure to vc still seems to be high in some countries ( e.g. , averages of approximately 1,300 mg / m(3 ) until 1987 in one factory ) , and exposure may also be high in others where vc is not regulated . by combining the most relevant epidemiologic studies from several countries , we observed a 5-fold excess of liver cancer , primarily because of a 45-fold excess risk from angiosarcoma of the liver ( asl ) .
the number of asl cases reported up to the end of 1998 was 197 worldwide .
the average latency for asl is 22 years .
some studies show a small excess risk for hepatocellular carcinoma , and others suggest a possible risk of brain tumors among highly exposed workers .
lung cancer , lymphomas , or leukemia do not seem to be related to vc exposure according to recent results .
the mutation spectra observed in rat and human liver tumors ( asl and/or hepatocellular carcinoma ) that are associated with exposure to vc are clearly distinct from those observed in sporadic liver tumors or hepatic tumors that are associated with other exposures . in rats , the substitution mutations found at a : t base pairs in the ras and p53 genes are consistent with the promutagenic properties of the dna adduct 1,n(6)-ethenoadenine formed from vc metabolites .
risk assessments derived from animal studies seem to overestimate the actual risk of cancer when comparing estimated and reported cases of asl.imagesfigure 1figure 2 | |
developmental variations and morphological abnormalities such as cervical enamel projections , enamel pearls , and palatogingival grooves contribute to localized periodontal disease progression .
the major variant of this tooth type is the presence of an additional third root or a supernumerary lingual root called radix entomolaris ( re ) .
the role of re in the dental treatment , especially in root canal therapy , has been discussed broadly .
this macrostructure when found lingually was first mentioned in the literature by carabelli ( 1844 ) and was labeled as radix entomolaris by bolk ( 1915 ) .
the additional root can also be found distolingually which was first mentioned by de moor et al . in india ,
presence of an additional third root has a prevalence of 0.2% which depicts its rarity .
it has a frequency of less than 4% in whites and 2.8% in african population , whereas in populations with mongoloid traits ( american indian , eskimos , and chinese ) , this additional root occurs with a frequency between 5% and more than 30% . in these populations ,
the re is considered to be normal morphologic variant and can be seen as an asiatic trait ( yew sc , chan k 1993 ) .
it was suggested that the three rooted mandibular molar has certain impact as a genetic marker and therefore has a certain anthropologic significance ( tratman er 1938 , walker rt 1988 ) .
more specifically , only curzon ( 1974 ) suggested that certain traits such as the three rooted molar had a high degree of genetic penetrance as its dominance was reflected in pure eskimo / caucasians mixes who had similar prevalence of trait .
although this observation suggests that the formation of the re could be related to penetrance of an atavistic gene or polygenetic system ( calberson 2007 ) , the etiology behind this trait is still unclear .
the [ table 1 ] below depict the prevalence of mandibular first molar in europeans and non- europeans populations . the prevalence of mandibular first molar in europeans and non - europeans populations there was no evidence of extra root in south africans bushmen or in australian aborigines ( drennan 1929 and campbell 1929 ) .
the re was found on the first , second , and third mandibular molars , occurring least frequently in second molars .
table 2 presents the incidence of 3 roots found in mongolian , caucasian , and negro populations among different molars .
the incidence of 3 roots found in mongolian , caucasian , and negro populations among different molars the incidence of mandibular molars with three roots according to race and sex is mentioned in table 3 .
a case of 14-year - old girl presented to the department of periodontics , in government dental college and hospital aurangabad ( india ) , with deep periodontal pockets with mandibular first molars bilaterally on distal aspect .
radiographs were taken at 90 and other with a 30 mesial shift on both mandibular molars which depicted presence of three roots in mandibular molars figures 1 and 2 .
patient was referred to endodontic department and teeth were found to be vital on pulp vitality test .
iopa of mandibular right posterior region depicts three rooted mandibular first molar iopa of mandibular left posterior region depicts three rooted mandibular first molar on surgical interventions under local anesthesia , conventional flaps were reflected and thorough debridement of root surfaces was done .
three walled defects were explored bilaterally figures 3 and 4 with grade ii furcation involvement between distobuccal and distolingual roots figures 5 and 6 .
regenerative therapy with hydroxyapatite grafting was carried out figures 7 and 8 and flaps were approximated with direct sutures .
on radiographic evaluation after six months , some bone gain was seen on right side , while on left side no change in level of bone figures 9 and 10 .
right lateral buccal view ( mirror view ) of posterior mandibular region , showing 10 mm of pocket distal to mandibular first molar ( measured by unc-15 probe ) left lateral lingual view ( mirror view ) of posterior mandibular region , showing 8 mm of pocket distal to mandibular first molar ( measured by unc-15 probe ) right lateral buccal view ( mirror view ) of posterior mandibular region , showing grade ii distal furcation with mandibular first molar left lateral lingual view ( mirror view ) of posterior mandibular region , showing grade ii distal furcation with mandibular first molar right lateral buccal view of posterior mandibular region , showing regenerative therapy with mandibular first molar left lateral lingual view of posterior mandibular region , showing regenerative therapy with mandibular first molar postoperative iopa of mandibular right side depicting some bone gain postoperative iopa of mandibular left side depicting no bone gain
morphological features of lingual root such as small sized and curved root shape were observed frequently .
the study by huang , lin , lee
et al , suggested that the presence of distolingual root contributes to the formation of a distal furcation , which can be complex .
the case presented here was of a non - european young female probably of caucasian genetic origin .
yew and chan ( 1993 ) considered re to be a normal morphological variant for mongoloids and called it as asiatic trait .
the repeated southeastern movement of caucasoid people from their original area of characterization has resulted in an extraordinary mixture of racial types . henceforth , we can say , that this female patient might be of caucasian mix race with this asiatic trait . according to quackenbush ( 2002 ) ,
the extra root occurred unilaterally in approximately 40% of all cases and predominantly on right side .
tratman ( 1938 ) suggested that there was a sex difference in which females were inclined to display the anomaly bilaterally symmetrical on both the sides , while males presented the anomaly asymmetrically frequently on the right side . according to schafer ( 2009 ) , the incidence was 1.52% in women and 1.15% in men .
this case was similar with the finding with that of tratman ( 1938 ) and schafer ( 2009 ) . according to ferraz ( 1992 ) ,
this trait is common in mandibular first molars and in females which is in accordance with this case report .
the present case was of type ii radix entomolaris which contributed to the chronic localized periodontitis with mandibular first molars .
the unfavorable distal furcation involvement became noticeable due to poor oral hygiene , food impaction , and short root trunk of mandibular first molar .
regenerative therapy was carried out in both the defects measuring 10 mm on right side and 8 mm on left side .
after six months on radiographical evaluation , some bone gain was observed with right mandibular first molar , while no further bone loss was evident with left mandibular first molar .
a deep understanding of the relationship between root morphology and periodontal destruction is crucial for periodontal care .
the anatomy of human teeth presents racial variations which can lead to therapy failure when not recognized . | a deep understanding of the relationship between root morphology and periodontal destruction is crucial to successful periodontal care .
most mandibular molars have two roots ( one mesial and the other distal ) and three canals .
the major variant of this tooth type is the presence of an additional third root distally or mesially or a supernumerary lingual root .
this article presents a case report of a 14-year - old girl with mandibular first molar having three roots bilaterally which is contributing to the periodontal destruction .
there were three walled defect distal to first molar with grade ii furcation involvement distally .
regenerative surgeries were performed and patient was kept under maintenance .
the relationship between additional third root and periodontal destruction seldom has been described clearly .
this paper has been presented in 34th national conference at dharwad . |
the burnout syndrome is a serious consequence of chronic exposure to work - related stressors .
the construct of burnout syndrome appeared for the first time around the early 1970s , aimed at explaining the process of physical and mental deterioration in professionals working in areas such as teaching , health care , social work , or emergency legal services .
subsequently , burnout syndrome was defined as a sustained response to chronic work stress comprising three dimensions : the experience of being emotionally exhausted ( emotional exhaustion ) , negative attitudes and feelings toward the recipients of the service ( depersonalization ) , and feelings of low accomplishment and professional failure otherwise known as lack of personal accomplishment . among nurses ,
the incidence of occupational stress - related burnout is high and factors such as age of the nurse , years of service , hierarchy of nurse , lack of adequate staff , difficult or demanding patients , younger age , male gender , and inadequate clinical supervision have been reported to be associated with burnout .
others are excess workload , emotional stress , unevaluated work and underpayment , poor leadership , death and dying , conflicts with staff , accepting responsibility , lack of social support , conflict with other nurses , conflict with physicians , presence of stressors related to private life , feeling that the job is threatened , and higher nursing grade .
similarly , kingdom noted that excess workload and number of people living in the household were the best predictors of physical health problems among nurses in japan , while likelihood to leave the current nursing position and lack of support in the workplace were the best predictors of mental problems .
stress is a factor that affects nurses on a daily basis and can result in nurses ' absenteeism and aggression as well as reduced productivity and efficiency .
burnout has a special significance in health care settings where staff experience both emotional and physical stress .
a growing recognition of job stress leading to dissatisfaction among registered nurses in nigerian hospitals has contributed to current problems with recruitment and retention of nurses in the country in general but more peculiar in some states .
if nurse administrators identify factors influencing nurses ' job satisfaction in government hospitals and implement strategies to address these factors , nurses ' turnover rate will decrease and recruitment and retention rates will increase . moreover ,
burnout among nurses has a negative effect on the quality of patient care [ 9 , 10 ] .
several studies have demonstrated various components of burnout among nurses in developed countries of the world [ 7 , 8 , 11 ] . however , not much has been done in a poor resource , multiethnic , and culturally laden country like nigeria .
there is a dearth of information on this subject matter , hence the need for this study .
ibadan is the capital of oyo state , nigeria , and it is the third largest city in nigeria .
the hospital is owned by the state government and it attends to all medical , surgical , and psychiatric cases .
there are two hundred and ninety - two ( 292 ) nurses working in the hospital .
the first was existing hospital records while the second was a survey . ethical approval was obtained in february , 2008 from the ethical review committee of the oyo state ministry of health . for the survey ,
individual letters were written to all nurses working at ring road state hospital ibadan in april , 2008 , to inform and request for their participation in the study .
information about the number of nurses and their status , attending doctors , coverage of all shifts by doctor on call , and patient - to - nurse ratio was obtained from the hospital documents .
part 1 included basic demographic data ( age , sex , marital status , number of children , religion , and time to reach their hospital ) and some questions about experiences during the past month ( number of night shifts and conflict with other nurses or with doctors ) .
part 2 consisted of the 12-item ghq and the maslach burnout inventory ( mbi ) .
all survey instruments were individually handed over to the participants during a monthly staff screening exercise after each of them had received individual code only known to the research team members .
this coding system enabled participants to be contacted after the study for those who required intervention .
although it does not yield a diagnosis , positive scores are indicative of psychological distress .
each item is rated either 0 or 1 on the basis of the frequency with which the subject has experienced the symptom in the recent past , yielding a maximum score of 12 .
a score of 1 or above is suggestive of psychological distress ; however , to increase specificity , a cut - off point of 2 was used and score of 2 was used as positive screen .
maslach burnout inventory ( mbi ) is a 22-item self - report inventory designed to measure the characteristics of burnout .
the frequency column has a 6-point likert - type format and the intensity column is 7 points .
direct scoring was used for the items of the emotional exhaustion ( ee ) and depersonalization ( d ) subscales by adding together the values of the ratings shaded while reverse scoring was used for the item of the personal accomplishment ( p ) subscale by adding together the reversed values of the shaded ratings .
the maslach burnout inventory has cross - cultural reliability and validity , and , in nigeria , a study aimed at establishing the psychometric properties of the mbi using a sample of health care workers obtained reliability coefficients as follows : cronbach 's alpha of 0.86 and split - alpha of 0.57 .
these survey instruments were pilot tested among 30 nurses from adeoyo maternity hospital , a sister hospital also being run by the same board as the study center .
the pilot study showed that the design of the study was appropriate to the study population .
all statistics were performed with spss 13.0 software . initially , bivariate analyses compared certain demographic variables such as gender , age , and hierarchy with high burnout .
pearson 's chi - square was used to compare categorical variables , while t - test was used to compare two continuous variables and two - way anova for more than two continuous variables . for multivariate risk analysis of high burnout variables that were significant during bivariate analysis
variables were entered in the binary form , that is , 0 , 1 , and were coded in a way to illustrate the effect of each level . to facilitate the interpretation of odds ratio , a reference category was always chosen for the independent variables with which other variables could be compared
they were 270 in all , 64.1% were women , all ( 100% ) were practicing a religion , 70% were married , and only 10% were staff nurses .
a high level of burnout was identified in 39.1% of the respondents in the area of emotional exhaustion ( ee ) , 29.2% in the area of depersonalization , and 40.0% in the area of reduced personal accomplishment ; this was significant = 8.4 df ( 2 ) p = 0.01 ( table 2 ) .
older nurses ( p < 0.01 , female gender ( p < 0.01 ) , being unmarried ( p < 0.01 ) , junior nursing hierarchy ( p < 0.01 ) , nurse / doctor conflict ( p < 0.01 ) , and too frequent night duties ( p < 0.01 ) were significantly associated with high burnout in all the three dimensions ( table 3 ) .
nonavailability of a doctor to work with ( p < 0.02 ) was significantly associated with burnout in the area of emotional exhaustion and depersonalization and also in the area of reduced personal accomplishment ( p < 0.01 ) ( table 3 ) .
poor wages were significantly associated with burnout in the area of emotional exhaustion ( p < 0.02 ) , in the area of depersonalization ( p = 0.04 ) , and in the area of reduced personal accomplishment ( p = 0.01 ) ( table 3 ) .
multivariate analysis showed that doctor / nurse conflict ( or = 3.1 , 95% ci : 1.96.3 ) , low doctor / nurse ratio ( or = 6.1 , 95% ci : 2.513.2 ) , inadequate nursing personnel ( or = 2.6 , 95% ci : 1.55.1 ) , and too frequent night duties ( or = 3.1 , 95% ci : 1.75.6 ) were predictors of burnout in the area of emotional exhaustion and doctor / nurse conflict ( or = 3.4 , 95% ci : 2.27.6 ) , low doctor / nurse ratio ( or = 2.4 , 95% ci : 1.44.1 ) , and too frequent night duties ( or = 2.4 , 95% ci : 1.54.8 ) in the area of depersonalization .
high nursing hierarchy ( or = 2.7 , 95% ci : 1.54.8 ) , poor wages ( or = 2.9 , 95% ci : 1.65,6 ) , and too frequent night duties ( or = 2.3 , 95% ci : 2.34.5 ) in the area of reduced personal accomplishment were associated with high burnout after model adjustment ( table 4 ) .
our study has demonstrated a significant difference in the prevalence of burnout across the three dimensions .
there were also demographic variations in the prevalence of burnout among these nurses across these burnout dimensions .
we found that high burnout was significantly reported by older nurses , a finding previously reported but which contrasts an earlier one .
as previously reported [ 1719 ] , we also found that junior nursing hierarchy was significantly associated with high burnout .
this appears paradoxical as the senior nurses are expected to have reported more burnout compare d with junior ones since the senior ones should be older in age .
this result may not be unassociated with bullying in nursing which is fast becoming a common practice [ 20 , 21 ] .
more women reported high burnout in this study contrast to that of edwards and colleagues although the same has been reported even among other professional such as physicians .
the present study was not able to corroborate an earlier one in japan which reported that number of people living in the household is the best predictor of burnout among nurses .
we found that nurse / doctor conflict and nonavailability of physicians to work with were associated with burnout .
stehle similarly noted that many of the stressors identified among nurses concerned working relationships with doctors .
the potential explanation for this association in the present study is that nonavailability of physicians to work with would require the nurses to pay additional attention to the patients in order to cover up for the doctor 's absence thereby creating additional strain for them .
this singular factor underscores the importance of team building in the prevention of burnout in nurses .
we did not find an association between burnout and number of children or other personal factors but found that organizational factors , too frequent night shifts , poor wages , and inadequate security at night , were associated with high burnout .
one of the reasons why inadequate security at night could have been associated with high burnout is increased risk to physical injuries and assaults . for maslach and colleagues ,
burnout is a response to overload and some of the factors retained after model adjustment during multivariate analysis were as a result of work overload .
it has been suggested that reducing hours may be the first step to reduce resident burnout and this is applicable to nurses . in our multivariate analysis , as previously reported [ 2628 ] , all the predictors of burnout among the nurses were essentially organizational factors ( doctor / nurse conflict , inadequate number of physicians to work with , inadequate nursing personnel , and too frequent night duties ) , suggesting the need for organizational restructuring of the health services of the oyo state if prevalence of burnout among the nurses is to reduce .
there is an increased likelihood for illness and injury among employees working in long - hour schedules and schedules involving unconventional shift work ( e.g. , night and evening shifts ) .
these issues are of utmost ethical import taking into consideration fatigue - related errors such employees working in these kind of demanding schedules may be liable . such errors could have serious and adverse repercussions for public safety .
thus , the result from this study indicating the association between too frequent night duties and burnout is of utmost ethical consideration considering the medical errors that could accrue from such practice .
the prevalence of psychiatric morbidity reported in this study high is close to that of burnout in the areas of emotional exhaustion and reduced personal accomplishment illustrating that although burnout is not a recognized clinical psychiatric or psychological disorder , there are some similar features between burnout and diagnosable conditions such as depression , anxiety disorders , or mood disorders
. similar high prevalence of psychiatric symptoms has been reported in other parts of the world among nurses [ 31 , 32 ] .
a major strength of this study is that this is the first study to our best knowledge of the largest nursing workforce in nigeria .
although the study was limited to one of the hospitals being managed by an umbrella board , findings from this study may be generalized to other general hospitals in the state .
the present study has a number of potential limitations : our aim was to focus on the individuals ( nurses ) rather than on the patients themselves , thus patients ' factors and interdepartmental differences were not included in the analysis . | objective . to evaluate the prevalence and associated factors of burnout among nurses in a nigerian general hospital . methods . a total sampling method was utilized . measurements .
burnout was evaluated using the maslach burnout inventory ; ghq-12 was used to determine the presence of psychiatric morbidity .
results . a high level of burnout was identified in 39.1% of the respondents in the area of emotional exhaustion ( ee ) , 29.2% in the area of depersonalization and 40.0% in the area of reduced personal accomplishment .
multivariate analysis showed that doctor / nurse conflict ( or = 3.1 , 95% ci : 1.96.3 ) , inadequate nursing personnel ( or = 2.6 , 95% ci : 1.55.1 ) , and too frequent night duties ( or = 3.1 , 95% ci : 1.75.6 ) were predictors of burnout in the area of ee , doctor / nurse conflict ( or = 3.4 , 95% ci : 2.27.6 ) and too frequent night duties ( or = 2.4 , 95% ci 1.54.8 ) in the area of d , high nursing hierarchy ( or = 2.7 , 95% ci : 1.54.8 ) , poor wages ( or = 2.9 , 95% ci : 1.65,6 ) , and too frequent night duties ( or = 2.3 , 95% ci : 2.34.5 ) in the area of rpa . conclusions .
prevalence of burnout among these nurses was high .
the government therefore needs to look into factors that will enhance nurses ' recruitment and retention for effective health care delivery system . |
patients with a first episode of graves ' hyperthyroidism ( gh ) are often treated initially with pharmacological therapy .
many clinicians use a therapeutic regimen commonly referred to as block and replacement therapy
this involves the administration of a thyroid - hormone- ( th- ) synthesis - blocking agent such as methimazole ( mmi ) or propylthiouracil ( ptu ) to which l - thyroxine ( t4 ) is added once serum th concentrations reach the euthyroid range . in other causes of hyperthyroidism , such as toxic multinodular goiter or in severe cases of gh , radio - iodine ablation or ( partial ) surgical thyroidectomy
may be used as an initial means to block thyroid hormone secretion , often combined with t4 supplementation at a later stage .
hyperthyroidism is associated with profound changes in energy homeostasis that usually resolve upon treatment with brt . as a result ,
the weight loss experienced during hyperthyroidism is usually regained during treatment . some older studies in hyperthyroid subjects
have reported no difference between premorbid body weight ( bw ) and bw after one year of treatment [ 1 , 2 ] , fitting with the notion of tight bw set - point regulation . however , a more recent study in 162 patients reported a continuing bw gain after treatment for hyperthyroidism .
bw had increased by ~4 kg after 1 year and increased by up to 10 kg after four years of treatment . in line with this observation , up to 84% of patients report weight gain exceeding their premorbid bw following treatment of hyperthyroidism .
although the etiology of this excessive weight gain is incompletely understood at present , some authors have proposed that it may result from subnormal energy expenditure due to iatrogenic suppression of th concentrations to the lower end of the normal range [ 1 , 5 ] .
it is remarkable that to date studies addressing this issue have focussed on the comparison between bw and energy homeostasis in untreated hyperthyroid patients with the euthyroid situation during brt .
approximately 50% of patients with graves hyperthyroidism remain euthyroid following cessation of brt after at least one year of treatment , reflecting the remission rate of autoimmune hyperthyroidism . in our outpatient clinic of endocrinology ,
this regimen offers a good opportunity to investigate changes in bw upon cessation of brt in euthyroid patients with graves disease and to identify determinants of bw and energy metabolism in this setting .
patients were recruited from the outpatient clinics of endocrinology and metabolism at the academic medical center of the university of amsterdam .
inclusion criteria were ( i ) a minimum of six months of mmi or ptu in combination with t4 pharmacotherapy for graves ' hyperthyroidism , diagnosed on the basis of standard biochemical and scintigraphic criteria , ( ii ) biochemical euthyroidism ( defined as serum free t4 ( ft4 ) between 9 and 23
pmol / l , serum t3 between 1.3 and 2.7 nmol / l and serum tsh
< 5 mu / l ) on both study occasions , and ( iii ) age between 20 and 60 years .
as low or even suppressed serum tsh values can be found in euthyroid patients treated for graves hyperthyroidism , probably resulting from binding of circulating tsh receptor ( tsh - r ) autoantibodies ( tbii ) to the tsh - r on the thyrotrophs , patients with serum tsh values below the lower limit of the reference range could participate in the study .
exclusion criteria were ( i ) pregnancy , ( ii ) abnormal liver function as apparent from serum alanine - aminotransferase > 45 u / l , aspartate - aminotransferase > 40 u / l or gamma - glutamyl - transferase > 60
u / l , and ( iii ) abnormal kidney function ( serum creatinine > 95
the study was approved by the medical ethical committee of the academic medical center of the university of amsterdam and accordingly , written informed consent was obtained from all subjects prior to inclusion . in this observational study
, the participants were studied in the morning after an overnight fast on two occasions , that is , ( i ) during biochemical euthyroidism while on brt ( visit 1 ) and ( ii ) during biochemical euthyroidism , 12 weeks after discontinuation of brt ( indicating remission of graves ' hyperthyroidism ) ( visit 2 ) . at visit 1
, a medical history was obtained regarding premorbid bw , initial presentation of graves hyperthyroidism .
data on bw progression during the course of treatment were obtained from clinic records for every individual patient . on both occasions , bw , resting energy expenditure ( ree ) , body composition , and serum t3 , t4 , ft3 , ft4 , reverse t3 ( rt3 ) , tsh , insulin , glucose , adrenalin , and noradrenalin were assessed .
all measurements were performed in the morning between 8.00 and 12.00 am . of all patients
that were included in the study , one patient used a -adrenergic blocker ( metoprolol ) , and one patient used a benzodiazepine ( diazepam ) .
both patients were instructed not to take any co - medication within at least 24 h prior to study measurements .
all patients were instructed not to take thyroxine in the morning of visit 1 , and smoking was not allowed within 12 h prior to measurements .
patients were instructed to prevent physical exercise at least 3 days before both study visits , to eat 3 meals per day , and not to change their eating or exercise habits between the visits .
importantly , patients were instructed to refrain from any specific diet during the study period .
body composition was measured using bioelectrical impedance analysis ( maltron bf-906 , rayleigh , uk ) .
oxygen consumption ( vo2 ) and co2 production ( vco2 ) were measured with 20 sec intervals for a total time of 30 min by indirect calorimetry using a ventilated hood system ( sensormedicsmodel 2900 ; sensormedics , anaheim , calif , usa ) .
patients were studied in the morning after an overnight fast . during and 30 min prior to the calorimetry measurements ,
subjects were instructed to rest in the supine position in a temperature - controlled room ( 23c ) .
ree , lipid , and glucose oxidation were calculated from vo2 and vco2 values using algorithms previously reported by frayn .
the final 25 min of each calorimetry measurement , during which stable vo2 and vco2 values are reached , was averaged for further analysis in every individual patient .
serum t3 ( total ) , t4 ( total ) , and rt3 were measured with in - house radioimmunoassay ( ria ) .
serum ft4 , ft3 , and tsh were measured by time - resolved fluoroimmunoassay ( delfia , wallac oy , turku , finland ) .
for ft4 the intra - assay variation was 4 - 5% , the inter - assay variation 6 - 7% , and the detection limit 2 pmol / l .
for ft3 the intra - assay variation was 6% , the inter - assay variation 9% , and the detection limit 1 pmol / l .
for tsh the intra - assay variation was 3 - 4% , the inter - assay variation 4 - 5% , and the detection limit 0.01 mu / l .
insulin was measured with a chemiluminescent immunometric assay ( immulite 2000 system , diagnostic products corporation , los angeles , calif , usa ) with an intra - assay variation of 36% , an inter - assay variation of 46% , and a detection limit of 15 pmol / l .
serum tbii was quantitatively determined by a second - generation luminescence receptor assay ( dynotest trak human assay , b.r.a.h.m.s . ) .
intra - assay variation for noradrenalin : 2% and adrenalin 9% ; inter - assay variation for noradrenalin : 10% and adrenalin : 1418% ; detection limit was 0.05 nmol / l for both hormones .
we performed comparisons between study occasions with the wilcoxon signed rank test and expressed correlations as spearman 's rank correlation coefficient ( r ) .
spss statistical software version 12.0.1 ( spss inc , chicago , ill , usa ) was used for statistical analysis .
thirty - nine biochemically euthyroid patients were initially included and studied before cessation of brt . of these patients ,
nine had developed a relapse of hyperthyroidism while two patients who had undergone radio - iodine ( i ) ablation therapy had developed hypothyroidism at visit 2 .
six patients were excluded from the final analysis on the basis of serum ft4 or t3 values outside the reference range , or serum tsh values above the upper limit of the reference range .
thus , the final analysis was performed in 22 patients who were biochemically euthyroid at both visits .
these patients were instructed not to smoke 12 hours prior to measurements and not to change their smoking habits between study visits .
patients reported that before the onset of the symptoms later attributed to hyperthyroidism that had urged them to seek medical advice , bw , that is , premorbid bw , was 64.0 ( 47.597.0 ) kg .
eighty - two percent ( 18/22 ) of patients reported that decreased bw was among the symptoms of hyperthyroidism . at the time of diagnosis of hyperthyroidism ,
the patients had lost 4.8 ( 0.025.0 ) kg of self - reported premorbid bw .
nineteen patients were started on methimazole , and 3 patients were started on ptu , to which thyroxine was added as soon as biochemical euthyroidism was reached . at visit 1 , when brt was discontinued , patients had been treated with brt for 13.5 ( 9.548.0 ) months . during
the difference between premorbid bw and bw at visit 1 ( i.e. , presumed weight gain ) was 2.4 ( 17.114.6 ) kg ( premorbid bw versus visit 1 bw , p = 0.001 ) .
there was no significant difference in median bw between visit 1 and visit 2 , 12 weeks after cessation of brt ( table 1 , p = 0.899 ) .
the median difference in bw between visit 1 and visit 2 calculated per individual patient was 0.0 ( 5.43.2 ) kg . furthermore , there was no difference in ree , fat and glucose oxidation , rq , or body fat mass between visit 1 and visit 2 ( table 2 ) .
serum t4 and ft4 were not different between visit 1 and 2 ( p = 0.362 and p = 0.676 , resp . , table 3 ) .
serum t3 tended to increase at visit 2 as compared with visit 1 ( 3% , p = 0.069 ) , whereas ft3 showed a statistically significant increase at visit 2 ( 20% , p = 0.005 ) .
serum tsh levels decreased by 59% and serum tbii concentrations by 15% at visit 2 compared with visit 1 ( p = 0.001 and p = 0.007 , resp . ) .
accordingly , the serum t3/t4 ratio increased by 10% from 1.79% ( visit 1 ) to 1.97% ( visit 2 , p = 0.033 ) , and the serum ft3/ft4 ratio increased by 16% from 31.8% ( visit 1 ) to 37.0% ( visit 2 , p = 0.007 , see figure 1 ) . at visit 1
, there was a positive correlation between serum ft4 and ree ( p = 0.044 , figure 2(a ) ) and a negative correlation between serum ft4 and % body fat mass ( p = 0.035 , figure 2(b ) ) .
in addition , there was a highly significant negative correlation between ree and % body fat mass ( r = 0.549 , p = 0.008 ) .
serum ft3 tended to correlate positively with ree ( p = 0.066 , figure 2(c ) ) , but showed no correlation with % body fat mass ( p = 0.288 , figure 2(d ) ) .
however , at visit 2 , these significant correlations were absent ( ft4 versus body fat % r = 0.247 , p = 0.270 , ft4 versus ree
r = 0.320 , p = 0.172 , ft3 versus body fat % r = 0.109 , p = 0.630 , ft3 versus ree , r = 0.135 , p = 0.550 ) .
intriguingly , the difference in ( ) ft3/ft4 ratio and ree between visit 1 and visit 2 showed a positive correlation ( r = 0.465 , p = 0.029 , see figure 3 ) .
there were no differences in serum glucose , insulin , adrenalin , or noradrenalin between the two visits ( table 3 ) .
patients with graves disease who are treated with a combination of a thyreostatic drug and thyroxine replacement , that is , block and replacement therapy ( brt ) , often experience a marked gain in bw , which has been reported to exceed self - reported premorbid bw .
however , there are some inconsistencies in the published data on this issue . in the present study , we found a median weight excess of 2.4 kg compared with self - reported premorbid bw in a group of 22 euthyroid patients with graves hyperthyroidism who had been treated with brt for a median of 13.5 months . to further investigate this phenomenon we assessed energy homeostasis and serum hormone concentrations in these patients both before and 12 weeks after cessation of brt .
somewhat unexpectedly , patients showed no change in bw or ree at 12 weeks after brt cessation .
with respect to the 2.4 kg bw excess , we are aware of the uncertainty regarding the reliability of self - reported premorbid bw .
symptoms such as a decrease in bw are slowly progressive and patients may find it hard to remember the precise onset of symptoms .
therefore , we can not exclude that underestimation of the self - reported premorbid bws may have led to an overestimation of the bw excess during brt in our patients . in any case , the present study does not support the clinical impression of a marked and excessive bw increase after ~1 year of brt following hyperthyroidism .
hyperthyroidism is associated with a marked increase in ree [ 12 , 13 ] . the simultaneous increase in appetite and caloric intake
does not completely prevent weight loss in most hyperthyroid patients . by inference , bw changes appear to be primarily determined by ee in these patients . in addition , it has been shown previously that caloric intake decreases rapidly to normal within 3 months of initiation of antithyroid treatment , whereas in the same period bw increases to premorbid levels . therefore , increased caloric intake is not a likely candidate to explain the bw gain after initiation of thyreostatic treatment .
our finding that ree and bw are similar during treatment with brt compared with 12 weeks after brt cessation suggests that brt does not induce a major change in energy homeostasis and , in turn , bw gain in excess of the premorbid situation .
the patients in our study were euthyroid on both occasions , following the design of the study .
this enabled us to investigate the relationship between subtle changes in serum th values within the euthyroid range on the one hand and measures of energy homeostasis on the other hand .
as anticipated , serum ft3/ft4 ratios significantly ( 16% ) increased after cessation of brt .
this can probably be attributed to reinstated thyroidal t3 secretion , whereas enzymatic conversion from exogenous t4 is probably the major source of serum t3 during brt .
interestingly , there was a significant and positive correlation between the difference in serum ft3/ft4 ratio and the difference in ree between the 2 study occasions , suggesting that the serum ft3/ft4 ratio is a positive determinant of ree under these circumstances ( figure 3 ) .
in addition , serum ft4 levels within the euthyroid range were a positive determinant of ree .
this illustrates the sensitivity of ree to even small fluctuations in circulating thyroid hormone concentrations , which is supported by previous reports [ 15 , 16 ] .
as anticipated , ree and % body fat mass showed a highly significant negative correlation .
therefore , it is likely that the negative correlation between serum ft4 and % body fat mass can be explained by the observation that ft4 positively determines ree .
it appears remarkable that these correlations reached statistical significance despite the relatively small number and heterogeneity ( i.e. , regarding age , sex , bmi , body fat mass ) of patients .
why this was only evident during brt treatment and not at visit 2 remains unclear .
it may be that after cessation of brt , the pathophysiological changes in the hpt - axis associated with graves disease ( e.g. , by circulating tbii ) prevent the detection of clear - cut associations between circulating thyroid hormones and ree .
another explanation may be that a period of 12 weeks is not sufficient to reinstate physiological thyroid hormone synthesis and release from the thyroid gland .
although ree and bw were unaltered 12 w after brt cessation , the data presented do raise a number of interesting possibilities .
our finding that changes in serum ft3/ft4 ratios positively determine ree in euthyroid patients ( figure 3 ) suggests that tissues capable of modulating energy metabolism sense even small changes in circulating ths and alter ree accordingly .
candidates include tissues significantly contributing to ree and expressing th receptors such as the heart and cns , while striated muscle and adipose tissue may contribute to a lesser extent .
another possibility to explain these findings may be th - dependent regulation of ree via the brain , as the hypothalamus plays a major role in the regulation of energy metabolism [ 18 , 19 ] .
interestingly , there is a high density of th receptors in the rat and human hypothalamic arcuate and paraventricular nuclei [ 20 , 21 ] , and these nuclei are both key players in bw regulation .
recently , we have shown that hypothalamic t3 administration stimulates hepatic glucose production via a neural pathway involving the hypothalamic paraventricular nucleus and the sympathetic nervous system .
thus , the hypothalamus is able to sense ths and in turn modulate hepatic glucose metabolism by means of altered sympathetic outflow to the liver .
this raises the possibility that it may similarly modulate energy metabolism by autonomic projections to other target tissues such as skeletal muscle and adipose tissue in response to th [ 23 , 24 ] .
serum tsh concentration was lower at visit 2 as compared with visit 1 , although it remained within reference intervals .
this may be explained either by subtle t4 under - treatment during brt despite serum t4and ft4 levels within the reference range .
alternatively , the decrease in serum tsh may point to a tendency to develop a relapse of hyperthyroidism in some patients .
however , this latter possibility appears to be less likely given the decrease of serum tbii , 12 w after brt cessation ( table 4 ) . in conclusion , we observed no changes in bw and resting energy metabolism 12 weeks after cessation of brt in euthyroid graves patients , so the present findings do not support the notion that brt per se induces excessive bw gain .
however , patients who stopped brt showed an increase in the serum ft3/ft4 ratio , and these changes in ft3/ft4 ratio were a positive determinant of changes in ree .
hence , it is well possible that the changes in circulating thyroid hormones after cessation of brt require a longer observational period in order to translate into clear - cut alterations in bw . | patients with graves ' hyperthyroidism ( gh ) treated with a combination of thyrostatic drugs and t4 , that is , block and replacement therapy ( brt ) , often report body weight ( bw ) gain .
we aimed to determine changes in bw and energy metabolism upon cessation of brt in these patients , and to identify possible endocrine determinants .
we analysed 22 patients with gh ( i ) during brt , and ( ii ) 12 weeks after brt cessation .
patients were euthyroid at both visits .
there were no differences in bw or resting energy expenditure ( ree ) between visits . at visit 1 , after 13.5 ( 9.548.0 ) months of brt , serum free ( f)t4 correlated positively with ree ( r = 0.433 , p = 0.044 ) and negatively with body fat % ( r = -0.450 , p = 0.035 ) .
plasma ft3 and ft3/ft4 ratio showed an increase 12 w after cessation of brt ( 20% , p < 0.0001 and 16% , p = 0.007 , resp . ) .
moreover , the relative change in ft3/ft4 ratio showed a significant , positive correlation with the relative change in ree between the 2 visits ( r = 0.465 , p = 0.029 ) . in conclusion ,
serum ft4 determines ree in euthyroid patients with gh treated with brt .
twelve weeks after brt cessation , bw and energy homeostasis are unaltered . however , as serum ft3/ft4 ratio increases after cessation of brt , which is a positive determinant of changes in ree , a longer term bw decrease is likely to occur . |
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