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most commonly affects people of african descent . in nigeria , sickle cell anaemia affects about 1 - 3% of the nigerian population .
individuals who have only one copy of the mutation are said to have sickle cell trait.12 these people are usually healthy but can transmit the disease to their children.3 sickle cell anaemia is characterised by acute episodes of pain ( in the abdomen , chest or joints ) and exhibits a range of severity.45 sickle cell disease is a condition which alters the shape of the red blood cells from round to sickle shape , causing them to block small blood vessels and interfere with normal blood flow .
children affected with sickle cell disease experience chronic episodes of pain and an increased susceptibility to potentially life - threatening conditions , including bacterial infections and organ failure . at the present time , there is no cure for sickle cell anaemia . although there are some altering therapies such as hydroxyurea and hematopoietic cell transplantation for the management of sickle cell disease , but these can have serious side effects and are very risky
the average life expectancy for people with sickle cell anaemia is less than 50 years .
the first deoxyribonucleic acid ( dna ) diagnostic procedure for prenatal purposes was reported more than thirty years ago.6 subsequently , it was recognised that the mutation itself affected the cleavage site of a restriction enzyme , ddei , that could recognize the dna sequence of ctnag ( n = a , t , c , or g ) .
while dna from a normal allele ( ctgag ) would be digested by the enzyme , dna from an affected allele in which a is substituted by t ( ctgtg ) would not.78 the resulting differences between dna fragment sizes can then be recognised by electrophoresis , thus forming the basis for diagnosis . with the advent of polymerase chain reaction ( pcr ) ,
rapid dna analysis methods have become available , and these techniques are now widely used for prenatal diagnosis.910111213 pre - implantation genetic diagnosis ( pgd ) is a procedure that has emerged against the backdrop of in vitro fertilisation ( ivf ) technology . in nigeria , this process of ivf was first introduced by ashiru et al.14 following their pioneering work , we were able to advance this technique for patients with infertility,1516 and now for patients with advanced maternal age and sickle cell disorders with the use of pgd .
with pgd , the genetic status of an embryo can be determined before transfer into the uterus after ivf , thus almost completely eliminating the risks of bearing a child with the disease17(pgd is not 100% accurate ) .
the most common form of pgd involves the extraction of one or two cells from the pre - implantation embryo , often around the 8-cell stage .
couples at risk for the transmission of genetic diseases can now choose ivf and pgd to avoid the problem of selective abortion that is associated with pre - natal diagnosis during pregnancy .
the patient was a 29-year - old woman and spouse with heterozygous genotype ( as ) .
couple already have a 2-year - old child who is affected with sickle cell anaemia .
prior to ovarian stimulation , hysterosonogram ( hsn ) was done which revealed normal uterine cavity .
the protocol was also approved by the research and ethics committee of the centre . following 3 weeks of oral contraceptive use , the patient was down - regulated using a gnrhanalog ( leuprolide ) .
super ovulation was achieved with the use of recombinant follicle stimulating hormone ( fsh ) . when three or more leading follicles measured > 18 mm in diameter , the patient was triggered with human chorionic gonadotropin ( hcg ) 5000 iu .
the surrounding cumulus and corona cells were then removed and the nuclear maturity of the oocytes was assessed under an inverted microscope . only metaphase ii oocytes were injected with morphologically normal motile spermatozoa .
further culture of injected oocytes was done in 20 l microdrops of culture medium under lightweight paraffin oil .
fertilisation was confirmed after 16 - 18 hours by the observation of two distinct pronuclei ( 2pn ) .
oocytes with 2pn were assessed on day 2 after injection for embryonic development . on day 3 ,
a single blastomere was biopsied for embryos at 6 - 8 cell stage with less than 50% fragmentation [ figures 1 and 2 ] .
blastomere showing nucleus fifteen biopsied cells were sent for genetic screening and analysed using pgd / pcr technique [ figure 3 ] .
results provided after 48 hours showed that 5 ( 33% ) of the biopsied cells were of genotypes unaffected by sickle cell anemia ; of these , three were heterozygous carriers of hb s ( as ) and two were homozygous for hba ( aa ) .
three unaffected embryos ( two heterozygous normal and one homozygous ) were transferred on day 5 at blastocyst stage .
endometrial lining was prepared for fet using increasing doses of estradiolvalerate from 2 mg daily up to 8 mg with weekly monitoring of the endometrium .
two vitrified normal embryos ( one hbas and one hbaa ) were thawed both at hatched blastocyst stage and transferred [ figure 4 ] .
the advent of ivf as a treatment for infertility has created the opportunity to study the chromosomal constitution of surplus human pre - implantation embryos .
cultured human pre - implantation embryos have been used to develop methods which allow pgd analyses by pcr on biopsied blastomeres from an embryo.18 beta - thalassaemia and sickle cell anaemia are -globin chain quantitative and structural disorders that lead to anaemia syndromes . until recently
, the only alternative for couples with a high genetic risk was to undergo prenatal diagnosis followed by termination of an affected pregnancy .
the pgd of -thalassaemia and sickle cell anaemia is an alternative that avoids therapeutic abortion by diagnosing embryos for -globin defects before implantation into the mother 's womb.19 on a world - wide scale , pgd for -thalassaemia and/or sickle cell anaemia has already been applied on single blastomeres20 and on the first and second polar bodies.21 the molecular strategies used were dna amplification followed by genetic diagnosis by denaturing gradient gel electrophoresis analysis,20 restriction enzyme digestion , the creation of a new restriction enzyme recognition sequence involving the ivs1 nt 110 mutation2122 and the use of fluorescence pcr.22 in the present study , pgd was applied clinically for sickle cell anaemia on a fertile carrier couple with previous experiences of therapeutic abortion for affected foetuses , and a sickle cell disease child . despite the fact that sickle cell anaemia is one of the most common genetic disorders and detailed genetic information is available,23 unaffected pregnancies following pgd for sickle cell anaemia previously have not been reported .
lack of previous success in this area presumably is due to the length of time and effort required to overcome technical difficulties inherent in these procedures , as well as lack of available research funding.17 our results demonstrate that sickle cell anaemia can be detected in single cells by pcr and restriction enzyme analysis and that unaffected pregnancies can be established by the transfer of embryos of known genetic makeup that have undergone biopsy . in total , one pgd cycle with a frozen embryo transfer cycle were carried out .
the goal of pgd is to reduce the risk of having children born with genetic diseases from a priori risk of 25% to a value significantly less .
patients undergoing pgd should be advised that if a pregnancy does occur from this treatment , they should consider undergoing chorionic villi sampling ( cvs ) or amniocentesis to confirm these micro - genomic results . in summary
, this is the first unaffected pregnancy and delivery after successful pgd for sickle cell anaemia .
our results demonstrate that pgd for the detection of sickle cell anaemia is a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected foetus .
the procedure , successfully used in this case , may also be applied to other monogenic disorders and further supports the notion that pgd is destined to be an integral aspect of assisted reproductive technology .
given the current methods and relatively high cost of the procedure , it is unlikely that pgd will totally replace prenatal testing .
however , it is conceivable that with further refinements , pgd will certainly become an invaluable and powerful diagnostic modality . | a couple , both carriers of the sickle cell anaemia trait ( genotype hbas ) with an offspring already affected with the genetic disease underwent a pre - implantation genetic diagnosis / polymerase chain reaction screening of biopsied blastomeres .
dna analysis of single blastomeres was carried out to find out indicated a viable intra - uterine pregnancy with embryos which carried the sickle cell mutation , which resulted in a livebirth ( hbas ) .
pgd / pcr in combination with ivf appears to be the most suitable treatment plan for patients who are at a higher risk of reproducing offspring affected with inheritable genetic diseases . |
pulse rate , also called heart rate , is the number of heartbeats per unit of time , commonly used as beats per minute ( bpm ) .
pulse rate is varied in accordance with the body 's need for oxygen changes , such as during exercise or sleep .
since various health - related phenotypes are closely correlated with pulse rate [ 1 - 3 ] , measuring pulse rate is commonly used for diagnosing and accessing medical conditions .
pulse rate is known to be positively correlated with triglycerides and negatively correlated with high - density lipoprotein cholesterol .
metabolic syndrome is associated with poor exercise capacity and poor heart rate recovery in patients who have coronary disease . moreover ,
high pulse rate is negatively correlated with lifespan in mammals , including humans . despite the importance of pulse rate as a risk factor for various diseases ,
however , few genes are known to be associated with pulse rate , and the underlying mechanism for pulse rate remains unraveled .
we previously reported pulse rate - associated loci from the korea association resource ( kare ) genomewide association study ( gwas ) , comprising 8,842 samples in the korean population . in this study
, we extended the previous study and reexamined the relevance of pulse rate - associated single - nucleotide polymorphisms ( snps ) from an additional gwas of 3,703 korean samples .
in the discovery stage , the association results for pulse rate were obtained from the kare study .
subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .
the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .
pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts , the first measurement of the participants , who rested for 5 min ,
the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,
genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,
santa clara , ca , usa ) . from 4,302 genotyped samples , we excluded samples with a low call rate ( 95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1 10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .
association analyses were performed using plink
( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .
the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .
the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .
meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .
in the discovery stage , the association results for pulse rate were obtained from the kare study .
subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .
the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .
pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts ,
the first measurement of the participants , who rested for 5 min , was used .
the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,
genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,
, we excluded samples with a low call rate ( 95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1 10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .
association analyses were performed using plink
( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .
the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .
the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .
meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .
the discovery gwas for pulse rate included 352,225 snps profiled with affymetrix genome - wide human snp array 5.0 in 8,842 individuals from the ansung and ansan cohorts as described previously .
characteristics of the study , including age , sex , and trait summaries , are presented in table 1 .
the association analysis identified several genomic locations as potentially associated with pulse rate . unlike the previous gwas
, we did not apply a threshold with a p < 0.01 in both ansung and ansan cohort in addition to a combined p < 1 10 for the snp selection for the replication study so that we could include more promising snps for their association with pulse rate .
we observed 61 significant snps by applying only a threshold with a combined p < 1 10 . after excluding snps by cluster plot inspection and removing singletons , 3 independent snps ( that is , with pairwise linkage disequilibrium statistics r < 0.2 and maf 0.05 within a 500-kb window of the genomic region )
were finally chosen to validate in the independent population - i.e. , the hexa cohort , comprising 3,703 individuals . in the replication study ,
all 3 snps were confirmed for their association with pulse rate at a p < 0.05 ( table 2 ) . the first pulse rate signal located to 1q32.2a ( rs12731740 , maf = 0.103 , poverall = 2.20 10 ) .
three genes , such as cd46 , clorf132 , and cd34 , appear near this locus .
the physiological relevance of the 3 genes in relation to pulse rate is not clear [ 8 , 9 ] .
thus , fine mapping surrounding this locus by targeted resequencing is highly recommended to discover the causal variants for pulse rate .
the second pulse rate snp is rs12110693 ( maf = 0.494 , poverall = 1.10 10 ) on 6q22.31c .
the only gene located near this snp is loc644502 , which has no known function .
the third locus associated with pulse rate is at 6q22.31c ( rs2892767 , maf = 0.261 , poverall = 2.50 10 ) .
this locus is near gja1 ( gap junction protein , alpha 1 , 43 kda ) , which is a member of the connexin gene family .
the protein encoded by gja1 is a component of gap junctions , which are intercellular channels that provide a route for the diffusion of low - molecular - weight materials from cell to cell .
in this study , we performed a genomewide scan for pulse rate and identified 3 loci accounting for the variation in pulse rate .
two of 3 loci were previously reported and replicated once again in an independent hexa - shared cohort .
the other one ( rs2892767 ) near gja1 was previously implicated for its association with pulse rate but was not chosen for the replication study , since the snp did not meet the selection criteria in the discovery stage . in the present study ,
snp rs2892767 near gja1 was taken forward to the replication study using the independent dataset from the hexa - shared cohort and was nicely confirmed for its association with pulse rate .
two snps , rs2892767 near gja1 and rs12110693 near loc644502 , are 408 kb apart from each other and are located in a separate ld , indicating that both are independent snps .
we performed multiple linear regression analyses , conditioning the snps to each other to find a significantly stronger proxy for pulse rate .
however , no dramatic decrease in statistical significance was observed , and no interaction was found between rs2892767 and rs12110693 ( table 3 ) .
gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development .
it is also known that gap junctions , clusters of cell - to - cell channels composed of connexins , mediate the orderly spread of electrical excitation throughout the heart .
the gja1-encoding protein has been reported to be involved in the development and function of the heart by allowing direct cell - to - cell exchange of molecules , which mediate multiple signaling events .
cho et al . previously reported the same locus near gja1 to be responsible for pulse rate without evidence of replication . in the present study
as indicated from the functional role of gja1 , it is a strong candidate gene for pulse rate .
thus , further efforts , such as fine mapping or imputation based on large - scale sequencing data near rs2892767 on 6q22.31 , would reveal the causal variant of this locus .
in addition , functional studies for gja1 would be worthwhile to facilitate a full understanding of its biological role in pulse rate .
our study was relatively underpowered to discover additional novel loci and demonstrated that large - scale genomewide meta - analysis comprising hundreds of thousands samples is required for understanding the missing heritability of pulse rate . | pulse rate is known to be related to diverse phenotypes , such as cardiovascular diseases , lifespan , arrhythmia , hypertension , lipids , diabetes , and menopause .
we have reported two genomewide significant genetic loci responsible for the variation in pulse rate as a part of the korea association resource ( kare ) project , the genomewide association study ( gwas ) that was conducted with 352,228 single nucleoride polymorphisms typed in 8,842 subjects in the korean population .
gja1 was implied as a functionally causal gene for pulse rate from the kare study , but lacked evidence of replication .
to re - evaluate the association of a locus near gja1 with pulse rate , we looked up this signal in another gwas conducted in a health examinee - shared cohort of 3,703 samples .
not only we were able to confirm two pulse rate loci ( 1q32.2a near cd46 and 6q22.13c near locl644502 ) identified in the kare gwas , we also replicated a locus ( 6q22.31c ) near gja1 by the lookup in the health examinee gwas . considering that the gja1-encoded protein is a major component of cardiac gap junctions ,
a functional study might be necessary to validate its genuine molecular biological role in the synchronized contraction of the heart . |
this complication usually occurs during abdominal aortic surgery and is an unusual complication following live donor nephrectomy .
live kidney donors are healthy individuals who willingly undergo major surgery to improve the well - being of another individual .
therefore , it is of utmost importance to minimize the risks of this procedure and thus maximize donor safety .
a 39-year - old mediterranean female with a bmi of 26 , underwent an elective , living - related , transperitoneal left - sided totally robot - assisted donor nephrectomy using the da vinci surgical system .
donor nephrectomy was performed by a transplant surgeon and assisted by a second transplant surgeon , as described elsewhere [ 2 , 3 ] .
briefly , the procedure was performed with the da vinci surgical system s using three arms with a fenestrated bipolar forceps ( endowrist bipolar cautery instrument ; intuitive surgical , sunnydale , usa ) in arm 1 , a monopolar cautery hook ( endowrist monopolar cautery instrument ; intuitive surgical , sunnydale , usa ) to dissect structures in arm 2 and the camera ( 3d vision system 12 mm 30 endoscope ; intuitive surgical , sunnydale , usa ) in arm 3 .
after full exposure of the kidney and vascular structures , a pfannenstiel incision was made and an endo - bag ( endocatch ; us surgical , norwalk , usa ) was introduced into the abdomen .
subsequently , the ureter was clipped and divided with scissors , and the renal artery and the vein were transected using an endoscopic stapler ( endogia ; us surgical , norwalk , usa ) .
the kidney was then placed in the endobag and extracted through the pfannenstiel incision . before removing all trocars ,
the renal bed and vascular stumps were inspected using the da vinci , and intra - abdominal hemostasis was performed with the fenestrated bipolar forceps .
the operation field was found to be dry after which all incisions were closed intracutaneously .
postoperatively , the patient was placed on dipidolor patient - controlled analgesia combined with paracetamol . during the postoperative recovery
she was discharged at postoperative day 3 with a normal appetite and no wound problems .
on postoperative day 11 , she visited the outpatient clinic for a follow - up visit and the nausea was completely gone .
however , she noted abdominal pain located on the right side near the iliac crest and reported two episodes of hematuria .
laboratory results showed a decreased c - reactive protein ( crp ) level and normal leukocyte count .
on postoperative day 19 , the patient noted bright red fluid leakage from the pfannenstiel incision without any further symptoms and contacted the nurse practioner .
it was decided that she would visit the outpatient clinic if the leakage would worsen or other complaints would emerge . on postoperative day 29 , the patient visited the outpatient clinic for her scheduled 1-month postoperative follow - up visit .
she still experienced pain in her abdomen which was mildly controlled with anti - analgesia .
the pain progressed when she laid on her right or left side and , at night , her abdomen was more distended than usual . on physical examination
, she had a distended abdomen , with tenderness on palpation on the right and the left side , and below the umbilicus , with a shifting dullness on percussion during movement .
an abdominal ultrasonography showed intraperitoneal fluid in all four quadrants of the abdomen and a diagnostic paracentesis demonstrated yellow fluid with a lipid aspect , suspected of being chylous ascites and confirmed by biochemical analysis showing positive triglycerides of 57.98 mmol / l .
she was admitted to the hospital for drainage and a medium - chain triglyceride - based diet was started . on day 4
the medium - chain triglyceride - based diet was continued for a total of 4 weeks after which a second abdominal ultrasonography was done , which showed no fluid in the abdomen .
in this case report , we describe the first reported complication of chylous ascites after total robot - assisted donor nephrectomy .
this complication is more common in major oncological surgical procedures , where lymph nodes are often resected .
therefore , from an oncological point of view these patients often casually accept this complication .
nevertheless , healthy live kidney donors are different from patients who undergo extensive dissection damaging lymphatic vessels necessitating postoperative treatment . in over three decades
, > 1500 live donor nephrectomies have been performed at our centre and never before have we encountered a case of postoperative chylous ascites after a donor nephrectomy .
a review of the literature resulted in a total of 56 reported cases of chylous ascites after donor nephrectomy ( supplement 1 ) . a huge
contrast has been compared with the thousands of live donor nephrectomies that have been published over the years .
the occurrence of chylous ascites is low and the expectation is that this is an underestimation .
there is no clear treatment pathway ; however , a median chain triglyceride diet combined with a therapeutic drain or octreotide seems to be the first step .
we could not identify any other reported cases of chylous ascites after robot - assisted donor nephrectomy [ 2 , 47 ] . during a robot - assisted donor nephrectomy , it is not necessary to dissect the lymfatic tissues around the aorta and the origo of the real artery .
larger lymphatics around the renal vessels are either clipped or sealed by modern energy devices . in our case , a fenestrated bipolar forceps and a monopolar cautery hook were used for dissection and sealing .
however , with monopolar energy , although control the lymphatics for a short time , necrosis can occur later on leading to leakage of large amount of chyle .
the complication is often diagnosed after a few weeks , causing a delay in treatment .
fortunately , the complication can be successfully treated with conservative measures within a few weeks as demonstrated in this case .
| we present the first case report of chylous ascites following total robot - assisted donor nephrectomy .
a 39-year - old female underwent a transperitoneal left - sided total robot - assisted donor nephrectomy .
the procedure was uneventful and the patient was discharged without any symptoms . at postoperative day 29 ,
the patient presented with abdominal pain , nausea and a distended , painful abdomen with shifting dullness .
she was diagnosed with chylous ascites by ultrasonography and puncture analysis , and treated with therapeutic drainage and dietary restriction .
after 4 weeks , she was free of symptoms .
the occurrence of this complication is rare after donor nephrectomy .
fortunately , the complication can be successfully treated within a few weeks with minimal discomfort for the patient as demonstrated in this case .
it is of utmost importance to minimize the risks and limit discomfort for live kidney donors who willingly undergo major surgery to improve the well - being of another individual . |
obesity is a worldwide phenomenon , with an incidence increasing in parallel with obesity - associated diseases such as insulin resistance , type 2 diabetes mellitus , and hypertension .
the combination of obesity and hypertension places patients at a higher risk of hypertensive end - organ damage and vascular events [ 2 , 3 ] .
blood pressure ( bp ) is subject to diurnal variation , and studies using ambulatory bp monitoring ( abpm ) have demonstrated the clinical significance of disturbances in the diurnal bp profile and have associated nondipping with a progression in end - organ damage [ 4 , 5 ] .
moreover , a nondipping pattern of bp has been reported to occur in several conditions that are frequently associated with obesity .
although body mass index ( bmi ) is associated with the severity of ambulatory hypertension and an increase in daytime bp , bmi does not reflect body fat distribution .
epicardial adipose tissue is related to visceral fat , rather than total adiposity , and shares microcirculation with myocardial tissue and coronary vessels [ 6 , 7 ] . because elevated bp is associated with ectopic fat accumulation in the intrathoracic and epicardial areas , a possibility of association of epicardial adipose tissue with hypertension as well as diurnal bp patterns [ 10 , 11 ] has been found in some recent studies [ 8 , 9 ] .
epidemiological studies have found that epicardial fat is two to three times more common among females than males and that excessive body weight increases the risk of diabetes mellitus ( dm ) and cardiovascular diseases approximately one - and - a - half to two times higher in women than it does in men .
furthermore , a higher prevalence of hypertension among overweight adults is found in women than in men , which may indicate different susceptibilities based on gender . given these facts
, we aimed to investigate the influence of obesity on the association between epicardial fat thickness ( eft ) and circadian bp changes in patients with recently diagnosed essential hypertension ( eh ) , with a gender subgroup analysis .
this cross - sectional observational single - center cohort study included patients who underwent 24 h abpm and echocardiography between january 2008 and october 2013 .
demographic characteristics recorded at the first visit included age , sex , height , weight , medication , smoking history , and other diseases .
blood was drawn for measurement of total serum cholesterol , triglycerides , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) cholesterol , blood glucose , creatinine , uric acid , and high sensitivity c - reactive protein ( hs - crp ) .
body mass index ( bmi ) was calculated as the ratio of dry weight in kilograms to height squared ( in square meters ) .
a total of 441 consecutive patients , who underwent office bp measurements , 24-hour abpm , and laboratory measurements for cardiovascular risk factors and echocardiography , were analyzed .
following the recommendations of the european society of hypertension , a normotensive state was defined as a mean daytime ambulatory systolic and diastolic bp < 135/85 mmhg by abpm , associated with
true ht was assigned if the average daytime bp was higher than 135/85 mmhg and the average nighttime bp was above 120/75 mmhg .
obesity was defined as a bmi above 25 kg / m , as per the korean society for the study of obesity .
the patients with the following diseases were excluded from the study : secondary hypertension , significant liver disease , neurologic disorder , malignant disease , valvular heart disease , heart failure , and history of acute coronary syndrome , myocardial infarction , coronary revascularization procedure , carotid revascularization procedure , ischemic leg ulcer , peripheral revascularization , or amputation .
because the presence of type 2 dm is known to be associated with increased eft regardless of obesity [ 6 , 7 ] , we also excluded overt type 2 dm from the analysis to avoid its possible confounding impact on the association between eft and circadian bp variability in our hypertensive patients with or without obesity .
office bp measurements were performed using an automated device ( easy x 800 ( r / l ) , jawon medical , seoul , korea ) .
measurements were taken after patients had rested for 10 min in a sitting position , with the arm comfortably placed at the heart level .
each set of two measurements was averaged to give the office systolic and diastolic bp .
abpm was performed on each patient 's nondominant arm using an automatically oscillometric device ( tonoport v , par medizintechnik , berlin , germany ) on a normal working day .
all subjects were instructed to rest or sleep between 10:00 pm and 7:00 am ( nighttime ) and to continue their usual activities between 7:00 am and 10:00 pm ( daytime ) .
the accuracy of the device was checked against the standard auscultatory method to ensure that the difference in bp measurements between methods did not exceed 5 mmhg .
the device was set to obtain bp readings at 20 min intervals during the day ( 7:00 am10:00 pm ) and at 40 min intervals during the night ( 10:00 pm7:00 am ) .
each abpm dataset was first automatically scanned to remove artifactual readings according to preselected editing criteria .
data were edited by omitting all readings of zero , all heart rate readings < 20 or > 200 , diastolic bp readings > 150 and < 40 mmhg , systolic bp readings > 240 and < 70 mmhg , and all readings where the difference between systolic and diastolic bps was less than 10 mmhg .
the following abpm parameters were evaluated : average ambulatory 24-hour systolic and diastolic bp levels , average ambulatory daytime systolic and diastolic bp levels , average ambulatory nighttime systolic and diastolic bp levels , and mean ambulatory 24-hour , daytime , and nighttime bps .
additionally , for both sbp and dbp , the magnitude of the nocturnal decline in bp ( nocturnal decline ) was calculated as follows : daytime average bp minus nighttime average ; the percentage change in bp from day to night ( % day night bp ) was calculated as ( daytime bp nighttime bp ) 100/daytime bp . with this latter data , a normal dipper pattern was diagnosed when the reduction in the average sbp and dbp during the nighttime was 10% of the average daytime values . the nocturnal bp status was also assessed and expressed as either nocturnal normotension or hypertension , with nocturnal hypertension defined as a nighttime bp of 120/70 mmhg .
standard two - dimensional ( d ) and strain echocardiography were performed on all subjects while lying in the left lateral decubitus position using a 3.5 mhz transducer ( philips ie33 , philips medical systems , bothell , wa , usa ) , and the echocardiography examiners were blinded to patient information
. measurements of the thickness of interventricular septum and posterior wall , the diameter of the lv cavity , and the lv mass index ( lvmi ) were performed according to american society of echocardiography criteria .
pulsed wave doppler of the transmitral lv inflow was performed in an apical 4-chamber view with the sample volume placed at the level of the mitral valve tips , and the following measurements of global lv diastolic function were determined : peak early ( e ) and late ( a ) diastolic mitral flow velocity , and their ratio , e / a ; early ( ea ) diastolic mitral annular velocity ; deceleration time of the e wave ; and lv isovolumetric relaxation time ( ivrt ) .
echocardiographic assessments of the eft were measured perpendicularly from the free wall of the right ventricle at end - systole in three cardiac cycles according to the method previously described by iacobellis et al .
epicardial fat was described as the echo - free space between the outer wall of the myocardium and the visceral layer of the pericardium .
the maximum eft was also measured from the point on the free wall of the right ventricle along the midline of the ultrasound beam perpendicular to the aortic annulus as the anatomic landmark ( figure 1(b ) ) and from the apical 4-chamber view focused on the right ventricle ( figure 1(c ) ) , because one of the critical issues in eft measurement is the inconsistency in the measurement location , and mean eft was averaged from the parasternal long axis , parasternal short axis , and apical 4-chamber view images .
the offline measurement of eft was performed by two cardiologists ( ik shim and ki cho ) who were unaware of the clinical data .
statistical analyses were performed with the commercially available computer program spss 18.0 for windows ( spss inc .
values are expressed as mean standard deviation or as a percentage ( % ) .
parameter differences among the 3 groups were evaluated using the one - way anova test for normally distributed variables and the kruskal - wallis test for non - normally distributed variables .
values are expressed as means ( standard deviation ) if numerical variables or as the number of subjects and their percentages ( % ) , if categorical .
pearson correlations analysis was performed to determine factors that potentially influenced bp and hr variability .
multivariate logistic regression models for bp variability were built to determine which variables were independently associated with this status .
a total of 441 patients with eh ( male / female , 236/205 ; mean age , 50.7 13.8 ) and 83 normotensive normal weight patients ( controls , male / female , 42/41 ; mean age , 51.0 13.3 ) were analyzed , and their clinical features and ambulatory blood pressure parameters according to gender are given in table 1 ( male ) and table 2 ( female ) .
circadian bp profile and bp variability assessed by 24-hour mean bps variation were increased in hypertensive patients , especially in hypertensive obese patients , without gender difference ( all p 's < 0.05 ) . moreover , the proportion of patients with a nondipping pattern was the highest among the hypertensive obese patients .
although there were no significant differences in hr variability among the groups , hypertensive obese patients had higher daytime and 24-hour mean hr and male hypertensive obese patients also had higher nighttime hr than control patients ( all p 's < 0.05 ) .
although total cholesterol levels were higher in hypertensive obese patients of both genders than in the controls , male hypertensive obese patients had higher triglycerides and female hypertensive obese patients had lower hdl cholesterol than controls ( all p 's < 0.05 ) .
although there was no significant difference in systolic function , hypertensive patients showed significantly increased wall thickness and enlarged left atrial diameter , which were more prominent among hypertensive obese patients , without gender difference ( tables 3 and 4 ) .
interestingly , eft was higher in female than in male patients ( 7.0 2.5 versus 5.9 2.2 mm , p < 0.001 ) . among women , eft was highest in the obese eh group ( 7.5 2.6 mm )
whereas that of the control was 6.4 2.8 mm and that of the nonobese eh group was 6.7 2.8 mm ; however , eft did not vary significantly among males ( 5.9 1.9 versus 6.0 2.7 versus 5.9 2.4 mm , resp .
, p = 0.937 ) . when we compared the 24-hour abmp parameters and eft between male and female patients , female patients had significantly higher eft ( 7.0 2.6 versus 5.9 2.2 mm , p < 0.001 ) and a greater daytime bp variation ( 15.1 4.72 versus 13.6 4.45 mmhg , p < 0.001 ) than male subjects
; males had higher 24-hour mean bp ( 138.8 14.7 versus 137.3 15.3 mmhg , p = 0.043 ) and hr variability ( 15.6 7.45 versus 14.3 6.02 , p = 0.023 ) than female subjects ( table 5 ) .
variable circadian bp profiles and la size were significantly related to bmi in both females and males ( all p 's < 0.05 , figures 2(a ) and 2(b ) , table 6 ) .
however , eft was significantly correlated with 24-hour mean bp ( both day and night ) as well as variability ( mainly night ) only in female patients ( figure 2(c ) ) .
when we investigated the association of quotient of eft / bmi with bp values , eft / bmi showed positive correlation with 24-hour systolic bp ( r = 0.172 ; p = 0.009 ) , 24-hour mean bp ( r = 0.134 ; p = 0.041 ) , day systolic bp ( r = 0.150 ; p = 0.022 ) , day systolic bp variation ( r = 0.133 ; p = 0.043 ) , night systolic bp ( r = 0.217 ; p = 0.001 ) , night systolic bp variation ( r = 0.181 ; p = 0.006 ) , night diastolic bp ( r = 0.132 ; p = 0.047 ) , and night diastolic bp variation ( r = 0.179 ; p = 0.007 ) .
however , no correlation was shown between eft / bmi and 24-hour abpm parameters in male .
multivariate logistic regression analysis demonstrated that the 24-hour mean bp variability was associated with sbp ( standardized coefficient = 0.199 ; p = 0.018 ) and eft ( standardized coefficient = 0.175 ; p = 0.016 ) in female patients , but not in male ones ( table 7 ) .
when we performed binary logistic regression analysis to identify the independent determinants of nocturnal nondipping bp pattern , eft was independent contributor to the nondipping pattern only in female ( odds ratio 7.034 , 95% confidence interval 2.258 to 21.909 , p = 0.001 ) ( table 8) .
the most relevant information obtained from this study is that relationships among eft , obesity , and circadian bp variability are affected by gender in different manners .
although circadian bp profile and bp variability were increased in hypertensive obese patients without gender differences , female patients had significantly higher eft and greater daytime bp variability than male subjects .
bp variability was associated with sbp and eft only in female patients ; therefore , eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men .
bmi is known to be associated with the severity of ambulatory hypertension and increased daytime bp . in the present study
, we investigated the relationship between the parameters derived from 24-hour abpm ( 24-hour sbp and dbp , and bp variability ) in nondiabetic hypertensive patients with or without obesity .
in general , bmi is correlated with bp level , and individuals with abdominal obesity have increased bp levels or are at risk for hypertension [ 3 , 5 ] . the mechanism behind obesity - related bp elevation has not been fully established .
obesity may induce autonomic dysfunction secondary to the elevation of plasma insulin concentration , because hyperinsulinemia has been associated with sympathetic activation [ 18 , 19 ] and marked increases in hr . vice versa , sympathetic hyperactivity is also an independent contributor to the insulin resistance associated with obesity [ 21 , 22 ] .
thus , it is possible that mean arterial pressure and nighttime hr might remain at daytime levels as a consequence of sustained elevation of plasma insulin concentration .
our hypertensive obese patients had higher proportions of nondipping patterns and higher daytime and 24-hour mean hr ; moreover , male hypertensive obese patients also had higher nighttime hr than the controls .
these findings are similar to the report of antic et al . , who showed that the normal dipping pattern of bp and hr is rapidly lost following a switch to a high fat diet , without affecting daytime values .
moreover , obesity suppresses nighttime parasympathetic activity and increases nighttime hr values . because diurnal variations in mean arterial pressure and hr appear to be autonomically mediated , the loss of nocturnal dipping of mean arterial pressure and hr in our obese patients must represent an underlying change in autonomic function .
since the nighttime mean bp was altered in obese patients , the presence of hypertension in obese patients might be missed by office bp measurements that were made only during the daytime .
thus , our results emphasize the importance of continuous 24-hour monitoring of cardiovascular variables , especially in obese patients .
regarding gender differences , obese females showed a higher tendency towards bp variability than obese males .
one interesting finding was that females showed a significant relationship between increased eft , obesity , and bp variability , but this was not the case in males .
still yet , there is no consensus in the literature on the impact of gender on the amount of epicardial fat .
iacobellis et al . showed bigger size of eft in males than in females , and our result showed the greater values of eft in females .
recent study by akilli et al . showed larger eft in females than in males .
thus , these findings may indicate different gender susceptibilities related to obesity and the regional differences in fat distribution , especially eft distribution .
several studies have reported gender differences regarding the effects of obesity and change in body weight on bp [ 13 , 26 ] and cardiovascular outcome [ 12 , 27 ] .
it has been reported that obese women have a higher relative risk of diabetes and heart failure with increasing abdominal circumference than men and that obesity has an association with diastolic dysfunction and lvmi only in women . from these results
, women can be considered to be more susceptible to cardiovascular adverse effects related to obesity . in our study , accentuated bp variability in obese hypertensive women may have indicated a role in cardiovascular vulnerability . however , the exact explanation for these gender differences is still unclear .
one possible mechanism is that such gender differences result from abrupt hormonal and body fat composition changes after menopause .
thus , obese females have a greater amount of visceral adipose tissue which may lead to insulin resistance .
insulin resistance and changes in the autonomic nervous system as a result of redistribution of body fat may have an impact on the cardiovascular system in obese women .
excess fat has traditionally been understood to be found in intra - abdominal organs such as the liver and in subcutaneous tissue .
recently , however , epicardial adipose tissue has been found to reflect visceral adiposity and has been proposed as a new cardiometabolic risk factor . increased plasma fatty acid levels
may stimulate the cardiac autonomic nervous system activity through an increase in plasma catecholamine concentrations , which may be related to impaired diurnal bp patterns .
several reports have found a relationship between bp pattern and eft [ 10 , 31 ] , and echocardiographically assessed elevated eft was independently associated with impaired diurnal blood pressure profiles in hypertensive individuals . also , increased eft has been reported to be associated with diastolic dysfunction and la dilatation due to local or systemic effects in untreated hypertensive patients . in our results , variable circadian bp profiles and la size
were significantly related tobmi in both males and females , but eft was significantly correlated with la diameter and 24-hour mean bp with variability as well as nocturnal nondipping bp pattern , only in female patients .
the present study suggests that there would be a sex - dependent regulation of bp associated with epicardial adipose tissue , and a recent study demonstrated a sex - dependent regulation of diet - induced lvh associated with sexual dimorphic expression of adipocytokines in epicardial adipose tissue .
although our patient sample seems sufficiently large compared to other studies in the literature , in this cross - sectional study we observed predictive factors and outcome variables simultaneously and had no follow - up data .
, although we suggested that the hyperinsulinemia would be the possible main cause of elevated nighttime hr levels , we did not measure the plasma insulin levels . because both pre- and postmenopausal women were included in the study , we can not assume the impact of estrogen on epicardial fat . finally , all participants were korean , and we thus can not generalize our results to populations of other ethnic groups . in conclusion ,
the relationship among eft , obesity , and circadian bp variability was affected by gender in different manners .
eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men . | this study aimed to investigate the effects of gender on the association between epicardial fat thickness ( eft ) and circadian blood pressure ( bp ) changes in patients with recently diagnosed essential hypertension ( eh ) .
a total of 441 patients with eh ( male / female : 236/205 , mean age : 50.7 13.8 ) and 83 control patients underwent 24-hour ambulatory bp monitoring and echocardiography .
obese eh patients had higher circadian bp profile with bp variability , wall thickness , and left ventricular mass than nonobese eh patients and controls ( all p 's < 0.05 ) without gender differences .
eft was higher in female than in male patients ( 7.0 2.5 versus 5.9 2.2 mm , p < 0.001 ) and higher in the obese female eh group ( 7.5 2.6 mm ) than in the control ( 6.4 2.8 mm ) or nonobese eh group ( 6.7 2.8 mm ) among women , whereas eft did not vary among males ( 5.9 1.9 versus 6.0 2.7 versus 5.9 2.4 mm , p = 0.937 ) .
multivariate logistic regression analysis demonstrated that the 24-hour mean bp variability was associated with sbp ( p = 0.018 ) and eft ( p = 0.016 ) in female patients , but not in male patients .
the relationships among circadian bp variability , obesity , and eft were affected by gender in different manners .
eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men . |
during the past 2 decades , laparoscopic surgery has gained worldwide popularity resulting from its well - known characteristics of less postoperative pain , fewer complications , earlier discharge , and better cosmesis .
smaller skin incisions in laparoscopic surgery were related to fewer unwanted side effects , including less postoperative pain . however , in recent studies comparing conventional and single - incision laparoscopic surgical procedures , the postoperative pain scores were not different .
keyless abdominal rope - lifting surgery ( kars ) is a novel , gasless ( isobaric ) , single - incision laparoscopic surgical technique used in various gynecologic operations and cholecystectomies . besides being a single - incision laparoscopic technique
, kars does not require carbon dioxide ( co2 ) use to inflate the abdominal wall and the intra - abdominal pressure is not elevated during the operation .
in addition , postoperative shoulder pain seems to be more frequently observed after laparoscopic surgical procedures in which the intra - abdominal pressure is elevated to between 12 and 18 mm hg by use of co2 . in this study we aimed to compare the postoperative pain levels for kars and conventional multiport laparoscopy performed for adnexal cysts .
although kars has been performed in our institute since the first half of 2010 , the study included benign ovarian cysts operated on between november 2011 and june 2013 .
beginning in november 2011 , intraoperative and postoperative pain management for all operations performed in our unit was standardized ; thus the uniform management approach allowed us to collect and analyze the data from different surgical techniques .
the study was approved by the local institutional ethical committee of kafkas university school of medicine .
the included women had cystic masses including simple cysts with or without septation , endometriomas , and benign cystic teratomas .
most of the participants complained of groin pain , menstrual bleeding disorders , dysmenorrhea , and infertility .
emergency cases including patients with hemodynamic instability , severe abdominal pain , and inaccurate preoperative diagnoses were excluded .
malignant cysts were excluded based on the patient 's history , clinical findings , ultrasonography and magnetic resonance imaging , and levels of cancer antigen 125 , alpha fetoprotein , carcinoembryonic antigen , and cancer antigen 199 .
women who were diagnosed with an adnexal cyst and in whom a laparoscopic operation was indicated were invited to choose one of the operative techniques .
because kars was performed only in our center , we could not design a randomized trial .
thus the groups of this cross - sectional prospective clinical study were formed according to the women 's choice as the kars or conventional co2 laparoscopy groups .
initial visual analog scale ( vas ) scores of 5.7 1.42 and 6.3 1.42 in patients undergoing kars and conventional laparoscopy , respectively , indicated that we needed at least 30 operations in each group to perform a study with a statistical power of 80% at an level of .05 .
the demographic and physical characteristics of the participating patients included their age , gravidity , parity , height , weight , and body mass index , as well as the number of abortions , ectopic pregnancies , and offspring .
oral intake was prohibited starting at 11 pm , and a 135-ml solution containing 19 g of sodium dihydrogen phosphate and 7 g of disodium hydrogen phosphate was administered rectally at 6 am on the operation day .
we used the operative laparoscopy study group 's classification system for staging intra - abdominal adhesions : 0 , none ; 1 , filmy and avascular ; 2 , dense and vascular ; and 3 , binding and cohesive .
the abdominal access time in kars patients included the time needed for the construction of the pathway into the abdominal cavity and the placement of the lifting ropes . in conventional laparoscopy patients ,
the abdominal access time included the time needed for the creation of the co2 pneumoperitoneum at 14 mm hg of pressure and the insertion of the 3 intra - abdominal access ports .
all included patients were considered class 1 or 2 according to the american society of anesthesiologists classification and received a standard anesthetic management regimen during the operations .
we used propofol , 1.5 to 2.5 mg / kg , to induce anesthesia and facilitated intubation with rocuronium , 0.4 to 0.6 mg / kg .
oxygen was supplemented before and after the intubation at rates of 100% and 50% ( mixed with the operating theater 's air ) , respectively .
we used 2% sevoflurane and 50 g of fentanyl for anesthesia maintenance . to replace the fluid deficit , we used sodium chloride or ringer lactate solution at 10 ml / kg on intravenous insertion at 2 ml / kg per hour .
unexpected bleeding was managed by use of additional crystalloid solution at 3 ml / kg per hour .
atropine , 1 to 1.2 mg intravenously , was used to reverse the neuromuscular blockade .
the postoperative pain management and vas pain score recordings were initiated in the obstetrics and gynecology service .
all patients , regardless of initial vas pain score , received 50 mg of meperidine intramuscularly at the beginning of the postoperative period and 75 mg of diclofenac sodium during the second postoperative hour .
vas pain scores were recorded at the beginning and second , fourth , and 24th hours of the postoperative period .
beginning from the fourth postoperative hour , patients with vas pain scores of > 4 points received an additional dose of meperidine , 25 mg intramuscularly .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger .
after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,
all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4
statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .
the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .
normally distributed variables of the 2 groups were compared by use of the student t test , and non normally distributed variables were compared by use of the mann - whitney u test .
the test was used to assess the probability of existence of shoulder pain in the 2 groups .
a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .
the hosmer - lemeshow test showed that the model fit ( = 13 762 , p = .88 , n = 77 ) .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,
all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4 - 0 intracutaneous absorbable sutures .
statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .
the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .
normally distributed variables of the 2 groups were compared by use of the student t test , and non normally distributed variables were compared by use of the mann - whitney u test .
the test was used to assess the probability of existence of shoulder pain in the 2 groups .
a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .
the hosmer - lemeshow test showed that the model fit ( = 13 762 , p = .88 , n = 77 ) .
during a 20-month period , 77 women with benign ovarian cysts were operated on by kars ( n = 32 ) or conventional co2 laparoscopy ( n = 45 ) .
the demographic data , physical characteristics , and preoperative findings of the participating women were summarized in table 1 .
the demographic findings , surgical histories , and preoperative hematologic statuses of the groups did not differ significantly ( p > .05 ) .
demographic data , physical characteristics , and preoperative findings of participating women in both groups data are presented as mean standard deviation unless otherwise indicated .
conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) .
minor bleeding and unintended cyst ruptures were the only observed intraoperative complications . in none of the cases was a blood transfusion or postoperative intensive care admission needed .
we did not need to convert to conventional laparoscopy or laparotomy in the kars group or to laparotomy or mini - laparotomy in the conventional laparoscopy group .
simple oral analgesic use after the 24th hour of the postoperative period was adequate for postoperative pain relief in both groups .
most of the intraoperative and postoperative findings were similar in the 2 groups ( p > .05 ) ; however , the abdominal cavity access and total operative times in the kars group were significantly longer than those in the conventional laparoscopy group ( p < .05 ) .
intraoperative and postoperative findings according to operative technique data are presented as mean standard deviation unless otherwise indicated .
the vas pain scores at the beginning and second , fourth , and 24th hours of the postoperative period were significantly lower in the kars group ( p < .05 ) , although the differences in mean vas scores never exceeded 1 point .
in addition , the women in the kars group needed less additional analgesia in comparison with those in the conventional laparoscopy group ( p < .05 ) .
moreover , a higher number and percentage of the patients in the conventional co2 laparoscopy group had shoulder pain ( p < .05 ) .
postoperative pain scores and additional analgesic requirements according to operative techniques data are presented as mean standard deviation unless otherwise indicated .
conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) . the test analyzing shoulder pain showed that postoperative shoulder pain was significantly less frequent in the kars group ( = 5.304 , p = .021 , n = 77 ) .
logistic regression for postoperative shoulder pain predicted that shoulder pain was affected only by the operation type and duration ( table 4 ) .
although the operation duration was longer in the kars group , the frequency of shoulder pain was higher in the conventional laparoscopy group .
the total operative time correlated positively with the abdominal access and rope - lifting process time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) . in the conventional laparoscopy group ,
the total operative time correlated positively with the abdominal access time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) .
in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .
, our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .
postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .
although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .
the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .
postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .
pain sensation is subjective and may even differ for the same individual under different conditions .
it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .
although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .
gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons . however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .
in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .
gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .
single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .
gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .
similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .
however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study
in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .
thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .
recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .
surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study
we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .
in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .
previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al
, the postoperative pain scores did not differ between single - incision and multiport laparoscopies .
however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study
the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up . in addition
, there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .
moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .
better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .
although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .
they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .
although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .
the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .
the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,
fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .
in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011
prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .
they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .
although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study , although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .
our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .
however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .
in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .
in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .
to our knowledge , our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .
postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .
although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .
the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .
postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .
pain sensation is subjective and may even differ for the same individual under different conditions .
it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .
although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .
gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons .
however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .
in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .
gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .
single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .
gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .
similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .
however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study
in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .
thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .
recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .
surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study
we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .
in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .
previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al , the postoperative pain scores did not differ between single - incision and multiport laparoscopies . however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study
the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up .
in addition , there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .
moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .
better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .
although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .
they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .
although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .
the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .
the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,
fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .
in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011
prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .
they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .
although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study
, although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .
our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .
however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .
in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .
kars seems to cause less postoperative pain during the management of benign ovarian cysts in comparison with conventional multiport co2 laparoscopy . | background and objectives : keyless abdominal rope - lifting surgery is a novel , gasless , single - incision laparoscopic surgical technique . in this study we aimed to compare the postoperative pain from keyless abdominal rope - lifting surgery with carbon dioxide laparoscopy performed for benign ovarian cysts.methods:during a 20-month period , 77 women underwent surgery for a benign ovarian cyst .
keyless abdominal rope - lifting surgery and conventional carbon dioxide laparoscopy techniques were used for the operations in 32 women and 45 women , respectively .
the 2 operative techniques were compared with regard to demographic characteristics ; preoperative , intraoperative , and postoperative data including early postoperative pain scores ; and frequency of shoulder pain and analgesic requirements.results:data regarding demographic characteristics , preoperative findings , cyst diameters and rupture rates , intra - abdominal adhesions , intraoperative blood loss , and postoperative hospital stay did not differ between groups ( p > .05 ) .
however , the mean operative and abdominal access times were significantly longer in the keyless abdominal rope - lifting surgery group ( p < .05 ) .
visual analog scale pain scores at initially and at the second , fourth , and 24th hours of the postoperative period were significantly lower in the keyless abdominal rope - lifting surgery group ( p < .05 ) .
similarly , keyless abdominal rope - lifting surgery caused significantly less shoulder pain and additional analgesic use ( p < .05).conclusion : keyless abdominal rope - lifting surgery seems to cause less pain in the management of benign ovarian cysts in comparison with conventional carbon dioxide laparoscopy . |
pancreatitis , the commonest adverse event following endoscopic retrograde cholangiopancreatography ( ercp ) , has been found to occur at widely varying rates , in 1% to 15.1% of pateints.14 post - ercp pancreatitis ( pep ) has been found at lower rates in recent studies because noninvasive methods , such as magnetic resonance cholangiopancreatography ( mrcp ) and endoscopic ultrasonography ( eus ) , have superseded diagnostic ercp.5 despite improvement of our knowledge , equipment , and methods for ercp , complications are still a significant hazard with this procedure .
pep is the most common severe complication.1,6,7 numerous studies have looked at risk factors for pep,14,616 with varied results , possibly because of different study designs , different candidate predictor variables , and differences between settings .
precise identification of risk factors is of great importance for recognition of high - risk cases in which ercp should be avoided if possible or protective modalities should be considered to minimize patients risk of morbidity and mortality .
we know that inflammatory biomarkers are useful at 2448 hours post procedure in predicting pep , and a few studies have evaluated pre - procedure inflammatory markers , especially c - reactive protein ( crp ) elevation , as predictors of pep.1723 none of the previous studies has focused on pre - ercp erythrocyte sedimentation rate ( esr ) as a potential risk factor for pep . despite the importance of identifying risk factors for pep ,
the present study examined prospectively the potential patient- and procedure - related risk factors , including esr , for pep in iranian population .
during a four - year period ( 20082012 ) , 780 patients who underwent ercp in a tertiary care hospital were analyzed in this prospective study .
the patients were referred to this center from different parts of iran , which enhances the ability to generalize the findings for the iranian population .
all patients gave their written informed consent to participate in this research before the ercp procedure , and the study protocol was approved by the ethics committee of the research center for gastroenterology and liver diseases , shahid beheshti university of medical sciences .
indications for ercp were determined by participating endoscopists before the procedure , on the basis of generally accepted diagnostic indications for ercp.5 laboratory evaluations , including those of alkaline phosphatase ( alp ) , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , bilirubin , and serum amylase , were performed on the first day of hospitalization , and the results were used as pre - ercp laboratory parameters .
patients in acceptable condition were discharged one to two days after ercp ; otherwise , they received longer inpatient care based on the severity of their complications and illness .
all the patients had a follow - up visit two weeks after discharge from hospital .
patient- and procedure - related data , including demographics , characteristics , clinical information and technical details , and findings from ercp procedures were recorded prospectively on a detailed data sheet .
exclusion criteria were a history of biliary sphincterotomy or pre - cut sphincterotomy , pre - procedure active pancreatitis , pregnancy , mental disability , and refusal to participate .
ercp procedures were performed by a total of eight expert endoscopists , almost always with a trainee performing at least part of the procedure . during the procedures ,
patients were under conscious sedation using a combination of intravenous lidocaine , midazolam , and propofol .
successful cannulation was defined as free and deep instrumentation of the biliary tree , and a cannulation attempt was defined as sustained contact with the cannulating device and the papilla for at least five seconds.17 difficult biliary cannulation was defined as the failure of biliary access despite 10 minutes of attempted biliary cannulation or more than five attempted unintentional pancreatic cannulations.18 any complications taking place during or following the procedure were also entered into the records .
pep was diagnosed according to the generally accepted criteria defined by cotton et al,6 ie , presence of upper abdominal pain 24 hours after an ercp procedure and a serum amylase level more than three times the upper limit of normal .
results were expressed as the mean standard deviation ( sd ) for quantitative variables and percentages for categorical variables .
multiple linear regression analysis was used to develop an optimal model for predicting pep with the presence of confounders .
the performance of the model was assessed with the hosmer lemeshow goodness - of - fit test .
all the statistical analyses were performed using spss version 16.0 ( spss inc . ) for windows .
between 2008 and 2012 , 780 patients ( male : female ratio 393:387 , mean age 57.5 years ) underwent diagnostic and therapeutic ercp with the primary diagnosis of hepatobiliary disorders .
of these 780 ercps , 105 procedures ( 13.5% ) were carried out in patients with invasive cancers ( pancreatic ductal adenocarcinoma , biliary carcinoma , metastastic tumor with biliary obstruction , hepatocellular carcinoma , ampullary carcinoma ) because of obstruction and 675 ( 86.5% ) were performed in patients with benign diseases , which included 313 procedures ( 40.1% ) to remove bile duct stones and 362 ( 46.4% ) for benign biliary stricture .
the patients included 393 men ( 50.4% ) , and 448 ( 57.5% ) were more than 65 years old .
previous cholecystectomy had been performed in 36.3% of patients and 9.0% had undergone previous ercp . a total of 80 patients had history of biliary stone diagnosed by a similar procedure .
also , history of confirmed pancreatitis and hepatitis was observed in 27 ( 3.5% ) and 10 ( 1.3% ) patients , respectively ( table 1 ) . regarding laboratory parameters ( table 2 ) ,
serum amylase was found to be elevated to more than 200 units / l in 102 patients ( 13.7% ) three hours after ercp and more than 800 units / l in 60 patients ( 7.7% ) . in all , 446 ( 70% ) participants underwent wire - guided cannulation and others ( 30% ) underwent sphincterotome biliary cannulation using contrast injection as the conventional method .
successful biliary cannulation was technically achieved in 82.5% of all patients at ercp , although in 13.0% of them , cannulation failed .
regarding pathologic changes in papilla , tumoral features and ulcerative changes were found in 3.3% and 0.6% of patients .
of the 780 patients who underwent diagnostic or therapeutic ercp , pancreatitis developed in 26 patients ( 3.3% ) .
significant bleeding occurred in two patients ; the only predictor in multivariable analysis was biliary sphincterotomy .
we diagnosed cholangitis ( using the criteria of transient worsening of clinical states and liver function tests consistent with cholangitis ) in four subjects , all of whom had common bile duct ( cbd ) stones with biliary stent placement .
table 3 shows the frequency of pep and raised esr according to indication for ercp . in the multivariable risk model for predicting pep ( table 4 ) , significant risk factors with adjusted odds ratios ( ors )
were age < 65 years ( or = 10.647 , p = 0.023 ) and esr > 30 ( or = 6.414 , p < 0.001 ) .
female gender , history of recurrent pancreatitis , pre - ercp hyperamylasemia , and difficult or failed cannulation could not predict pep . regarding the cannulation techniques
, there was no difference in the incidence of pep between guide wire - assisted ercp or conventional contrast - assisted cannulation .
we have identified two risk factors for the development of pep : age < 65 years and esr > 30 .
several studies have focused on younger age as an independent predictor of pep.14,617 so in younger individuals , it is important to perform ercp only when the indication is very strong and there is no other noninvasive substitute for ercp .
another risk factor for the development of pep in our study was increased esr level before ercp ( esr > 30 ) .
we found that raised esr > 30 mm / hour is a significant factor that independently predicts increased risk of pep .
although some studies have evaluated the level of post - ercp inflammatory markers , especially post - procedure crp elevation , as predictors of development of pep,1923 it seems that none of the previous studies have focused on pre - ercp esr level as a potential risk factor for pep .
however , the findings of previous studies suggest that inflammatory biomarkers are helpful at 2448 hours post - procedure , so they are not early predictors .
although it seems that our study is the first study to evaluate pre - ercp esr level as a risk factor for developing pep , many other studies have evaluated the role of pharmacologic agents in reducing the incidence or severity of pep.2,10,2227 several agents , most of which have an anti - inflammatory effect , have been tested in clinical trials . in terms of attenuating the inflammatory response ,
the most promising results have been with non - steroidal anti - inflammatory drugs ( nsaids ) , and some standard guidelines recommend the routine use of nsaids to prevent pep.28 most of these studies are experimental clinical trials , but it is possible that the main beneficial effect of these drugs is limiting systemic inflammatory response and lowering the level of esr as a potential risk factor for pep .
this study could be a guide for future clinical trials on rectal nsaids for the prevention of pep ; the effect of these drugs in prevention of pancreatitis , with attention to the level of esr before ercp , should be evaluated .
other risk factors such as female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct were not assessed because ercp procedures were done by an expert and because of the absence of manometry .
also , other factors such as pre - ercp bacterial infections might increase esr level .
some studies on both human and animal models , performed to evaluate the potential role of antibiotics in preventing pep , found reduced rates of pep in patients receiving antibiotic prophylaxis prior to ercp when compared to placebo.26 however , most studies did not find any role for infections in pep , except in cases of severe pancreatitis with necrosis .
the main limitation of this study was the absence of combined evaluation of crp and esr before ercp .
esr will be raised in almost all patients with any systemic inflammation , so our study is also limited because patients with systemic inflammation were not excluded . to date , many risk factors for pep have been reported from high - volume centers .
these have included female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct .
not all of these factors were analyzed in our study , and our results were not fully consistent with the previous results .
performing ercp may be safer in the elderly and in patients with low levels of serum esr because they may be less at risk of pep .
using pre - ercp anti - inflammatory pharmacologic agents like nsaids might be beneficial , by lowering esr level and reducing related risk of pep . | backgroundpancreatitis remains the most common complication of endoscopic retrograde cholangiopancreatography ( ercp ) , resulting in substantial morbidity and occasional mortality .
there are notable controversies and conflicting reports about risk factors of post - ercp pancreatitis ( pep).aimto evaluate the potential risk factors for pep at a referral tertiary center , as a sample of the iranian population.materials and methodsbaseline characteristics and clinical as well as paraclinical information of 780 patients undergoing diagnostic and therapeutic ercp at taleghani hospital in tehran between 2008 and 2012 were reviewed .
data were collected prior to the ercp , at the time of the procedure , and 2472 hours after discharge .
pep was diagnosed according to consensus criteria.resultsof the 780 patients who underwent diagnostic ercp , pancreatitis developed in 26 patients ( 3.3% ) . in the multivariable risk model , significant risk factors with adjusted odds ratios ( ors ) were age < 65 years ( or = 10.647 , p = 0.023 ) and erythrocyte sedimentation rate ( esr ) > 30 ( or = 6.414 , p < 0.001 ) .
female gender , history of recurrent pancreatitis , pre - ercp hyperamylasemia , and difficult or failed cannulation could not predict pep .
there was no significant difference in the rate of pep in wire - guided cannulation versus biliary cannulation using a sphincterotome and contrast injection as the conventional method.conclusionsperforming ercp may be safer in the elderly .
patients with high esr may be at greater risk of pep , which warrants close observation of these patients for signs of pancreatitis after ercp . |
sole aim and the prerequisite for dentistry is an effective pain control during dental procedures . to achieve this goal
local anesthesia is being used since long . in 1943 , lofgren synthesized the first modern anaesthesia.1 it is lidocaine , which was an amide derivative of diethyl amino acetic acid .
it is because of the high density and the firm adherence of palatal mucosa to the underlying bone .
twenty - five years after lidocaine , articaine was first synthesized by muschaneau in 1969 .
it has a thiophene ring in its molecule instead of usual benzene ring.2,3 this is most commonly used in germany .
commercially articaine for dental use is available in 4% solution with epinephrine 1:200000 or 1:100000 .
it also contains maximum 0.6 mg na - sulfite in 1.0 ml and sodium chloride .
once injected , absorption starts from the site of injection into the vascular compartment.1 the unbound local anesthetic is distributed throughout all the body tissue . due to the presence of thiophene ring
, it is inactivated in the liver as well as by hydrolyzation in the tissue and blood .
the aim of the study was to evaluate and compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride for the orthodontic extraction .
the study was carried out on 50 patients at outpatient department of oral and maxillofacial surgery who needed bilateral maxillary premolar extractions for orthodontic purpose .
patients included in this study were in the age group of 15 - 25 years , both genders and systemically healthy .
bleeding disorders , hypertensive , diabetic , pregnant , allergic to local anesthetics , reluctant and medically compromised patients were excluded from the study .
all the patients were explained about visual analog scale ( vas ) before injecting local anesthesia .
experimental sites ( group 1 ) were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .
no palatal anesthesia was injected , but the desired anesthetic effect was achieved with the above . on the other hand , control sites ( group 2 ) were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .
when the objective symptoms were checked , it was found that palatal anesthesia was absent hence additional 0.5 ml was injected to obtain a desired result . after assessing the signs and symptoms of obtaining complete anesthesia
all the parameters , i.e. , drug volume , time of onset , duration of anesthesia and pain rating were recorded for entire patients .
the mean administered volume of articaine and lignocaine were 0.779 0.1305 and 1.337 0.2369 respectively .
it should be noted that the articaine volume administered was almost half of the lignocaine ( table 1 ) .
the mean onset time of lignocaine anesthesia was 1.337 0.2369 , whereas in articaine group the mean time was 1.012 0.2058 min .
this indicates that onset time of articaine was significantly less than lidocaine ( p < 0.0005 ) ( table 2 ) .
pain rating showed that there was no significant difference in pain score in articaine palatal and buccal group ( p > 0.8892 ) , whereas a significant difference was noted in lignocaine palatal and buccal group ( tables 3 and 4 ) .
duration of pain in group 1 was 69.08 18.247 and 55.66 6.414 in group 2 patients .
duration of anesthesia is articaine group is more than the lignocaine group . in the entire study
, it goes biotransformation in both liver and plasma and hence gets cleared much quickly .
recent studies have shown that articaine carries lot of advantages over other anesthetic agents.4 in this study , we observed that the palatal infiltration was required in approximately 98% of cases when lignocaine was used , whereas in articaine group palatal anesthesia was never required .
thiophene ring in its molecular structure , which makes it more lipophilic and this accounts for its diffusion properties across all the tissues.5 articaine is metabolized in the liver , tissues and blood and hence it is cleared out very fast from the body .
this is the only anesthetic agent , which is inactivated from our body in two ways .
zlkowska et al . has reported that like all other anesthetic agents articaine is safe in epileptic patients.6 this study showed no adverse effects and no complications .
this study is in accordance with study by malamed et al . suggesting 4% articaine with 1:100000 adrenaline is safe and have a low risk of toxicity.2 statistical analysis in this study showed no significant difference in extraction pain on vas for test and control sites .
this inference relates to the study done by fan et al.7 oertel et al.8 uckan et al.9 and evans et al.10 when articaine is injected the local concentration of active drug is nearly twice of that obtained with lignocaine .
oertel et al . in his study showed this by determining the concentration of 4% articaine and 2% lidocaine in alveolar blood using high - performance liquid chromatography.11,12 thiophene derivative articaine blocks ionic channels at lower concentration than benzene derivative lidocaine.13 potocnik et al . in vitro study on rat surap nerve concluded that 2% and 4% of articaine is more effective than 2% and 4 % of lidocaine in depressing compound action potential of the a fibres.14 - 16 this efficacy and safety factors are observed in this study too .
it is a well - known fact that palatal anesthesia is a very painful experience even though surface anesthesia is used .
hence , if articaine is used , patients can be relieved from the painful palatal anesthesia without compromising with safety and efficacy .
certain added advantages like shorter time of onset , longer duration of action and greater diffusion property makes it an ideal anesthetic agent to be used in dentistry . | background : articaine in an anesthetic agent , which is used less frequently in dentistry .
it differs from other agents due to the presence of a thiophene ring in its molecular structure .
few groups of researchers claim that it is superior to lignocaine .
hence , the purpose of this study was to compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride in the orthodontic extraction.materials and methods : the study was carried out in 50 patients who needed the orthodontic extraction in the age group from 15 to 25 years .
experimental sites were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule without palatal anaesthesia .
control sites were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .
all the parameters , that is volume , duration , time of anesthesia and pain rating were noted and statistically compared.result:when statistically compared mean volume of articaine ( 0.779 0.1305 ) was less than lignocaine ( 1.337 0.2369 ) .
mean time of onset of articaine was 1.012 0.2058 min , whereas that of was 1.337 0.2369 .
pain rating showed not much difference , but in the lignocaine group palatal anesthesia was required in all the patients .
finally , the mean duration of anesthesia in articaine group was 69.08 18.247 , whereas in the lignocaine group was 55.66 6.414.conclusion:articaine has proved its usefulness in all regards .
literatures have proved its usefulness . like other anesthetic , it is safe and more effective .
it surpasses the need of additional palatal anesthesia .
rapid inactivation in liver and plasma reduces the risk of the drug overdose .
all the above factors make it an ideal anesthetic agent to be used in dentistry . |
the ras association domain family ( rassf ) consists of 10 members : rassf1 - 10 .
importantly , all isoforms contain a ras association ( ra ) domain either in their c - terminal ( rassf1 - 6 ) or n - terminal ( rassf7 - 10 ) regions . to date , no known catalytic activity has been described for this family , and the general consensus supposes that rassf proteins function as scaffolds to localize signaling in the cell .
rassf1 isoform a ( rassf1a ) is the most characterized member of the rassf family .
the rassf1 gene encodes multiple splice variants , including the two predominant isoforms , rassf1a and c. the rassf1a isoform is the longest variant of the rassf1 gene .
structurally , rassf1a is a product of exons 1 , 2/ , 3 , 4 , 5 , and 6 , while rassf1c consists of exons 2 , 3 , 4 , 5 , and 6 . both isoforms contain a c - terminal ra domain ; however , rassf1a has an additional c1 domain that is not present in rassf1c .
this short arm of chromosome 3 is known to exhibit loss of heterozygosity in many tumor models and is thought to harbor tumor suppressor genes . as the literature has shown , rassf1a fits this description .
the rassf1a promoter contains a cpg island that shows a high frequency of hypermethylation in tumors , thereby silencing rassf1a expression in many human cancers including lung , breast , ovarian , renal , and bladder [ 47 ] .
rassf1a expression is also lost in numerous cancer cell lines , while rassf1c expression is seemingly unaffected .
interestingly , recent work suggests that rassf1c may actually promote tumor progression [ 8 , 9 ] , further distinguishing these two splice variants .
all rassf proteins have an ra domain , which is thought to necessitate their binding to activated , gtp - bound ras proteins . while rassf5 ( nore1 ) is thought to bind ras directly , whether rassf1a is able to associate with ras is less clear .
it has been shown that rassf1a binds k - ras in vitro , and an interaction between ectopically expressed rassf1a and activated k - ras has been observed in hek293 cells [ 11 , 12 ] .
however , other work has found that this interaction only occurs in the presence of nore1 , arguing for an indirect association .
importantly , to our knowledge , there are no reports demonstrating the interaction of endogenous rassf1a and ras proteins .
it has been implicated in the negative regulation of cell cycle progression , cell proliferation , and cell survival .
rassf1a has been shown to localize to microtubules of proliferating cells , increasing microtubule stability and inhibiting cell division [ 14 , 15 ] .
this may be mediated through direct binding or though interaction with microtubule - associated proteins such as c19orf5 .
rassf1a has also been shown to inhibit proliferation by inhibiting the accumulation of cyclin d1 and arresting cell division [ 17 , 18 ] .
rassf1a also promotes apoptosis , which can reportedly occur through multiple mechanisms and is likely cell - type dependent .
one mechanism that mediates the apoptotic function of rassf1a involves protein interaction with modulator of apoptosis-1 ( moap-1 or map-1 ) .
moap-1 is normally sequestered in an inactive form in healthy cells . upon death receptor stimulation ,
rassf1a binds moap-1 , causing its activation and subsequent association with bax , which leads to apoptosis .
previous work has also demonstrated enhancement of rassf1a / mst - mediated cell death by the scaffold cnk1 .
rassf1a can also elicit inhibitory effects on growth and survival through engagement of the hippo pathway .
the hippo signaling pathway is a highly conserved kinase cascade that was originally discovered in drosophila and has been shown to be a critical regulator of cell proliferation , survival , and organ growth .
three members of this pathway , drassf , salvador and hippo , contain the sarah ( salvador - rassf - hippo ) domain , which is conserved in its mammalian counterparts rassf1 - 6 , ww45 , and mst1/2 , respectively .
while the drosophila ortholog drassf is known to antagonize hippo activation in the fly , it has been demonstrated that rassf1a promotes phosphorylation and activation of mst 1/2 by inhibiting the phosphatase pp2a in mammalian systems [ 29 , 30 ] .
the biological relevance of rassf1a - mediated activation of hippo signaling has also been investigated .
reported a rassf1a - mst2-yap - p73-puma signaling axis that promotes apoptosis in mammalian cells .
hippo signaling is also important for maintaining intestinal homeostasis and tissue regeneration in response to injury .
mouse models with conditional disruption of either mst1/2 or sav1 in the intestinal epithelium displayed hyperactivation of yes - associated protein ( yap ) , increased intestinal stem cell ( isc ) proliferation , and increased polyp formation following dextran sodium sulfate ( dss ) treatment [ 32 , 33 ] .
similarly , loss - of - function mutations of hippo components in the fly midgut caused increased isc proliferation .
these findings suggest that perhaps hippo signaling serves a more global role in regulating organ integrity , structure , and response to injury , and that perturbation of this pathway can lead to aberrant growth and dysfunction .
in 2005 , two independent groups generated and published findings regarding the systemic deletion of the rassf1a gene variant in mice [ 35 , 36 ] .
both described similar phenotypes involving the spontaneous generation of tumors , particularly in aged mice , thus further supporting the notion that rassf1a is a bona fide tumor suppressor [ 35 , 36 ] .
not surprisingly , nearly all studies involving rassf1a to date are related to cancer biology with few reports related to the cardiovascular field .
rassf1a is ubiquitously expressed and has been detected in heart tissue [ 3 , 37 , 38 ] .
initial investigation into the role of rassf1 gene products in a cardiac context came from the neyses laboratory .
their findings demonstrated that both rassf1a and rassf1c could associate with the sarcolemmal calcium pump , pmca4b , in neonatal rat cardiac myocytes .
this interaction was shown to mediate the inhibition of erk , and subsequent elk transcription and suggested the possibility that rassf1a could modulate cardiac myocyte growth .
five years later , the same group demonstrated that rassf1a does in fact negatively regulate cardiac hypertrophy in vivo using rassf1a mice .
although these mice have increased susceptibility to spontaneous tumorigenesis , no apparent cardiovascular phenotype was observed under basal conditions , that is , no differences in heart size , morphology , or function compared to wt .
however , when rassf1a mice were challenged with pressure overload , they responded with an exaggerated hypertrophic response , evidenced by significantly greater increases in heart weight / body weight and hypertrophic gene expression ( anp , bnp , -mhc ) .
cardiac myocytes of rassf1a mice were significantly larger , which explains the augmented heart growth .
chamber dilation of rassf1a mouse hearts was observed by echocardiography , consistent with eccentric hypertrophic remodeling .
hemodynamic analysis of wt and rassf1a mice showed a rightward shift in pv loops following pressure overload in rassf1a hearts , yet dp / dtmax , dp / dtmin , and fractional shortening were not altered in rassf1a mice compared to wt . to examine rassf1a function in cardiac myocytes , oceandy et al .
utilized a neonatal rat cardiac myocyte ( nrcm ) culture and the forced expression of rassf1a through adenoviral gene transfer .
increased rassf1a expression inhibited phenylephrine-(pe- ) induced cardiac myocyte growth and suppressed raf-1 and erk1/2 activation by pe treatment .
conversely , both raf-1 and erk1/2 phosphorylation were increased in rassf1a hearts following pressure overload , suggesting negative regulation of mapk signaling by rassf1a .
deletion mutants of rassf1a revealed an important function of the n - terminus of rassf1a that disrupts the binding of active ras and raf-1 , thus preventing erk activation and cardiac myocyte growth . to better understand the function of rassf1a in cardiac myocytes in vivo , we crossed genetically altered mice harboring a floxed rassf1a allele with mice harboring the cre recombinase transgene driven by the -mhc promoter .
this strategy disrupted endogenous rassf1a gene expression and ensured cardiac myocyte specificity [ 38 , 40 ] .
similar to the rassf1a mice , rassf1a - cre mice had no obvious baseline cardiac phenotype .
although we also found exaggerated heart growth in the rassf1a mice in response to pressure overload , the rassf1a - cre mice unexpectedly had attenuated hypertrophy , that is , smaller hearts and cardiac myocytes , compared to rassf1a and -mhc - cre controls .
furthermore , rassf1a - cre mice had significantly less fibrosis and myocyte apoptosis , and better cardiac function following pressure overload .
this was in stark contrast to the rassf1a mice , which presented significantly more fibrosis and a decline in cardiac function comparable to the levels found in wt mice .
as an alternative approach we also generated two different cardiac - specific transgenic mouse lines : the first expressing wild - type rassf1a and the second expressing a rassf1a sarah domain point mutant ( l308p ) that renders it unable to bind mst1 .
interestingly , we found that increased rassf1a expression in the heart caused increases in mst1 activation , cardiac myocyte apoptosis , and fibrosis , and led to worsened function following pressure overload .
conversely , rassf1a l308p tg mice had significant reductions in mst1 activation , apoptosis and fibrosis , while cardiac function was preserved after stress .
these opposing phenotypes strongly implicate mst1 as a critical effector of rassf1a - mediated myocardial dysfunction .
in cultured nrcms , increased rassf1a expression elicited activation of mst1 and caused mst1-mediated apoptosis .
however , in primary rat cardiac fibroblasts , rassf1a had a more pronounced effect on inhibition of cell proliferation rather than survival .
additionally , rassf1a depletion led to an upregulation of nf-b - dependent tnf- expression and secretion in cardiac fibroblasts , while no change in il-1 , il-6 , or tgf-1 was observed . through conditioned medium transfer experiments , we demonstrated that tnf- secretion from fibroblasts promotes cardiac myocyte growth .
furthermore , treatment of rassf1a mice with a neutralizing antibody against tnf- was able to rescue the augmented heart growth and fibrosis observed following pressure overload .
these data strongly implicated tnf- as a critical paracrine factor influencing the cardiac myocyte growth response to stress in vivo .
this work also demonstrated the cell - type specificity of rassf1a signaling in the heart and highlighted a novel signaling pathway downstream of rassf1a / mst1 that mediates a paracrine effect in vivo ( see figure 1 ) .
this mechanism involving multiple cell types , and paracrine signaling among them is rather unique and contrasts with more established signaling paradigms of cardiac hypertrophy including calcineurin / nfat , hdac / mef2 and mek / erk pathways , which have been elucidated in the cardiac myocyte .
using genetically altered mouse models we showed that increased expression of mst1 , and subsequent activation of the hippo pathway , caused increased apoptosis , dilated cardiomyopathy , and premature death .
interestingly , expression of mst1 also attenuated cardiac myocyte hypertrophy thereby impairing the heart 's ability to appropriately respond to stress .
in contrast , expression of a kinase - inactive mst1 mutant ( dn - mst1 ) prevented cell death and protected the heart from insult .
lats1/2 kinases ( mammalian homologs of warts ) are targets of mst1/2 that can phosphorylate and inactivate yap , thereby inhibiting yap - mediated gene transcription .
similar to our findings related to mst1 , we demonstrated that transgenic expression of lats2 in the heart led to inhibited growth and worsened function .
conversely , kinase - inactive lats2 ( dn - lats2 ) transgenic mice had larger hearts both at baseline and following pressure overload and displayed attenuated cardiac myocyte apoptosis in response to stress
. taken together , these results provide further evidence that activation of hippo signaling , via increased mst1 or lats2 expression , inhibits cardiac myocyte growth and promotes apoptosis in the adult heart
. furthermore , selective inhibition of hippo signaling in the cardiac myocyte ( dn - mst1 or dn - lats2 tg ) confers protection against insult , similar to what we observed in the cardiac myocyte - specific rassf1a deleted mice .
however , the hypertrophic response in these two models was opposite , which may result from a hippo - independent pathway(s ) downstream of rassf1a .
it should be pointed out that studies of adult mouse models using cardiac myocyte - restricted deletion of mst1/2 , lats1/2 or yap have not been published .
findings from these models should be helpful in further elucidating the role of hippo signaling components in the adult murine heart .
recent work from the martin laboratory demonstrated the importance of mammalian hippo signaling during cardiac development and cardiac myocyte proliferation .
conditional deletion of salvador ( sav1 ) in the embryonic heart , driven by nkx2.5-cre expression , caused increased myocyte proliferation and cardiac enlargement and was mediated by hyperactivation of yap and subsequent wnt/-catenin - regulated gene expression . in a similar vein , direct targeting of yap expression in the developing mouse heart further demonstrated its role in governing both myocyte proliferation and heart growth .
interestingly , both reports described an interaction between yap and wnt signaling , highlighting additional hippo signaling crosstalk in the heart .
fueled by the initial reports described herein , investigation into the role of rassf1a in cardiovascular biology has begun to accelerate . yet many questions remain outstanding . among them
, what are the upstream inputs that regulate rassf1a function ? what is the mechanism responsible for rassf1a cell - type - specific
what are the molecular constituents of the rassf1a complex ? does rassf1a have additional mst1-independent functions in the heart , as has been demonstrated in tumor cell lines ?
recent work identified activated k - ras as a promoter of rassf1a signaling in colorectal cancer cells .
this finding begs the question of whether k - ras or additional ras isoforms regulate rassf1a in other systems and cell types . based on our findings in rassf1a - deleted mice
, we speculate that the difference in proliferative capacity between cardiac myocytes and fibroblasts may explain the distinct effects of rassf1a signaling in the heart . there may also be differences in the expression or localization of signaling components , thereby modulating their ability to effectively signal in certain cell types .
exposure to diverse signals and cues in the extracellular milieu may also contribute to varied outcomes downstream rassf1a . as we continue to elucidate the role of rassf1a and hippo signaling in the heart
our work has shed light on the importance of cell type specificity rassf1a in determining pathological outcomes .
we also defined a paracrine mechanism functioning downstream of rassf1a in response to cardiac stress .
it is likely that additional complexities remain to be uncovered and will ultimately influence possible interventions to manipulate rassf1a and treat heart disease .
rassf1a signaling is diverse and our knowledge regarding rassf1a function is rapidly expanding . given that a bridge from cancer to cardiovascular biology is in place , it is likely that as additional rassf1a mechanisms of action are discovered , its impact on cardiac biology will continue to grow . | the rassf proteins are a family of polypeptides , each containing a conserved ras association domain , suggesting that these scaffold proteins may be effectors of activated ras or ras - related small gtpases .
rassf proteins are characterized by their ability to inhibit cell growth and proliferation while promoting cell death .
rassf1 isoform a is an established tumor suppressor and is frequently silenced in a variety of tumors and human cancer cell lines .
however , our understanding of its function in terminally differentiated cell types , such as cardiac myocytes , is relatively nascent
. herein , we review the role of rassf1a in cardiac physiology and disease and highlight signaling pathways that mediate its function . |
it is one of the most commonly used medicines in the treatment of rheumatoid arthritis or psoriatic arthritis .
we report the case of an adult woman with psoriatic arthritis who developed acute severe hepatitis following long - term treatment with mtx .
the periodic biological monitoring of liver enzymes : alanine aminotransferase ( alat ) , aspartate aminotransferase ( asat ) , gamma - glutamyl transpeptidase ( gamma - gt ) performed every 6 months did not reveal any abnormalities .
mtx was stopped in the year 2005 due to remission of the disease . in 2009 ,
it was reintroduced at a dose of 15 mg / week due to decompensation of the disease .
monitoring of liver enzymes performed every 6 months showed an elevation < 2 times the normal levels on average of asat and alat since 2010 . assessing the risk - benefit ratio and given the severe decompensation of the disease , mtx was continued . in may 2013 , patient presented with jaundice following the development of asthenia and weight loss .
laboratory investigation showed the following results : prothrombin time ( 71% ) , asat 580 u / l , alat 620 u / l , serum gamma - gt 146 iu / l , serum alkaline phosphatase 170 iu / l and total serum bilirubin 12 mg / dl .
antimitochondrial antibodies ( titer > 1/40 ) and antinuclear antibodies ( titer of 1/640 ) were detected in serum .
routine laboratory investigations for infection with hepatitis a , b , and c viruses and serology for cytomegalovirus and epstein - barr virus were negative .
methotrexate was stopped , and therapy with oral methylprednisolone ( 80 mg / day ) was initiated .
a significant improvement in the following parameters was observed a week later : prothrombin time ( 88% ) , asat 270 u / l , alat 340 u / l , s. gamma - gt 122 iu / l , and s. alkaline phosphatase 173 iu / l .
after 4 months , all laboratory parameters had normalized , and serum was negative for antimitochondrial and antinuclear antibodies .
patients taking mtx are more likely to discontinue therapy because of adverse drug effects rather than because of lack of efficacy .
adverse effects of mtx are , usually , mild and self - limiting , but may include serious adverse drug reactions such as hematopoietic suppression , pulmonary , and hepatotoxicity .
hepatic fibrosis is a usual adverse reaction reported with long - term use of mtx therapy in psoriatic patients .
causality assessment of the adverse drug event ( ade ) was carried out using who - umc criteria , naranjo 's scale , and roussel uclaf causality assessment method ( rucam ) scale . in this case ,
patient improved on withdrawal of the drug , and there were no other confounding factors that could have caused this adverse effect .
hence , the ade was probably caused by mtx ( who - umc criteria : probable ; naranjo 's score : 7 , probable ; and rucam scale : 5 ) .
in addition , sufficient and continuous exposure to the drug , lack of previous evidence of autoimmune disease and complete resolution of the condition within 4 months of discontinuation of the suspected drug confirmed the diagnostic criteria of drug - induced autoimmunity .
the first case of autoimmune hepatitis associated with mtx was described in 2011 in a 57-year - old man after 11 years of treatment with mtx .
a complete normalization of laboratory parameters was obtained 5 months after stopping this drug . to the best of our knowledge ,
it , usually , occurs at higher doses and also positively correlates with the cumulative dose of drugs .
other risk factors for the development of elevated liver enzymes and changes in liver biopsy include obesity , higher alcohol intake prior to commencement of mtx and persistent hepatitis b or c infection . in the present case ,
appearance of autoantibodies and transitory autoimmune disease associated with infliximab in a patient with psoriatic arthritis has also been documented and the preexistent serological signs of autoimmunity are believed to be a risk factor for the development of similar autoimmune reactions .
this report confirms the need to monitor liver enzymes carefully in patients with psoriatic arthritis using long - term treatment with mtx .
clinicians must collaborate with the pharmacovigilance center to detect and notify serious adverse effects of mtx .
further research is needed to identify the exact mechanism and establish , if possible , primary means of prevention of this serious adverse effect . | methotrexate ( mtx ) is one of the most commonly used medicines in the treatment of psoriatic arthritis .
the drug can produce steatosis and cirrhosis .
autoimmune hepatitis is a rare and serious adverse effect .
we describe the case of a 53-year - old woman who developed autoimmune hepatitis after a long - term use of mtx for psoriatic arthritis .
hepatitis was completely resolved 4 months after stopping this drug .
the pathophysiologic mechanisms of a drug - induced autoimmunity are unclear and complex .
this report confirms the need to monitor liver enzymes carefully in patients using long - term treatment with mtx for psoriasis or rheumatoid arthritis . |
mirizzi syndrome is a rare complication of cholecystolithiasis characterized by jaundice due to compression of the common hepatic duct
. the diagnosis may not be immediately apparent , and management is controversial with open surgery still recommended by some authors .
a case is detailed herein of a 67-year - old man who presented with abdominal pain , fever , and jaundice .
a dilated bile duct was found on ultrasound , but the gallbladder could not be seen .
the diagnosis of mirizzi syndrome was made at ercp , and a stent was placed through the papilla .
laparoscopic retrograde ( fundus first ) cholecystectomy was carried out utilizing a laparoscopic liver retractor .
in this particular case , it was not possible at ercp to get a guidewire and stent past the obstruction . a stent was left through the papilla , below the obstruction and this allowed primary duct closure during surgery .
acute mirizzi syndrome should be suspected when a patient presents with acute cholecystitis and jaundice with dilated intrahepatic ducts on ultrasound .
ercp is useful to confirm the diagnosis and allows stenting to alleviate the jaundice and facilitate the subsequent operation .
laparoscopic ultrasound is useful to locate the impacted stone and to partially replicate the touch of the surgeon 's hand , which is not available in laparoscopic surgery .
mirizzi syndrome ( ms ) is biliary obstruction from compression of the common hepatic duct ( chd ) by a gallstone impacted in the cystic duct .
mcsherry classified the syndrome into 2 groups : type i where extrinsic compression of the bile duct occurs and type ii where erosion occurs of the wall of the chd by the stone with formation of a cholecysto - choledochal fistula .
csendes further subdivided type ii depending on the size of the fistula and the degree of destruction of the wall of the chd .
the syndrome was initially described in 1948 by pablo luis mirizzi , professor of surgery in cordoba , argentina , and he erroneously postulated that the extrinsic pressure and inflammation induced spasm of the chd .
precise diagnosis may be difficult initially because the condition may be confused with choledocholithiasis and cholangitis .
the classical ultrasound findings are of a contracted gallbladder , dilated intrahepatic ducts , and a normal common bile duct ( cbd ) .
although a rare condition , many controversial aspects still surround the definition and management of ms . to illustrate the true ms and the value of a combination of endoscopic retrograde cholangiopancreatography ( ercp ) and laparoscopic surgery , a case
a 67-year - old man was admitted to the emergency department with a 3-day history of gradually worsening upper abdominal pain associated initially with vomiting and anorexia . the patient had a history of hypertension and polymyalgia rheumatica .
he was noted to be jaundiced with a temperature of 38 celsius and right upper quadrant tenderness and guarding .
his liver function tests were markedly deranged with bilirubin 106 mol / l [ range , 3 to 22 ] , alkaline phosphatase 159 iu / l ( range , 20 to 110 ) , and alanine transaminase 770 iu / l ( range , 5 to 65 ) .
this showed dilated intrahepatic ducts with the cbd reported as also being dilated and measuring 9 mm in diameter .
subsequent imaging showed that the ultrasound had actually detected a dilated chd as the cbd was not dilated . at ercp , contrast filled a nondilated cbd up to an obstruction due to a stone ( figure 1 ) .
attempts were made to pass a guidewire beyond the obstruction up into the chd and intrahepatic ducts but without success .
the guidewire kept going into the gallbladder , and it became apparent that there was an ms with the obstructing stone in the cystic duct causing compression of the chd ( figure 2 ) .
after the guidewire had been manipulated for a time , some pus came out of the papilla signifying a gallbladder empyema .
a 10 french straight plastic stent was placed through the papilla without papillotomy with its proximal tip lying below the obstruction and , as expected , no bile or intrahepatic contrast drained .
the stone is impacted in the cystic duct , which is anterior and parallel to the common hepatic duct .
note the narrow common bile duct and the dilatation of the common hepatic and intrahepatic ducts . a computed tomogram ( ct ) scan was done and showed a contracted gallbladder with a calcified stone causing biliary obstruction ( figure 3 ) . at laparoscopic cholecystectomy , an acutely inflamed
a liver retractor was used to elevate the liver and maintain exposure of the bile duct ( diamond - flex , cardinal health , snowden - dencer mis products , tucker , ga , usa ) ( figure 4 ) .
it was possible to feel the stone via a grasper , and its position was definitely confirmed by laparoscopic ultrasound ( figure 5 ) .
the duct was incised to release the impacted stone with a single - use cbd blade ( espiner medical ltd .
the cbd was examined with a 5-mm flexible choledochoscope showing that the duct was clear distally with the stent in place ( figure 7 ) .
it was not possible to retroflex the choledochoscope to examine the common hepatic or intrahepatic ducts .
the gallbladder was excised , the opening in the cystic duct sutured with continuous 40 vicryl ( ethicon inc . ,
somerville , nj , usa ) and a 20 french nonsuction tube drain placed in the subhepatic space near the bile duct .
the stent was removed at ercp 6 weeks later and a check cholangiogram showed a normal bile duct with no filling of any residual cystic duct and no stricture . during 18 months of follow - up , no delayed sequelae have occurred .
computed tomograpic scan showing a calcified stone causing bile duct obstruction with the proximal end of the stent just below the stone .
residual contrast from the ercp is present in the gallbladder ( somatom volume zoom , 4 slice , siemens ag , erlangen , germany ) .
the gallbladder ( gb ) has been dissected from the liver retrograde , and the liver retractor is in place .
the arrow points to surgicel ( ethicon , somerville , nj , usa ) in the gallbladder bed of the liver .
laparoscopic ultrasound image showing the impacted stone ( arrow ) in a thickwalled duct ( aloka inc .
choledochoscopy using a 5-mm scope showing a clear bile duct with the stent visible ( insert ) .
ms is a rare cause of jaundice due to extrinsic compression of the chd and is present in approximately 0.35% of cholecystectomies .
many series report much higher rates ; however , this may represent differing views on what constitutes a case of ms .
kwon and inui recently reported a prevalence of 1.2% , but only a third of their patients were jaundiced , and despite this the majority still underwent open surgery .
similarly , planned open operation for mirizzi syndrome is accepted and , in fact , is still advocated by many .
many surgeons do not view conversion as detrimental and therefore do not persist laparoscopically when cholecystectomy is difficult .
conversion or an open operation allows the use of proprioception or the touch of the surgeon 's hand and is generally accepted as a way to improve the safety of any operation , especially one in which severe inflammation is present . to replicate this ,
however , open surgery is associated with significant short- and long - term morbidity , and a difficult operation is not necessarily easier or safer when performed open .
a degree of tactility is possible via instruments although not by currently available robotic systems , and laparoscopic ultrasound is very useful in stone disease .
it is generally accepted that an increased risk of bile duct injury exists during surgery for ms , and laparoscopic surgery may increase this risk .
operative cholangiography is advocated to improve the safety of cholecystectomy , but an accurate transcystic cholangiogram will not be possible in ms . a standard technique in open surgery for the difficult laparoscopic cholecystectomy was the fundus - first approach .
this can be replicated in laparoscopic surgery by the use of a liver retractor and means that exposure does not rely on traction on the fundus of the gallbladder . in ms ,
the gallbladder is often fibrosed and contracted so that fundic traction gives relatively poor exposure of the hepatobiliary triangle .
also once the gallbladder is freed from the liver , the obliterated calot 's triangle can be more easily evaluated . the magnified view combined with modern instrumentation
ercp may provide definitive treatment for ms ; however , the impacted stone can create real difficulties .
more often , ercp is used to make the diagnosis and insert a stent to alleviate the jaundice and allow planning of an elective operation .
if ercp is to be used as definitive treatment , sophisticated techniques may be needed for these cases , including the use of a mother and baby scope and electrohydraulic or laser lithotripsy . in the case
described herein , i could have tried to break up the stone with an over the wire mechanical lithotripter , although engaging the stone in the basket may have been difficult .
alternatively , a mother and baby scope and contact lithotripsy ( laser or electrohydraulic ) could have been tried but would not have been possible in my opinion because the actual cbd was not dilated and may not have taken the 5-mm baby scope .
because i was planning to remove the gallbladder anyway , i preferred to leave the choledochal sphincter intact to avoid longer term risk of choledocholithiasis from a colonized biliary tract and papillary stenosis . in this case , i could not stent the obstruction from below but a percutaneous transhepatic approach could have been used .
this would have been relatively straightforward as the hepatic ducts were dilated and might have been a useful strategy if this patient had been unfit for surgery .
there is purportedly a 5 times higher rate of gallbladder malignancy in mirizzi sydrome compared with that in uncomplicated gallstone disease .
prasad et al found 5.3% of patients with ms had gallbladder cancer compared with 1% in non - ms cases , and most were diagnosed on histology after cholecystectomy .
if the patient is fit for surgery , the optimal management of ms should include cholecystectomy .
the technique described leaves the choledochal sphincter intact and does not require intraoperative antegrade stenting or use of t - tubes . in ms as described herein , it would not have been easy to insert these tubes properly as the opening is into the cystic duct rather than the bile duct proper .
the preoperative endoscopically placed transpapillary stent temporarily overcame the outflow resistance of the sphincter and allowed safe primary duct closure although periductal drainage was needed .
in ms , even if an operation is planned , preoperative ercp and stenting should be considered because this approach should simplify and improve the safety of the operation .
in addition , the fitness of the patient for surgery and the complexity of the pathology are important for planning the therapeutic approach . in my opinion , ms should ideally be treated by a combination of ercp and stenting with laparosocopic
stenting overcomes the resistance of the choledochal sphincter , and even if accurate closure of the opening in a friable and inflamed duct is not possible , it should avoid the development of a significant biliary fistula .
careful planning combined with modern equipment should allow most cases to be managed without t - tubes , destruction of the choledochal sphincter , or open surgery . | background : mirizzi syndrome is a rare complication of cholecystolithiasis characterized by jaundice due to compression of the common hepatic duct . the diagnosis may not be immediately apparent , and management is controversial with open surgery still recommended by some authors.method:a case is detailed herein of a 67-year - old man who presented with abdominal pain , fever , and jaundice .
a dilated bile duct was found on ultrasound , but the gallbladder could not be seen .
the diagnosis of mirizzi syndrome was made at ercp , and a stent was placed through the papilla .
laparoscopic retrograde ( fundus first ) cholecystectomy was carried out utilizing a laparoscopic liver retractor.results:in this particular case , it was not possible at ercp to get a guidewire and stent past the obstruction .
a stent was left through the papilla , below the obstruction and this allowed primary duct closure during surgery.conclusion:acute mirizzi syndrome should be suspected when a patient presents with acute cholecystitis and jaundice with dilated intrahepatic ducts on ultrasound .
ercp is useful to confirm the diagnosis and allows stenting to alleviate the jaundice and facilitate the subsequent operation .
laparoscopic ultrasound is useful to locate the impacted stone and to partially replicate the touch of the surgeon 's hand , which is not available in laparoscopic surgery . |
dirofilaria immitis ( the canine heartworm ) is a filarial nematode naturally hosted by dogs , cats , foxes , muskrat , raccoons , and bears .
adult worms live in the right heart of these animals and produce a debilitating disease .
this filarial nematode is transmited by mosquitoes . when an infected mosquito takes blood from a human , the larvae of d. immitis invade and develop for a time in cutaneous tissues , migrate to the right heart ventricle where they eventually die .
the fragments of worms may reach the pulmonary arteries and lodge in a small caliber vessel to cause infarction or embolism events .
patients with pulmonary dirofilariasis are usually asymptomatic ; however , occurrence of cough , chest pain , hemoptysis , and wheezing has been described in some patients .
eosinophilia , commonly relevant in parasite infections , are present only in 15% of patients with pulmonary dirofilariasis . usually ,
a non - calcified , peripheral coin lesion or nodule is seen in chest x - ray or computed tomography ( ct ) .
pathologically , spherical subpleural infarct with a central thrombosed artery containing the parasite in stage of degeneration is most commonly seen [ 4 - 6 ] . in the republic of korea ( = korea ) , the first case of human pulmonary dirofilariasis was reported in 2000 . in this report
a 48-year - old male was referred to seoul national university hospital after a routine check - up at a local hospital due to abnormal findings seen in chest ct .
he was an ex - smoker ( quit 10 years ago ) and was a social drinker .
he had a pet dog , but the dog died 3 years ago from unidentified parasite infection .
complete blood cell count showed a white blood cell count of 6.8610/l ( normal : 4.0 - 10.010/l ) , a hemoglobin concentration of 14.5 g / dl ( normal : 14 - 18 g / dl ) , and a platelet count 26710/l ( normal : 130 - 45010/l ) .
the differential count showed neutrophils at 36.1% , lymphocytes at 51.2% , and eosinophils at 1.5% .
the c - reactive protein level was 0.02 mg / dl ( normal : 0.5 mg / dl ) .
serum sodium , potassium , creatinine , magnesium , calcium , and liver function test were all within normal limits .
the spirometry was normal ; fvc 4.95l ( 105% of predicted ) , fev1 3.77l ( 107% of predicted ) , and fev1/fvc ( 76% ) .
the chest ct showed a 1.21.0 cm sized subpleural solitary pulmonary nodule at the right lower lobe .
although it looked as a benign inflammatory nodule such as cryptococcal infection , malignancy could not be excluded ( fig .
on positron emission tomography ( pet ) image , the small subpleural nodule at the right lower lobe showed minimal increased fludeoxyglucose ( fdg ) uptake ( standardized uptake value [ suv ] 1.25 ) ( fig .
at first we considered a percutaneous needle biopsy for diagnosis , but biopsy was considered not technically feasible due to the location of the nodule .
macroscopic examination of the resected mass revealed a gray , firm , and well - demarcated tumor , measuring 1.21.00.7 cm , with slight pleural indentation and central necrosis , located in the right lower lobe of the lung .
microscopically , chronic granulomatous inflammation which was composed of coagulation necrosis with rim of fibrous tissues and granulations was recognized . in the center of the necrotic nodules ,
the parasite was a nematode larva and had prominent internal cuticular ridges and thick cuticle , a well - developed muscle layer , an intestinal tube , and uterine tubules .
the diameter of the worm was approximately 240 m . based on these morphologic features ,
after surgical resection , the patient was discharged without any complications and is being followed up in healthy conditions .
human pulmonary dirofilariasis was first reported in 1887 , when de magalhes documented the discovery of a single male and a single female filarial worm in the left ventricle of a boy from rio de janeiro , brazil . in 1961 , dashiell reported the first case involving the lung .
usually d. immitis invades the lungs in human cases , but reports of human dirofilariasis involving other organs like the subconjunctiva , liver , and subcutaneous sites are also available .
another case of dirofilariasis in the liver reported in korea was actually a misdiagnosis of hepatic capillariasis .
the chest ct images and pathologic findings of our case were similar to the first korean dirofilariasis case .
however , our case is the first to show additional pet imaging . in our case ,
pet - ct was performed for differential diagnosis of malignancy , and the pet - ct showed a small subpleural nodule at the right lower lobe with minimal increased fdg uptake ( suv 1.25 ) . in the life cycle of d. immitis , the dog is the principal host , though other suitable hosts include cats , wolves , coyotes , and foxes .
are the normal vector - intermediate host , but more than 60 species of mosquitoes in 6 genera are capable of propagating the development of d. immitis . in the usual life cycle ,
the sexually mature female worm resides in the dog 's right ventricle and sheds thousands of microfilariae into the blood stream .
when a mosquito bites the dog , it ingests the microfilariae . over the next 10 to 16 days ,
human are the " dead - end host " since larvae can not develop into adults in humans . in our case ,
human pulmonary dirofilariasis is usually discovered during routine health examinations because it does not cause notable symptoms .
it is seen as a well - circumscribed , non - calcified , peripheral coin lesion or nodule .
usually , the pulmonary lesion is a spherical nodule that upon gross examination appears as a well - circumscribed , spherical , grayish - yellow nodule 1 to 3 cm in diameter , surrounded by normal lung parenchyma . on microscopic examinations
, there is a central core of necrosis surrounded by a granulomatous zone of tissues .
in our case , the nodule was chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations .
dirofilaria infections can be diagnosed on the basis of worm morphology , such as the thick multilayer cuticle with lateral internal ridges , large lateral cords , and coelomyarian musculature . in cross sections , d. immitis measures 100 - 300
m in diameter and possesses a multilayered curicle with a thickness of 10 - 25 m .
the present case showed worm structures such as prominent internal cuticular ridges , no external cuticular ridges , thick cuticle , well - developed muscle layer , and presence of an intestinal tube .
the diameter of the worm was 240 m , which was compatible with a d. immitis immature female worm . | dirofilariasis is a rare disease in humans .
we report here a case of a 48-year - old male who was diagnosed with pulmonary dirofilariasis in korea . on chest radiographs ,
a coin lesion of 1 cm in diameter was shown .
although it looked like a benign inflammatory nodule , malignancy could not be excluded .
so , the nodule was resected by video - assisted thoracic surgery .
pathologically , chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations was seen . in the center of the necrotic nodules ,
a degenerating parasitic organism was found .
the parasite had prominent internal cuticular ridges and thick cuticle , a well - developed muscle layer , an intestinal tube , and uterine tubules .
the parasite was diagnosed as an immature female worm of dirofilaria immitis .
this is the second reported case of human pulmonary dirofilariasis in korea . |
the maxillary posterior area contains cancellous bones with low bone density and thin cortical bones , the quantity and quality of which are lower than those of the mandibular bone .
this is due to the small implant - to - bone contact area and the inferior bone quality.123 moreover , the severe bone loss caused by sinus pneumatization or chronic periodontitis , which leads to a reduction in the height of the remaining residual ridge , can further increase the implant failure rate.4 in addition , the occlusal load in the posterior area is 3 - 5 times higher than that in the anterior area.5 to address these problems , various bone graft techniques to augment the insufficient bone volume and thereby ensure the success of maxillary posterior implants have been proposed.6 such surgical methods have been accepted as clinically meaningful procedures .
however , these procedures have a number of disadvantages , such as the invasiveness of the surgery , high cost , and long treatment duration after bone graft and implant installation.6 in addition , because bone resorption occurs after a bone graft , the increase in vertical height is unpredictable
. microscopic studies by wallace et al.7 suggested that it was difficult to obtain bones with optimal strength from a bone graft .
rosen et al.8 asserted that the success rate of the implant placement after the bone graft depended on the residual ridge height .
this was supported by aka et al.9 who showed that stress applied on the implant was concentrated on the upper part of the implant and that , hence , the stress was not transferred to the grafted bone .
given the above facts and the disadvantages , success rate of bone grafting in a severely atrophied maxilla is not very promising .
studies have reported that this method may allow new bone formation without the need for bone graft.1011 however , winter et al.12 stated that the amount of new bone generated by sinus augmentation alone was insufficient and the results were not satisfactory considering the complexity of the technique .
this approach offers many advantages , which include a shorter treatment time , less cost , fewer complications in patients , and minimal surgical invasiveness . since the introduction of short implant for installation in atrophied residual ridge,13 many previous studies have defined a short implant as measuring 7 mm or less.141516 although short implants have been reported to have a lower survival rate than standard implants due to the small bone - contact area,1415 their survival rates have increased gradually with advancements in surface treatment technologies.16 as a finite element analysis ( fea ) shows that stress transfer at the implant - bone interface is limited to the upper 2 - 3 mm , the long implant is thought to be biomechanically unnecessary.917 therefore , a short implant may be used for achieving primary implant stability in the case of unfavorable bone quantity and may be used as an alternative for surgical approaches such as bone graft or sinus augmentation .
replacement of a missing molar with a single implant may cause various mechanical failures such as screw loosening , screw fracture , and implant fracture.1819 in a severely atrophied maxillary posterior area , using a single wide implant or splinting multiple implants is a way to overcome the unfavorable crown / root ratio of short implants.202122 two - implant - supported maxillary molars with implants installed in buccal and palatal positions were suggested by balshi and wolfinger.23 the 2 short implants ( 2si , fig .
1 ) technique can be used to reconstruct a multi - rooted molar by installing 2 short narrow implants without the need for an additional surgical approach .
the 2si approach is a minimally invasive procedure that solely utilizes residual bones to overcome unfavorable bone quantity and quality in the maxillary posterior area .
it can yield stable treatment results while minimizing the need for additional procedures and reducing the cost and treatment time .
a 5-year clinical study demonstrated the clinical success of 2sis.24 the purpose of this study was to compare the stress distribution of various types of 2sis that were installed and restored in severely atrophied maxillary molar sites .
an internal connection implant with an 11-degree taper interface was used ( gs , osstem , seoul , korea ) . internal connection implants ( length , 7.0 mm ) with narrow platform ( np 3.5 mm ) , regular platform ( rp 4.0 mm ) , and wide platform ( wp 5.0 mm ) were modeled .
three - dimensional image data of the human maxillary first molar from ct images were transformed into fea meshes and the connecting area between the crown and implant was smoothened .
a gold crown on the abutment ( rigid , osstem , seoul , korea ) was connected with the implant .
abutment for each implant diameter was connected , and the occlusal surface area of the definitive prosthesis was constructed to have the same shape . an implant was designed to be installed on the missing maxillary first molar area on the maxilla with a pneumatized sinus , 0.5 mm cortical bone on top and bottom , and 3 mm cancellous bone ( fig .
2 ) . for the boundary conditions , bottom and mesio - distal parts of the maxillary bone
the mechanical properties of the bone , gold , and titanium were set as previously described ( table 1).2526 the implant bone interface was constructed with 100% contact for complete stress transfer to the surrounding bone .
they were broadly categorized into 3 groups of 2si and 4 groups of single implants .
the 2si groups were categorized into 1 ) np oblique ( two narrow implants were obliquely installed ) , 2 ) np vertical ( two narrow implants were installed in palatal residual bone due to the resorption of buccal bone ) , and 3 ) np horizontal ( two narrow implants were installed in distal residual bone due to the mesial bone resorption ) .
single implant installation groups were set to 1 ) rp - cantilever ( a rp implant was installed in stepped bone due to resorption of the mesial bone ) , 2 ) wp - cantilever ( a wp implant was installed in stepped bone due to resorption of the mesial bone ) , 3 ) rp - center ( a rp implant was installed in a flat bone ) , and 4 ) wp - center ( a wp implant was installed in a flat bone ) . meshing and pre - processing were performed using hyper - mesh 10.0 ( altair engineering inc . , troy , mi , usa ) and visual crash for pam version 10.5 ( esi group , paris , france ) .
the average occlusal force , a 250 n static axial load , was applied to the center of the occlusal surface ( axial loading ) . to simulate an oblique loading condition , a 250 n
oblique load with a 45 angulation from the implant axis was applied on the center of the buccal cusp ( oblique loading ) .
the von mises stress was measured in the interface of implant - bone , implant / abutment complex , and at the bone of the peri - implant area in all models . to assess the stress distribution
the processing and post - processing procedures were performed using pam - medysa v2014 ( esi group , paris , france ) and hyper - view v10.0 ( altair engineering inc . , troy , mi , usa ) .
analyses of stress distribution in the implant - bone interface of an implant in the buccal side ( np-1 ) showed an evenly distributed stress pattern in the np - horizontal and np - oblique groups , whereas the np - vertical group exhibited a relatively high stress pattern in the top and center areas of the interface ( fig .
similar stress distribution was observed in the np - horizontal and np - oblique groups , while a different stress pattern was observed in the np - vertical group ( fig .
stress distribution at the bone of the peri - implant area showed a different pattern to that observed in the interface .
the np - horizontal and np - vertical groups showed a large stress concentration in stepped areas between different bone levels , while an even stress distribution was observed in the np - oblique group with flat bone level ( fig .
few differences in stress distribution of the implant / abutment complex were observed in each group and all stress was concentrated on the interface between the abutment and inner surface of implant ( fig .
4d ) . when oblique loading was applied , similar stress distribution was observed at the bone - implant interface of the buccal ( np-1 ) and palatal ( np-2 ) implants in the np - horizontal and np - oblique groups ( fig .
although the np - vertical group showed slightly higher stress concentration , the difference was not as evident as with axial loading ( fig .
an evenly distributed stress pattern at the peri - implant bone was noticed in the np - oblique group compared with the np - vertical or np - horizontal groups ( fig .
the stress distribution of the implant / abutment complex showed virtually no difference in each group , and the amount and location of stress concentrations were similar to those in axial loading ( fig .
when axial loading was applied , the wp implant showed lower and evenly distributed stress at the implant - bone interface than the rp implant ( fig .
6a ) . however , the stress difference was not large between the center and cantilever positions .
as for the stress on the peri - implant bone , a more even stress distribution was observed in the center position implant than in the cantilever position implant .
lower stress was observed in the wp implant than in the rp implant ( fig .
stress patterns at the implant / abutment complex of single implants were similar to that of the 2si groups ( fig .
when oblique loading was applied , the largest von mises stress was observed in the rp implant .
the wp implant showed a relatively even stress distribution pattern and had a similar level of von mises stress to that of 2si groups ( fig .
the stress distribution on the peri - implant bones was similar to that in axial loading but the level of stress was higher .
stress exerted on the implant / abutment complex was very large ; a high amount of stress was placed on the abutment and implant in both rp and wp implants .
additionally , larger and wider stress distribution was observed in the implant / abutment contact area on the opposite side of the loading point ( fig .
the stress distribution at the implant - bone interface was compared by categorizing the bone levels into two ( flat and stepped ) .
when axial loading was applied , higher von mises stress was observed on the implant - bone interface in the rp implant than in the wp implant or 2si implants .
the highest stress was concentrated on the region with discontinuity between the different bone levels ( stepped area ) ( fig .
the 2si groups showed the lowest von mises stress compared with the rp and wp implants .
the area between the implants showed lower stress distribution than the outer side of the implant in 2si group .
the 2sis showed the most even stress distribution and rp implant showed the largest stress pattern on the peri - implant bones ( fig .
when applying oblique loading , the largest stress pattern was noted at the buccal side .
higher stress was observed in the rp implant than in the wp implant or 2 np implants ( fig .
stress distribution of the peri - implant bones was relatively small and even in the 2si groups .
a high level of stress was concentrated in the stepped area of the bone ( fig .
differences in stress distribution based on implant diameter , number , or position could be observed , but no differences related to types of bone levels were observed ( flat bone vs. stepped bone ) ( fig .
to identify the biomechanical effectiveness of 2sis , various bone levels and bone morphologies were assumed and implants were positioned on the basis of the bone condition .
the residual maxillary bone bed was designed with a total length of 4.0 mm and 0.5 mm of cortical bone on the top and bottom for bicortical engagement .
hence , when load was applied on the implant prosthesis , a large stress concentration was observed in certain regions on the bottom cortical bone located on the maxillary sinus side , unlike the result in another fea study.9 similarly , in clinical situations , stress may be concentrated on the bottom cortical bone as well as on the top portion in the case of severely atrophied bone . in this study ,
the von mises stress distribution on the bone away from the implant - bone interface was also considered .
this is because it was difficult to estimate the effect of stress distribution solely based on the stress pattern at the localized implant - bone interface in case of a severely atrophied bone .
in addition , stress on the implant - abutment complex was analyzed to estimate the risk of fracture in a narrow implant .
the single rp implant group showed the highest level of stress at the implant - bone interface .
as the diameter of implant used for restoring the maxillary molar is larger in wp , stress is more evenly distributed and general stress amount ( in mpa ) tends to be lower .
however , two np implants exhibited more favorable stress distribution than a single wide implant ; this confirms the biomechanical advantage of 2sis . in the 2si groups , the level of stress was not much different between palatal and buccal positioned implants .
meanwhile , np - vertical , which is a buccal - only or palatal - only positioned condition , exhibited a higher level of stress in the form of a bending moment when axial loading was applied .
however , under oblique loading , a similar level of stress was exhibited by the three implant positions ( np - oblique , np - horizontal , and np - vertical ) .
these trends arose because oblique loading induced lopsided loading on the buccal or palatal sides .
therefore , the bending moment could be generated in a variously positioned implant , whose diameter is smaller than the roots of a natural tooth .
the stress distributions in the center and cantilever positions of the rp and wp implants did not show a large difference .
it may be because adjacent teeth were not modeled and both axial and oblique loading were applied on the mesio - distal center .
these results are consistent with previous studies , which showed that the buccal cantilever was the most unfavorable and the mesio - distal cantilever was unfavorable only in terms of oral hygiene.2728 stress distribution of the peri - implant bone showed similar trends , where von mises stress was lower in implants of 2si groups than in rp or wp implants .
this may be explained by the contact area between the implant and bone tissue , as placing multiple np implants is an effective way to increase the contact area .
since installation of a wp implant is difficult in atrophied bones , 2sis can provide better initial osseointegration and its subsequent maintenance with effective stress distribution .
stress on the implant / abutment complex was lower than that in the implant - bone interface or peri - implant bones .
the stress was concentrated on the contacting surface of abutment and the inner part of implant when oblique loading was applied on rp or wp implants .
clinically , fractures are common in the geometric discontinuity between the upper conical part and the lower hex part of the abutment in internal conical connection implants.29 moreover , multiple implants can be advantageous since single molar implants exhibit occasional mechanical failures such as screw loosening , screw fracture , and implant fracture,28 and the oblique loading generated even in physiologic mastication can be detrimental to single maxillary implants . in a previous clinical study on 2sis ,
none of the np implants using the 2si method was fractured.24 the trend of the presence of higher von mises stress around rp implant than around wp or np implants did not differ by the bone levels .
prominently , the highest level of stress was exhibited on the stepped area of the bones .
this can be viewed as a limitation of the fea study ; stress concentration on the discontinuous area of features is unavoidable in the interpretation of the fea . in clinical situations ,
however , these phenomena are not likely to occur as bone loss occurs continuously . in preliminary modeling ,
however , real bone images can distort the result when comparing the effect of implant diameter and number .
therefore , over - interpretation of the importance of stress concentration at the discontinuous region should be avoided .
as an alternative to bone graft on a severely atrophied maxillary posterior area , single wide implant or splinting of multiple implants can be performed.2123 the results from the this study showed that , multiple implants , even with a short length and narrow diameter , offer a favorable implant - bone interface , peri - implant bone , and implant - abutment complex .
the use of 2sis , which place np implants in the most dense bone area in the buccal and palatal sides and utilize residual bones per se without surgical difficulties , is a procedure allowing enhanced osseointegration and favorable stress distribution .
however , 2si could not be used in the case with narrow mesio - distal space .
the results from the fea study can be the consequence of restricted stress transfer within the 2 - 3 mm of the top of the bone.9 further , as adjacent teeth were not predetermined , the replication was not identical to the actual situation .
moreover , clinical efficacy can not be validated solely by a biomechanical study , as only a relative comparison is possible . however , this study is important in that it supports the findings of the clinical study that showed favorable clinical success rates of 180 2sis.24
the stress distributions based on various bone levels and implant installation conditions in a severely atrophied maxillary posterior area were analyzed .
when axial loading or oblique loading was applied , the highest level of stress was observed on the implant - bone interface and peri - implant bones in a single rp implant and the lowest stress distribution was exhibited in 2si conditions .
the highest level of stress was concentrated on the implant / abutment - contacting surface of the single wide implant and the lowest level of stress was on the implant / abutment complex of 2sis . | purposethe aim of this study was to investigate the stress distribution of 2-short implants ( 2sis ) installed in a severely atrophic maxillary molar site.materials and methodsthree different diameters of internal connection implants were modeled : narrow platform ( np ) , regular platform ( rp ) , and wide platform ( wp ) .
the maxillary first molars were restored with one implant or two short implants .
three 2si models ( np - oblique , np - vertical , and np - horizontal ) and four single implant models ( rp and wp in a centered or cantilevered position ) were used .
axial and oblique loadings were applied on the occlusal surface of the crown .
the von mises stress values were measured at the bone - implant , peri - implant bone , and implant / abutment complex.resultsthe highest stress distribution at the bone - implant interface and the peri - implant bone was noticed in the rp group , and the lowest stress distribution was observed in the 2si groups .
cantilevered position showed unfavorable stress distribution with axial loading .
2si types did not affect the stress distribution in oblique loading .
the number and installation positions of the implant , rather than the bone level , influenced the stress distribution of 2sis .
the implant / abutment complex of wp presented the highest stress concentration while that of 2sis showed the lowest stress concentration.conclusion2sis may be useful for achieving stable stress distribution on the surrounding bone and implant - abutment complex in the atrophic posterior maxilla . |
hydatid disease ( echionococcosis ; hydatidosis ) is a parasitic disease , endemic in many sheep raising countries ( australia , south america , mediterranean countries , russia , middle east , and central asia ) .
adult cestodes of echinococcus granulosus inhabit the small intestine of a definitive host ( dogs , foxes , and wolves ) and produce infective eggs . through the feces , either cestode segments or free eggs
these are ungulates ( sheep , goats , pigs , and horses ) but in some cases accidental ingestion of eggs , usually shed by dogs , could lead to human cystic echinococcosis ( ce ) .
contaminated food , water , or hand - to - mouth transmission are usual modes of infection in humans [ 2 - 4 ] . so far ,
once ingested by the intermediate host , in its intestinal tract , parasite eggs transform into the oncosphere .
oncospheres penetrate the intestinal wall , enters the portal vein , and reaches the liver where they usually are trapped and become the hydatid cyst .
liver involvement has been described as the most common location of ce , varying from 59% to 78% of all cases [ 1,5 - 8 ] .
some larvae pass the liver and reach the lungs where they develop into pulmonary hydatidosis .
lung involvement is present in 15 - 27% of all cases [ 1,5 - 8 ] .
not so often , larvae may pass these filters and form cysts in the brain , skeletal muscles , bones , kidneys , spleen , or other tissues .
theoretically , they can occur at any site except the teeth , nails , and hairs .
epidemiological studies have been conducted in some countries , while in serbia , montenegro , bih , and albania no information is available about the precise incidence in humans .
the aim of this retrospective study was to investigate the skeletal manifestations of hydatid disease in serbia , it 's demographic distribution , site involvement , complications , and bone tissue comorbidities .
the study was conducted by reviewing the medical database of institute for pathology ( faculty of medicine in belgrade ) which is a reference institution in serbia for bone pathology .
materials in the study comprised all the cases with histologically verified bone hydatidosis during the period between 1971 and 2010 in the territory of serbia .
data obtained from medical records included the age at the moment of diagnosis , sex , localization of hydatid cyst , copresence of hydatid cyst in localizations other than the bone tissue , presence of skeletal complications , bone comorbidities , and diagnosis based on radiographies . in order to standardize the data ,
the radiological assessment was made on standard radiography , having in mind the length of the study period and diagnostic procedures available for that time .
changes in the bone structure were classified as tumefaction ( sharply or unshapely demarcated area of altered bone structure ) , cysts ( sharply demarcated lytic bone lesion with geographical borders ) , or fractures ( in cases where fracture line occured ) . in some patients , altered bone structure was able to be classified as cyst or tumefaction preceding the pathological fracture , while in other cases , no alteration of the bone structure was radiologically visible aside of the fracture line .
the age of the patients at the time of diagnosis was from 7 to 77 years .
the mean age of all patients was 40.918.8 years . in order to explore the age distribution of ce ,
we grouped patients into 5 age categories : i ( 1 - 18 years ) , ii ( 19 - 36 years ) , iii ( 37 - 54 years ) , iv ( 55 - 73 years ) , and v ( > 73 years ) . in group
the mean age of adult patients ( more than 18 years old ) was 44.716.6 years .
a total of 24 male patients presented 58.5% of all cases , while 17 female patients presented 41.5% .
the spine was the most commonly involved site with the predominance of the thoracic segment .
analysis of standard radiographies revealed that fracture was by far the predominant radiological diagnosis , followed by tumefaction and cyst ( table 2 ) .
pain was the symptom in 41.5% of patients , while some patients demonstrated complications such as paraplegia ( 22.0% ) , pathologic fracture ( 48.8% ) , and scoliosis ( 9.8% ) .
the pathological fracture was most frequently found in the spine ( 75.0% ) followed by the femur ( 20.0% ) and tibia ( 5.0% ) . however
in addition to the bone involvement , 2 patients demonstrated extraskeletal hydatid cysts : 1 in the liver and 1 in the lungs .
bone tissue comorbidities refer to bone tuberculosis in a patient with spinal hydatid disease ( 2.4% ) and to presence of fistula detected in 2 patients ( 4.9% ) , 1 with spinal and 1 with pelvic hydatidosis .
serbia is recognized as an endemic country for ce , but so far , no epidemiological study has been conducted . since echionococcosis is subjected to an obligatory disease for reporting , the data on the number of reported patients were available through the annual report on infectious diseases on the territory of republic of serbia published by serbian institute for public health , serbia . in the study period , a total of 727 patients with ce
the frequency was relatively high compared to the reports from other endemic countries , being 0.5 - 4.0% .
this frequency can be explained at least partially by a larger total number of patients due to careless reporting of echionococcosis by physicians , which was even noted in the official annual reports from 1978 to 1989 of serbian institute for public health , serbia .
studies in other endemic countries showed that the prevalence of ce increases with age , resulting that the most of the patients belong to the adult population .
the mean age at the moment of diagnosis , including all localizations , was from 33 to 52 years [ 13 - 18 ] . in our study ,
the mean age of the patients with skeletal manifestation of ce was 40.918.8 years , but among the adults the mean age was 44.716.6 .
higher frequency in aged people could be due to the fact that older people have more chances to be infected with e. granulosus during life , and also , the clinical signs of ce are more probable to appear later in life , as the disease progresses . as it was reported in previous studies [ 15,19 - 21 ] , no significant difference between sex was found . in our study ,
12.2% of patients were under 18 years , with the mean age being 13.04.2 years .
it was consistent with the results of a previous report on the frequency of echinococcosis among children in serbia .
although boys to girls ratio was 1.5:1.0 , no significant difference in the positive rate between sex was found . some other studies reported slight predominance of boys , which could probably be explained by their outdoor playing , closer contact with animals , and low level of personal hygiene , compared to girls .
symptoms associated with bone ce are not specific and often missing , making the diagnosis difficult and sometimes overlooked .
our results showed that 19.5% of patients did not report any symptoms and did not develop any complications .
these asymptomatic patients are not surprising in consideration that hydatid disease is anywhere in the body and often discovered as an incidental finding .
radiological diagnosis of osseous hydatid disease is also difficult because of non - specific findings .
radiography usually reveals single or multiple osteolytic lesions and in some cases shows cortical thinning , osteosclerosis ( fig .
intraosseous foci of hydatid disease predominate in spongious bone tissues and consist of small , separate , and thin - walled cysts .
while these cysts expand , they destroy the surrounding trabeculae and can reach a considerable size . due to cyst enlargement and consequent cortical thinning
, pathological fractures may occur . in the case of connective tissue proliferation and bone sclerosis ,
presentation of ce can be unclear , consisting of mixed radiological signs ( table 2 ) .
therefore , preoperative diagnosis of skeletal hydatid disease is difficult , and conclusive diagnosis is often made intraoperatively and by histological verification [ 26 - 28 ] .
histological examination of the cyst wall usually reveals an outer chitinous ( or fibrous laminar ) and an inner germinal layer , surrounded by either granulation tissue or fibrous capsule ( fig .
osseous ce is almost invariably related to primary infection and is not the result of extension from neighboring soft tissue lesions . however , some authors report that around 30 - 45% of bone hydatidosis is co - present with the hydatid cysts in other localizations .
we have reported just in 4.9% of cases the presence of hydatid disease in the liver or lungs ; however , there is no statement in our database about the radiological surveys for the possible diagnoses of hepatic or pulmonary hydatidosis .
therefore , it is impossible to exclude the presence of secondary cysts without a whole body scan . in patients with bone ce ,
spine is the most commonly affected site with the frequency of appearance 35 - 50% [ 24,29 , 33 - 36 ] .
our findings ( table 1 ) are in concordance with the ones reported by other authors , with the exception of the cervical region .
it is considered to be relatively silent , slowly progressive disease with a latent period of many years .
early symptoms appear when the small cyst expands and produces significant mechanical compression on surrounding vital structures . at this stage ,
intermittent pain can be present as a symptom . as the disease progresses and the cyst enlarges , patient starts suffering from persistent backache , gradually leading to severe neurologic deficit and varying degrees of weakness of limbs .
pain was present in 50% of patients with spinal hydatidosis in our study ( table 3 ) with the frequency of appearance similar to the ones reported by other authors .
paraplegia is the most serious complication of vertebral hydatidosis appearing in up to 75% of patients with spinal ec .
this compressive effect of spinal ce was present in 37.5% of patients in our study ( table 3 ) .
paraplegia is a neurological complication , occurring as a result of invasive intradural and extradural growth of the cyst .
cysts invade the spinal canal either by direct compression or by ischemic changes in the spinal cord .
radicular pain usually precedes paraparesis by few weeks , progressing rapidly to paraplegia in the thoracic spine , but slowly in the lumbar spine .
our results show that , in some patients , paraplegia can be developed as a compressive effect not just of the cyst , but of the spinal pathological fracture that ce can lead to , as well .
all these data indicate that once formed in the spinal column , hydatid cyst is likely to result in paraplegia during the progression of the disease . in long - term hydatidosis , apart from pain and paraplegia , some cases can result in secondary infections and fistula formation .
. their presence can resolve the disease in the term of surgical treatment , while bacterial superinfection can lead to sterilization of the parasite , limiting the bone destruction .
interestingly , in our study , 1 male patient was reported with bone ce and tuberculosis super - infection . despite ce
shows the spinal tropism causing severe complications , the therapy of spinal ce is still poor .
they are applied in order to retard the recurrences and control the disease but radical surgery is still the keystone .
data from the literature reported that the recurrence rate of bone ce , even after several operations , varied from 30% to 100% .
moreover , the mortality rate of spinal ce is unacceptably high , varying 14 - 58% . when ce is affecting long bones , it is generally the metaphyseal region , extending later to the diaphyses and nearby bones .
the incidence of long bone involvement in our study ( table 1 ) was similar to the results reported by other authors .
therapy is surgical resection , performed if lesion is segmental , otherwise , amputation or disarticulation are the only solutions .
our results ( table 1 ) did not differ from the ones obtained by other authors .
it 's progression is infiltrating and diffuse and may lead to pain , swelling , and stiffness of the hip joint .
this disease is hematogenously seeded along the trabeculae and through the medullar canal , leading to extensive bone lesions .
our results reported that these lesions were mostly complicated with pathological fracture ( 48.8% of all cases ) .
our results reported primary involvement of the spine ( 75% ) , with the predominance of lumbar over thoracic segment . in patients with bone ce
, bone tissue destruction develops through 3 mechanisms ; as the consequence of the cystic lesion expansion and its compression on surrounding tissues , ischemic processes through compression and obstruction of nutrient vessels , and cellular processes like osteoclast proliferation around the compressed bone tissue . in the absence of pericyst formation ,
the wall of the cyst is thin and can not overcome the rigid bone nature .
therefore , the cyst can not assume it typical , spherical shape , and it enlarges irregularly along the path of least resistance . in patients with skeletal ce ,
bone destruction lead to its thinning , extension into soft tissues and , as the worse case , can lead to fracture .
therefore , some authors suggest that all the pathological fractures in endemic countries should be considered as a possible case of ce . | although serbia is recognized as an endemic country for echinococcosis , no information about precise incidence in humans has been available .
the aim of this study was to investigate the skeletal manifestations of hydatid disease in serbia .
this retrospective study was conducted by reviewing the medical database of institute for pathology ( faculty of medicine in belgrade ) , a reference institution for bone pathology in serbia .
we reported a total of 41 patients with bone cystic echinococcosis ( ce ) during the study period .
the mean age of 41 patients was 40.918.8 years . in 39% of patients ,
the fracture line was the only visible radiological sign , followed by cyst and tumefaction .
the spine was the most commonly involved skeletal site ( 55.8% ) , followed by the femur ( 18.6% ) , pelvis ( 13.9% ) , humerus ( 7.0% ) , rib ( 2.3% ) , and tibia ( 2.3% ) .
pain was the symptom in 41.5% of patients , while some patients demonstrated complications such as paraplegia ( 22.0% ) , pathologic fracture ( 48.8% ) , and scoliosis ( 9.8% ) .
the pathological fracture most frequently affected the spine ( 75.0% ) followed by the femur ( 20.0% ) and tibia ( 5.0% ) . however
, 19.5% of patients did n't develop any complication or symptom . in this study
, we showed that bone ce is not uncommon in serbian population .
as reported in the literature , therapy of bone ce is controversial and its results are poor . in order to improve the therapy outcome , early diagnosis ,
before symptoms and complications occur , can be contributive . |
workers ' health may be protected by his / her own preventive behavior , despite numerous health hazards in the workplace .
however , worker behavior is constrained by various workplace pressures such as working conditions , including what is considered to be accepted working behavior and overall morale of the working group .
based on the employment contract , every employer has a certain amount of control / power and the expectation that workers will follow work - related requests .
this may lead to adverse health effects in workers ; therefore , the government has implemented regulations to protect worker health , such as the occupational safety and health ( osh ) act .
adverse health effects induced by unexpected incidents may present as occupational injuries , but occupational diseases can appear due to long term continuous exposure to harmful chemicals , dust , noise , etc . to prevent occupational diseases , it is important to manage worker health via health monitoring for early detection of abnormal conditions .
it is very important for worker health management to monitor the level of worker exposure to hazards in the workplace ; for example , health check - ups including absorption of hazard materials , so called ' biological monitoring ' .
noise levels in the workplace should also be measured in order to prevent worker 's hearing loss .
if a worker is exposed to high levels of noise , he / she should be transferred to another work area without noise or provided with proper personal protective equipment ( ppe ) such as earplugs , etc .
as above , information needed for workers ' health management can be classified into three categories according to 3 questions : 1 ) what is the exact nature of the occupational hazards ?
3 ) what health problems are induced by these hazards ? these questions should be answered in order to manage workers ' health effectively and efficiently .
health management for workers is accomplished by two different strategies : 1 ) focusing on reduction of exposure to hazard factors in the workplace based on risk assessment using exposure information ; 2 ) the provision of health care services and compensation for workers with adverse health effects .
recently , psychosocial factors such as work - related stressors have been included as hazard factors in the workplace , and health promotion activities have become an important component of worker health management before symptoms and signs of occupational diseases manifest . in this article , we reviewed the various kinds of surveillance systems for occupational diseases in korea and resultant data , and suggest reformation of the workers ' health management system .
the workers ' compensation insurance system is one of the old systems and ensures a worker 's life and his / her family is secure after work - related accident .
workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .
now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,
only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .
industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .
occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .
work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .
data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act
the proportion of reported cases is very small ( under 1% of total occupational diseases ) .
kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases . until now
, some workers have been excluded from workers ' compensation insurance :
self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers
self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .
since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular , occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .
the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .
the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .
non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend .
specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .
the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .
there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .
yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation .
in particular , brain and heart disease and musculoskeletal disorders have increased since the 1990s and 2000s , respectively .
recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .
another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .
the first one was done only one time in 1998 for workers in manufacturing sectors .
the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .
the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .
the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .
every survey has included some questions about worker 's experiences of occupational disease . according to the wcs
, the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .
the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .
workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .
now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,
only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .
industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .
occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .
work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .
data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act
the proportion of reported cases is very small ( under 1% of total occupational diseases ) .
kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases .
until now , some workers have been excluded from workers ' compensation insurance :
self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers
self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .
since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular ,
occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .
the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .
the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .
non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend . specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .
the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .
there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .
yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation . in particular , brain and heart disease and musculoskeletal disorders
recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .
another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .
the first one was done only one time in 1998 for workers in manufacturing sectors .
the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .
the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .
the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .
according to the wcs , the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .
the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .
these institutions are examine workers by the request of employers based on the osh act mandatorily .
for example , health examinations are regulated by government in order to protect worker health through early detection of occupational diseases .
health examinations for worker health protection are classified into two different types : special health examination and general health examination .
the special health examinations are comprised of health screening tests for early detection of selective occupational diseases in workers exposed to specific hazard factors such as noise , dust , organic solvent , etc . the general health examination is a mass screening test for finding health problems including occupational diseases of workers without exposure to hazard factors .
the health examination is focused on early detection of occupational diseases , and its results are used as information for workplace management in order to protect worker health .
cases diagnosed as ' suspicious cases of occupational disease ' are recommended to be transferred to non - hazardous workplaces . some of them may be compensated based on diagnosis of occupational diseases through the workers ' compensation insurance system
. detection rate of ' suspicious cases of occupational diseases ' in the special health examination was 0.46% in 2008 , an increase from 2006 .
the detection rate of ' cases observed for occupational disease ' was 12.85% in 2008 with an increasing trend .
the health examination is defined as a screening test for occupational diseases , so there is no confirmatory diagnosis in the health examination process . '
cases to be observed for occupational diseases ' are the result of an examination showing marginal figures for ' suspicious cases for occupational diseases ' . based on results from the health examination ,
suspicious cases will be recommended to undergo medical treatment , change jobs or tasks for reduction of exposure to hazard workplace , etc .
cases to be observed are not in need of any immediate intervention ; however , these cases may be also recommended similar treatment , such as wearing personal protective equipment ( ppe ) , changing jobs or tasks , etc . among suspicious cases and cases
to be observed detected by the special health examination , hearing loss due to noise was the most prevalent ( 3 ) ( table 4 ) .
detection rate of ' suspicious cases of occupational diseases ' during the general health examination was 0.001% in 2008 , and has been very steady since 2004 . in contrast , the detection rate of ' suspicious cases of general diseases ' has shown fluctuations between 4 and 5% in the last ten years ( 3 ) ( table 5 ) .
health examinations are very effective in finding asymptomatic chronic diseases , such as pneumoconiosis , hearing loss and some poisonings .
however , it can barely detect symptomatic diseases with no signs , like asthma , and easily curable diseases like dermatitis because of the characteristics of the periodic screening test .
therefore , statistics from the health examination include mostly pneumoconiosis and noise - induced hearing loss ( 4 ) ( table 4 ) .
the working environment measurement institution , regulated by the osh act , is in charge of assessing and , ultimately , reducing the level of worker exposure to hazard factors in the workplace .
every employer should do working environment measurement in order to identify hazard factors in the workplace and the level of worker exposure to these factors .
the special health examination is based on working environment measurement , such as selection of the examinee .
if the level of worker exposure is higher than the occupational exposure level in korea , the employer should immediately act to reduce it .
the magnitude of occupational diseases and associated fatality rate in korea have gradually increased since 2007 .
the trend in fatality rate can be reflected by the trend in fatal pneumoconiosis and brain and heart disease , as these two categories of occupational diseases predominate total occupational diseases .
most pneumoconiosis cases were coal workers ' pneumoconiosis ; however , nowadays most coal mines have closed due to restructuring of the energy production system in korea .
other dominant fatal occupational diseases , such as brain and heart diseases , will also decrease because general health programs have focused on prevention of hypertension , one of the most important causal factors of brain and heart diseases in workers . in the general population , the morbidity rate of brain and heart diseases has shown a recent downward trend .
one is the causal factors of occupational diseases for prevention , and the other is affected workers of occupational diseases in order to rehabilitate them through recovery of the labor force .
occupational diseases are induced by worker exposure to various hazard factors in the workplace , such as physical , chemical , biological and psychosocial factors .
workers with abnormal conditions can detect acute occupational diseases through self - diagnosis just after exposure to high levels of hazard factors , such as lead , mercury , etc .
however , it is not so easy to identify abnormal conditions of chronic occupational diseases , such as asbestos - induced diseases .
furthermore , exposure to low levels of hazard factors may have long - term cumulative effects on worker health ( fig .
if there is no evidence of work - relatedness , no ill - health can be confirmed as an occupational disease .
work - relatedness may be classified into two different types : direct and indirect causality .
first , based on direct causality between work or work environment and ill - health , it is easy to determine work - relatedness , but it is very difficult to evaluate indirect work - relatedness .
the same condition diagnosed as occupational disease by a physician can also be categorized as non - work - related because a particular hazard factor can not be detected in the working history .
final determination of work - related illness or death in any individual case requires detailed occupational and non - occupational information ( 5 ) . in the united states
, there exists occupational health surveillance , such as death certificates , hospital discharge data , physician reports , laboratory reports , workers ' compensation reports , national surveys , employer surveillance programs and occupational health clinics ( 5 ) .
since 1998 , there were some suggestions for the introduction of a surveillance system for occupational diseases in korea because worker health examinations and work environment measurements had limitations for management of occupational diseases ( 6 , 7 ) .
the magnitude of occupational diseases in korea is assessed mainly by the workers ' compensation system .
statistics might exclude unclaimed occupational diseases and those of workers from the public sector and workers who are not covered by compensation insurance ( 4 ) .
this means that non - compensated cases can be neglected and latent cases can always emerge as compensated cases in the immediate future .
moreover , statistics based on compensated cases of occupational diseases are not stable because different acceptance criteria for work - relatedness have been applied by the korea labor welfare corporation ( former name of comwel ) and judiciary branch ( 8) . in korea , there are two different kinds of health insurance : national health insurance and workers ' compensation insurance .
every worker with occupational ill - health can be covered by either of these two .
if some occupational diseases cases are covered by the national health insurance system , official statistics on occupational diseases can not include these cases . in order to determine the correct number , all occupational diseases covered by the two different insurances
unfortunately , the national health insurance system has no screening and confirming system for occupational diseases .
occupational disease compensation will focus on the treatment , compensation , and rehabilitation as secondary or tertiary prevention .
national surveys of worker health can provide useful information about ill - health workers without treatment or compensation .
this information can be used to prevent occupational diseases by detection of at - risk workers as a target population for management of occupational diseases .
the primary prevention of occupational diseases and injuries depends more directly on hazard surveillance , since the identification of hazards enables their reduction prior to the onset of ill - health .
occupational health surveillance is less well developed than its health counterpart due to scarcity of data sources and , with the exception of the occupational safety and health administration and the national institute for occupational safety and health surveys in the usa , the proprietary and inaccessible nature of hazard data collected by employers ( 5 ) .
occupational health surveillance entails the systematic monitoring of health events and exposures in working populations in order to prevent and control occupational hazards and their associated diseases and injuries ( 5 ) .
medical surveillance refers to the ongoing application of medical tests and procedures to individual workers who may be at risk for occupational morbidity to determine whether an occupational disorder may be present ( 5 ) .
if an individual or population is exposed to a toxin with known effects , and the tests and procedures are highly targeted to detect the likely presence of one or more effects in these persons , then this surveillance activity is more aptly described as medical screening ( 9 ) . in occupational health , there are two kinds of surveillance activities , one is public health and medical surveillance and the other is hazard surveillance .
the main purpose of occupational health surveillance is to identify the incidence and prevalence of known occupational diseases and injuries ( 5 ) .
a second broad function is to identify individual cases of occupational disease and injury in order to find and evaluate other individuals from the same workplaces who may be at risk for similar disease and injury .
another purpose of case identification may be to assure that the affected individual receives appropriate clinical follow - up , an important consideration in view of the scarcity of clinical occupational medicine specialists ( 10 , 11 ) .
finally , occupational health surveillance is an important means of discovering new associations between occupational agents and accompanying diseases . discovery of rare diseases , patterns of common diseases , or suspicious exposure - disease associations through surveillance activities in the workplace can provide vital leads for a more conclusive scientific evaluation of the problem and possible verification of new occupational diseases ( 5 ) .
periodic medical screening and surveillance examinations are another frequent service provided by occupational medicine physicians .
the term screening focuses on the individual ; screening tests are tools for secondary prevention with the goal of early diagnosis and treatment of disease in exposed workers .
true ' surveillance ' aggregates information about individuals in order to examine patterns within a population . properly applied ,
surveillance is a tool for primary prevention through the identification and elimination of the cause of disease ( 12 ) .
screening examinations may be target - organ specific or substance specific , and thus tend to be more focused than preplacement evaluations ( 12 - 14 ) .
based on considerable information , occupational disease management can be accomplished in order to protect and promote worker health .
many kinds of information are required to suggest the etiology of poor worker health , such as hazard , exposure level and related information in workplace .
the present system of surveillance for management of occupational diseases mainly depends upon the workers ' compensation and health examination systems .
these two systems can not cover all kinds of occupational diseases . in order to focus on primary prevention of occupational diseases , various data sources on hazards and exposure levels in workplaces
especially , prevention of occupational diseases may be focused on the reduction of worker exposure to the occupational hazards .
the workers ' compensation insurance system reviews each claim case for evaluating its work - relatedness .
this means that hazard factors and worker exposure to those hazard factors may be assessed .
these review data can be some of the most important information for prevention of ill - health by surveillance .
most of the current surveillance system in korea depends upon the osh act . from the viewpoint of surveillance , the occupational management system in korea is well designed except for the national survey system . in the future , national surveys for detection of occupational hazards and ill - health outcomes of workers should be developed .
the existing surveillance system of occupational disease can be improved by providing more refined information through statistical analysis of surveillance data .
recently , data mining has been used in order to estimate the national burden of occupational disease with other international statistics . in korea
, this technique can be also introduced for development of the management system of occupational diseases . | the management system of occupational diseases in korea can be assessed from the perspective of a surveillance system .
workers ' compensation insurance reports are used to produce official statistics on occupational diseases in korea .
national working conditions surveys are used to monitor the magnitude of work - related symptoms and signs in the labor force .
a health examination program was introduced to detect occupational diseases through both selective and mass screening programs .
the working environment measurement institution assesses workers ' exposure to hazards in the workplace .
government regulates that the employer should do health examinations and working conditions measurement through contracted private agencies and following the occupational safety and health act .
it is hoped that these institutions may be able to effectively detect and monitor occupational diseases and hazards in the workplace . in view of this , the occupational management system in korea is well designed , except for the national survey system . in the future , national surveys for detection of hazards and ill - health outcomes in workers should be developed .
the existing surveillance system for occupational disease can be improved by providing more refined information through statistical analysis of surveillance data . |
a cross - sectional retrospective study was carried out according to world health organization guidelines over 6 months and included 197 general practitioners ( gps ) and 19,700 prescriptions.this study revealed that the overall index of rational drug prescribing ( irdp ) indicator for gps was below the optimal prescribing practice level.we found disparities in the irdp between gps at different health facilities .
gps at national insurance fund facilities scored lower than those at state ministry of health facilities or other health facilities such as those owned by private interests , universities , and non - governmental organizations .
the irrational use of medicines is a major problem worldwide and represents the primary source of medical waste and harm .
the medicines component of the health services package is more variable and dynamic than other service components , particularly in terms of coverage , quality , and cost . in recent years
the overuse of medicines is diverse , and drug - prescribing indicators in developing countries are suboptimal , with core indicators varying between countries .
for instance , antibiotics and injectables are overused and generic medicines are underused in sudan .
the overuse of medicines not only leads to the development of drug resistance and wastes resources but also increases morbidity and mortality [ 3 , 4 ] .
thus , the appropriate use of medicines is an essential factor in the provision of quality health services , patient safety , and the rational use of health resources .
the world health organization ( who ) and the international network of rational use of drugs ( inrud ) have developed widely used core prescribing indicators to assess the prescribing quality of primary healthcare facilities and to enable comparisons between health facilities and prescribers . to our knowledge , no prescriber - based study has been conducted in sudan ; one study of the national health insurance fund ( nhif ) has been conducted in health facilities .
previous studies have been based on the facilities sampling method ( table 1 ) and have revealed antibiotic overuse [ 8 , 9 ] , underuse of generic medicines , and overprescribing of injectables .
other studies have revealed a relatively large average number of drugs per prescription and poor adherence to the essential medicines list ( eml ) .
these previous studies were conducted to investigate prescription patterns ; however , our study investigated the prescribing practices of general practitioners ( gps ) and used the largest sample and a unique study unit .
we used prescribing indicators as a benchmark among gps at primary healthcare centers ( phccs ) and as a baseline for regular assessment of prescribing quality . in sudan ,
the objective of this study was to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .
we used the assessment regimen created by who / inrud to evaluate prescribing performance according to the adopted standards to monitor the improvement of rationality in prescribing practice . before rational prescribing
can be promoted , the prescribing practices of those who provide 80 % of the services must be ascertained.table 1previous prescribing quality studies in sudanyeararealevelmean no .
drugs / prescription% of generic% of antibiotics% of injectable% from eml1991 n. provincephccs1.46363361996 kh statephccs1.9487322981998 kh statehospital and phccs2.1415929992004 kh stateteaching hospitals ( 2)1.9436510.52007 six statesphccs2.344.6662773.52010 kh statephccs243.271.813.792.72010 kh statefour pediatric hospitals249.381.33.52012 five nhif statesphccs2.654.2641499.32012 kh statehospitals and pharmacies2.837.354.338.672.8
eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers previous prescribing quality studies in sudan
eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers
the study was conducted in gezira state , which represents 27 % of nhif ; we targeted all gps ( 220 ) providing services for nhif subscribers at the primary healthcare level .
these gps provide services at three types of health facilities : ( 1 ) those owned by the nhif , ( 2 ) those owned by the state ministry of health ( smoh ) , and ( 3 ) those owned by others ( private interests , universities , and non - governmental organizations [ ngos ] ) .
insured patients pay 25 % of prescription costs , and pharmaceutical services are provided through a network of pharmacies owned by the nhif , smoh , and others .
we collected 100 prescriptions for each gp according to who / inrud guidelines for comparisons between prescribers .
the reference period of retrospective data collection was 6 months , representing the last quarter of 2014 and the first quarter of 2015 .
prescription collection was based on systematic random sampling , with an interval determined by dividing the gp s total prescriptions by 100 .
of the 220 gps , 197 ( 90 % ) had valid prescriptions over the 6 months , and the total valid number of observations was 19,700 prescriptions . trained technical professionals collected the data .
data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .
the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .
we used the who / inrud standard method for calculating prescribing indicators ( table 2 ) .
the unit of analysis used was gp ( 100 prescriptions for each gp).table 2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy)
inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators
inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .
the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .
the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each
. moreover , for each type of health facility , the irdp was applied for comparison .
finally , we analyzed 19,690 prescriptions from 197 gps ( 10 invalid prescriptions were excluded ) .
data at the gp level were clustered to represent the three types of health facilities .
data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .
the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .
we used the who / inrud standard method for calculating prescribing indicators ( table 2 ) .
the unit of analysis used was gp ( 100 prescriptions for each gp).table 2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy)
inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators
inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .
the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .
the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each gp . moreover , for each type of health facility , the irdp was applied for comparison .
finally , we analyzed 19,690 prescriptions from 197 gps ( 10 invalid prescriptions were excluded ) .
data at the gp level were clustered to represent the three types of health facilities .
the average number of medicines per prescription ( 2.55 : maximum 15 , minimum 1 ) indicated a poly - pharmacy pattern , with prescriptions involving antibiotics ( 54.71 % ) and injectable formulations ( 12.84 % ) .
a total of 81.19 % of the prescribed medicines were from the eml , and generics accounted for 46.34 % of prescriptions , which is some distance from the optimal required level of 100 % .
the average cost per prescription was 40.57 sudanese pounds ( sdgs ) ( table 3 ) .
values for nhif facilities were farther from the prescribing indicator standards than were smoh and other health facilities , except for the percentage of prescriptions containing antibiotics ( nhif 45.91 vs. smoh 57.24 vs. others 56.84 % ) ( table 3).table 3prescribing indicators and health facility typestype of health facility owner or operatorsmohnhifotheroverallnumber of gps1264328197average number of drugs per prescription2.442.972.42.55medicines prescribed by generic name ( % ) 48.9638.4749.2846.34prescriptions containing antibiotics ( % ) 57.2445.9156.8454.71prescriptions containing injectable formulations ( % ) 12.5515.589.9712.84drugs prescribed from gp s medicines list ( % ) 82.9772.6589.2781.19average cost of prescription ( sdgs)36.0759.5431.6940.57irdp3.463.083.753.39
gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health prescribing indicators and health facility types
gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health the correlation between gps and irdp was statistically significant at p < 0.001 .
the optimal index is 5 ; however , the overall irdp score for gps was 3.39 ( range 2.14.8 ) .
the prescribing quality indices revealed that prescribing practice was nowhere near the optimal index of 5 , with nhif facilities having a lower index ( 3.08 ) than smoh facilities ( 3.46 ) and health facilities owned by others ( 3.75 ) ( table 3 ) .
the objectives of this study were to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .
we conducted a cross - sectional retrospective study over 6 months that involved 197 gps with valid prescriptions , representing 90 % of the total study population ( 220 ) .
the study revealed that the mean number of medications per prescription was 2.55 1.32 drugs , which represents poly - pharmacy according to the benchmark we applied of more than two drugs .
the mean number of drug prescriptions was 2.55 ; this was much closer to the 2.6 found by a study conducted in nhif facilities in 2012 in five other states .
the mean number of drugs per prescription was higher than most previously reported sudanese studies when we excluded the studies by mustafa and mahmoud et al . , who reported 2.6 and 2.8 drugs per prescription , respectively ( table 1).when compared with other developing countries , the mean number of drugs per prescription in sudan ( 2.55 ) was less than in mali ( 3.2 ) , yemen ( 3.0 ) , uganda ( 2.9 ) , thailand ( 2.85 ) , and pakistan ( 2.7 ) [ 1923 ] .
however , gps in sudan prescribed a higher mean number of drugs per prescription than those in india ( 2.4 ) , tanzania ( 2.2 ) , saudi arabia ( 2.08 ) , and malaysia ( 2.0 ) [ 2427 ] .
collectively , nhif facilities had a higher mean number of medications than other facilities ( table 3 ) . the major implications of poly - pharmacy are additional avoidable costs and an increased probability of adverse drug reactions .
a limitation of the study was that inter - country comparisons were not adjusted according to the prevalence of chronic diseases in the population .
the percentage of generic medicines prescribed was 46.34 % , which is considerably lower than the standard of 100 % .
the calculated percentage represents a severe underuse of generic medicines that was also lower than the middle eastern mediterranean region s average use of generics ( 57.1 % ) .
this result is consistent with previous studies conducted in different districts in sudan ( table 1 ) . when compared with those of neighboring countries , the percentage of generic drugs per prescription in sudan ( 46.34 % ) was lower than in egypt ( 95.4 % ) , ethiopia ( 99.16 % ) , mali ( 70.4 % ) , uganda ( 91.3 % ) , and yemen ( 67.1 % ) [ 1921 , 29 , 30 ] .
however , the percentage of generic drugs prescribed was remarkably higher in sudan than in bahrain ( 14.3 % ) and jordan ( 5.1 % ) [ 29 , 30 ] .
gps in nhif facilities prescribed considerably fewer generics ( 38.47 % ) than those in smoh ( 48.96 % ) and other facilities ( 49.28 % ) .
the implications of low generic use are primarily the wastage of scarce health resources and a decrease in access to pharmaceuticals because of an affordability barrier .
the percentage of prescriptions containing antibiotics was 54.71 % , a considerably high result according to the who guideline benchmark of 30 % antibiotic use .
it was consistent with percentages assessed previously in other states of sudan ( table 1 ) .
the percentage in the who eastern mediterranean region was 53.6 % , slightly less than found for sudan in the current study .
the use of antibiotics was lower in nhif facilities ( 45.91 % ) than in smoh ( 57.24 % ) and other facilities ( 56.84 % ) .
antibiotic overuse can have a devastating impact in terms of the development of multi - drug - resistant bacteria , which can lead to unmanageable infectious diseases .
the percentage of prescriptions containing injections was 12.84 % , which is considered relatively over the optimal level , although no strict standard exists , as the who benchmark is < 10 % .
the average prevalence of injection use in the eastern mediterranean region is higher ( 27.1 % ) .
studies conducted in sudan reveal progress in the rational use of injections ( table 1 ) .
this reduction is attributable to a new malaria management protocol that focuses strongly on ingestible formulations rather than injection , which was the initial dominant medication formulation .
the overall percentage of medications prescribed from the eml was 81.19 % , whereas the prescribing quality indicator standard is 100 % .
eml adherence seems high ; however , in actuality , this figure is misleading because the best achievement in core prescribing indicators was this indicator worldwide . according to the who prescribing database , on average , the middle eastern mediterranean region s percentage of medicines prescribed from the eml was 90.8 % .
previous studies in sudan have often found this prescribing core indicator to be higher than observed in this study ( table 1 ) .
interestingly , we found eml adherence was lower in nhif facilities ( 72.65 % ) than in smoh ( 82.97 % ) and other facilities ( 89.27 % ) .
a total of 197 gps reported an irdp of 2.14.88 , which is relatively low compared with other neighboring countries . for instance , ten health facilities in saudi arabia reported an irdp of 4.375 . in egypt ,
the same processes were conducted in ten phccs and revealed high irdp rankings , ranging from 3.92 to 4.88 . although the irdp was not high , it was higher than the 3.32 reported in china .
the index values for nhif facilities ( 3.08 ) were lower than for smoh ( 3.46 ) and other ( 3.75 ) facilities ( table 3 ) .
the current study revealed that the average prescription cost was 40.57 sdg , almost double the proxy 20.31 sdg .
a study conducted in five states in 2012 reported an average prescription cost of 20.5 sdg , almost half that observed in the current study .
the primary cause of the cost increase is attributed to the devaluation of the national currency , particularly over the last 4 years . when comparing average costs with those in other countries , proper economic adjustment of currency values is essential .
prescriptions from nhif facilities cost more than those from other facilities , with the average prescription costing 59.54 sdg ( nhif ) , 36.07 ( smoh ) , and 31.69 sdg ( other facilities ) .
these disparities were attributed to the relatively high percentage of patients with chronic diseases in nhif facilities ( 36 % ) , with smoh facilities having fewer such patients ( 18 % ) .
the authors consider the main cause of the observed suboptimal prescription quality to be low adherence to prescribing guidelines .
nhif facilities performed worse than other facilities , which could be attributed to the accountability of non - nhif facilities ( all services provided by these facilities are reimbursed according to adherence to regulations ) , while the nhif facilities have not been subjected to that review .
appropriate medicine use is essential to the provision of quality health services , patient safety , and the rational use of health resources .
the present study used the largest sample to date and a unique study unit to investigate prescribing rationality among gps at different types of health facilities providing health services for insured patients in gezira state , sudan .
the study revealed that the overall irdp score for gps was less than optimal and characterized by significant disparities between gps at different health facilities , with nhif facilities receiving a lower index .
further studies should be conducted to determine the factors causing the considerable discrepancies ( 2.14.88 irdp ) between gps at health facilities owned by the nhif , the smoh , and others ( private interests , universities , and ngos ) .
the opinions expressed by the authors do not necessarily reflect the opinions of the chulalongkorn university , thailand , or the national health insurance fund , sudan .
| backgroundthe appropriate use of medicines is essential for the provision of quality health services , patient safety , and the rational use of health resources . in sudan ,
general practitioners ( gps ) provide 80 % of insured patients health services .
pharmaceutical service costs have been increasing since 2010.objectiveswe aimed to use the world health organization ( who ) and international network for the rational use of drugs prescribing indicators to assess prescription quality among gps in different types of primary healthcare centers ( phccs ) within the national health insurance fund ( nhif ) in gezira state , sudan.methodwe followed established who guidelines to conduct a cross - sectional retrospective study .
the study was carried out over 6 months and involved 197 gps with valid prescriptions , representing 90 % of the total study population of 220 gps .
we collected a systematic random sample of 100 prescriptions for each gp and used stata 12 to analyze the 19,700 prescriptions.resultsthe mean standard deviation number of medications was 2.55 1.32 per patient ; 46.32 % of drugs prescribed were generics ; 54.71 % of prescriptions were for antibiotics and 12.84 % were for injectable formulations ; and 81.19 % of prescribed medicines were from the nhif medicines list .
the overall index of rational drug prescribing ( irdp ) indicator was 3.39 , and the average cost per prescription was 40.57 sudanese pounds ( sdg ) . disregarding prescriptions for antibiotics ,
the prescribing quality of gps in nhif facilities was farther from optimal prescribing practice than those in state ministry of health - owned facilities and facilities owned by private groups , universities , and non - governmental organizations.conclusionthe present study provides strong evidence of irrational prescribing practice among gps , with significant disparities , particularly in terms of antibiotic overuse , generic drug underuse , and adherence to the nhif medicines list . |
blunt traumatic infrarenal aortic injuries are rare , with a few case reports in the literature .
a more common occurrence is an intimal flap which may form after blunt injury to the aorta , and most of these will resolve with anticoagulation alone [ 1 , 2 ] .
blunt aortic injury which initially presents as an intimal flap ( grade i ) may progress and evolve into an intramural hematoma ( grade ii ) or a pseudoaneurysm ( grade iii ) .
aortic intimal flap progressing to dissection or pseudoaneurysm is an occurrence identified in 2% or less of the patients , and the majority of these occur in the thoracic aorta [ 1 , 2 ] .
this article presents the case of a young woman found to have an intimal flap in the infrarenal abdominal aorta after a car accident , and the injury progressed to a pseudoaneurysm over 3 months .
a 47-year - old woman with a history of hypertension and smoking was involved in a high - speed motor vehicle accident and presented to the emergency department with back pain .
computed tomographic ( ct ) scans were obtained of her abdomen and pelvis . on a single image from the abdominal scan ,
what appeared to be either an intimal flap or a small amount of contrast extravasation outside of the infrarenal aorta could be seen .
her care providers had differing opinions as to what was being seen on the image .
since the abnormality was only seen on a single image , the exact etiology could not be delineated ( fig .
the patient was observed in the hospital overnight , had minimal complaints the next day , and was discharged on aspirin .
figure 1:initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .
initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .
a follow - up ct scan was obtained 3 months later . at the location of the previous aortic abnormality
, there was a 3 1 cm pseudoaneurysm that had formed and could now be clearly seen ( figs 2 and 3 ) .
figure 2:pseudoaneurysm now clearly seen 3 months later .
figure 3:black arrow pointing to pseudoaneurysm with enhancing flow outside of the aortic lumen .
operative repair was recommended , but the patient s aorta measured only 18 mm at the aortic bifurcation and was too small for even the smallest endovascular stent graft available at our institution .
therefore , an open replacement of her infrarenal aorta was performed using a dacron 22 mm graft . in the operating room ,
after the aorta was clamped and opened , a 4 mm tear in the intima had formed a mature channel into the pseudoaneurysm .
the patient recovered very well and was discharged from the hospital without complications . at her
2-year follow - up , she was in good health and had no further problems , but continues to smoke .
blunt abdominal aortic trauma occurs in only 0.040.1% of all nonpenetrating traumas [ 3 , 4 ] . and
the vast majority of these ( 92% ) are associated with multiple other injuries [ 3 , 5 ] .
our patient had no other injuries , and there was no retroperitoneal hematoma or other signs of vascular injury in the area , as is usually seen on ct when the aorta is injured [ 35 ] .
the lack of other radiographic traumatic findings added to the initial confusion as to whether there was a true injury to the aorta , or if the single image was showing artifact or an enlarged vertebral vessel .
since the majority of intimal flaps heal with anticoagulation alone ( 55% ) or remain stable ( 40% ) , a conservative plan including daily aspirin and re - imaging in 3 months was chosen . when repeat imaging showed a 3 cm pseudoaneurysm , operative repair was necessary .
endovascular repair was initially chosen for this case , but after measuring the patient s aorta , femoral vessels and the degree of taper at the bifurcation , there was not an appropriate endovascular prosthesis available in such a small size .
only one study has compared conventional open repair with endovascular repair for blunt injuries of the abdominal aorta and found similar results in both groups .
endovascular repair is generally favored since the injuries tend to be focal and discrete , making them quite amenable to a short graft .
further , the patients usually have other significant injuries that would complicate a long , and potentially morbid , open operation on the aorta .
although all blunt infrarenal aortic injuries are unusual , most of them consist of intimal flaps or dissections [ 3 , 58 ] .
a post - traumatic pseudoaneurysm , as this patient had , is even more unusual after blunt trauma .
one prior publication did document the delayed occurrence of a post - traumatic infrarenal aortic pseudoaneurysm that was diagnosed 3 years after injury .
endovascular repair for pseudoaneurysms must include post - procedure angiography to document no evidence of a type i or type ii endoleak , which could allow filling of the aneurysm sac and later rupture .
this case report highlights the need for delayed imaging for even minor intimal flaps or small aortic injuries after trauma , since progression does occur .
while the vast majority of these injuries improve over time or remain stable , a small percentage will worsen and require intervention .
| blunt traumatic infrarenal aortic injuries are unusual , and the formation of a delayed pseudoaneurysm of the aorta is even more rare . in this report
, a young woman developed a small intimal flap of the infrarenal aorta after a motor vehicle accident which progressed into a 3 cm pseudoaneurysm after 3 months .
operative repair was successful and the patient recovered .
this case illustrates the importance of repeat imaging of small blunt aortic injuries since progression can occur . |
strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .
cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .
frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .
ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .
cell extracts were cleared at 30,000 g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .
resins were collected at 900 g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .
bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .
eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .
strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .
cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .
frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .
ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .
cell extracts were cleared at 30,000 g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .
resins were collected at 900 g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .
bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .
eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .
| substrates of the proteasome are recognized and unfolded by the regulatory particle ( rp ) , then translocated into the core particle ( cp ) to be degraded1 .
a hetero - hexameric atpase ring , containing subunits rpt1-rpt6 , is situated within the base subassembly of the rp1 .
the atpase ring sits atop the cp , with the rpt c - termini inserted into pockets in the cp26 .
we have identified a novel function of the rpt proteins in proteasome biogenesis through deleting the c - terminal residue from each rpt .
our results indicate that assembly of the hexameric atpase ring is templated on the cp .
we have also identified an apparent intermediate in base assembly , bp1 , which contains rpn1 , three rpts , and hsm3 , a chaperone for base assembly .
the rpt proteins with the strongest assembly phenotypes , rpt4 and rpt6 , were absent from bp1 .
we propose that rpt4 and rpt6 form a nucleating complex to initiate base assembly , and that this complex is subsequently joined by bp1 to complete the rpt ring .
our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process . |
worldwide , head injury is the single largest cause of death and disability following an injury .
the burden of head injury is greatest in low- and middle - income countries ( lamic ) , where 85% of the world 's population live .
the world health organization estimates that almost 90% of deaths due to injuries occur in these settings .
head injury is the leading cause of disability in people under 40 , severely disabling 150200 people per million annually . in 2005
, road traffic injuries resulted in the death of an estimated 110,000 persons , 2.5 million hospitalizations , 89 million minor injuries and economic losses to the tune of 3% of the gross domestic product in india .
the accident rate of 35/1000 vehicles in india is also amongst the highest in the world .
however , resources have not been diverted adequately by governments toward prevention , management and rehabilitation of head injuries in lamic such as india .
most patients with severe traumatic brain injuries ( tbi ) in developing countries are discharged to home - based care due to lack of rehabilitative facilities and health insurance .
those patients who are discharged in an unconscious state are particularly difficult to manage at home and are prone to decubitus ulcers , respiratory infections , inadequate nutrition , and physiotherapy .
however , data is lacking on the actual load of vegetative patients and their outcomes , especially from developing countries .
patients , who remain unresponsive to the environment even though their eyes may be open , are considered to be in a vegetative state ( vs ) . a persistent
vs has been defined as a vs still present 1 month after acute traumatic or nontraumatic brain damage . as by definition
, vs requires a minimum period of 1 month of remaining unconscious , most patients who remain unconscious are not labeled as vegetative at discharge
. however , there are no studies assessing conversion to vs , specifically in this group of patients .
in this retrospective study carried at one trauma center in india , a prospectively maintained neurotrauma registry was queried from may 2010 to february 2013 for patients who had severe traumatic brain injury glasgow coma score ( gcs 8) at admission and had a motor response of m5 or lower on the gcs at discharge .
unconsciousness was described as patients with a motor score of m5 or lower on the gcs irrespective of the verbal ( v ) or eye ( e ) response .
demographic and clinical data was analyzed , and outcome assessed at 6 months after injury using a telephonic questionnaire .
the questions asked were : ( 1 ) is the patient alive ? ( 2 ) if alive , is the patient responding to commands ( conscious ) ? ( 3 ) if the patient is conscious , is he able to perform activities of daily living like feeding himself , going to toilet independently and going to preinjury occupation ? if the family members replied that patient had gone back to preinjury occupation it was considered as glasgow outcome scale ( gos ) 1 , if patient was able to perform activities of daily living , but not able to go back to work it was considered as gos 2 , if patient was conscious but not able to do activities of daily living , it was considered as gos 3 . if a patient remained unconscious , it was gos 4 and if the patient had died it was gos 5 .
gos 13 was considered as the unfavorable outcome and gos 45 was considered as a good outcome .
of these 166 ( 10.9% ) were unconscious ( motor response m5 on the gcs or lower ) at discharge from the hospital .
average hospital stay was 24.31 days ( range : 864 days ) and was 16.71 days for m5 , 20.63 days for m4 , 26.33 days for m3 , 30.59 days for m2 and 16.3 days for m1 patients [ table 1 ] .
mortality and outcome at 6 months with respect to motor scores at the time of discharge the discharge motor score was m5 in 32 ( 19.3% ) , m4 in 44 ( 26.5% ) , m3 in 59 ( 35.5% ) , m2 in 44 ( 26.5% ) and m1 in 9 ( 5.4% ) .
54 ( 52.9% ) patients had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months .
100% of m1 , 90.6% of m2 , 94.3% of m3 , 72.2% of m4 , and 60% of m5 patients had an unfavorable outcome at 6 months .
only 16 patients ( 15.7% ) had a good outcome ( gos 12 ) at 6 months post injury .
incidence , as well as the severity of head injuries , the , is rising in developing countries due to rapid industrialization with a lag in legal , healthcare , and safety reforms .
delhi with a population of around 15 million has the dubious distinction of having the largest number of road traffic accidents of any city in india .
the enormity of burden can be assessed by the fact that our study had more than 1500 severe head injured patients in <3 years and was conducted at only one hospital !
one of the reasons for the step - motherly attitude by policy makers toward head injury prevention , management , and rehabilitation , is the lack of hard statistics of the actual burden of disease which head injuries carry . in spite of the improvement in care for head injured patients ,
there has not been a marked decline in the mortality rates for severe head injuries .
kagan and baker found that mortality rates were between 26.7% and 41.4% for severe tbi patients in level 1 trauma centers .
fakhry et al . in their study found 28.8% mortality rate of severe tbi patients .
it has also been shown that developing ( low and middle income ) countries have a pooled mortality rate of 51% for severe tbi as compared to 30% for high - income countries .
nevertheless , in - hospital mortality tells only part of the story on the outcome of these patients .
six months outcome is often poorer due to lack of rehabilitative care and facilities , especially in developing countries .
most of the patients are discharged to home - based rehabilitation with tracheostomy and oro - gastric tube in situ . in our center ,
unconscious patients with severe tbi are discharged with tracheostomy tube in situ after ensuring that patient can maintain oxygen saturation on room air and after training relatives regarding care of tracheostomy tube , suctioning , and home - based rehabilitation .
interestingly , however , there are no studies on the outcome of patients who are discharged from the acute care facility in this unconscious state .
furthermore , a significant number ( 10.9% in our study ) are discharged in the unconscious state . as all studies are based on admission gcs , it is also often not possible to assess how many patients remained unconscious at the time of discharge and became vegetative at 6 months .
the outcomes of these patients have also not been studied properly . in our study , patients with m2 motor response had the longest hospital duration with unfavorable outcome occurring in 90% of these patients at 6 months .
gcs has remained a robust scale for assessment and prognostication in tbi and the motor score ( m response ) continues to have the highest predictive value within the gcs .
we , therefore , decided to use motor response as criteria to differentiate unconscious and conscious patients .
it may be sometimes difficult to differentiate unconsciousness from dysphasia as both disease states may show a motor response of 5 . however , left sided contusion in the posterior frontal and/or temporal regions along with the presence of gaze fixation are clues towards dysphasia .
our study had a reasonable follow - up of 61.4% of the patients at 6 months and showed the poor outcomes in this subgroup of patients .
54 ( 52.9% ) patients ( who were unconscious at discharge ) had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months with only 16 patients ( 15.7% ) having good outcome ( gos 12 ) at 6 months .
this study also shows that the majority of the patients ( nearly 85% ) who are discharged in an unconscious state will either die or become vegetative .
this is the only study of its kind on patients who remain unconscious at discharge following severe tbi and reveals that around 50% will die , and another 30% become vegetative within 6 months of discharge . | introduction : it is well - known that severe traumatic brain injuries ( tbi ) have a poor outcome .
however , what is not well - known is the outcome for those who survive but remain unconscious at the time of discharge from the hospital.aims and objectives : to assess the outcome of severe tbi patients who have a motor response of m5 or lower on the glasgow coma score ( gcs ) at discharge from a single centre in india.materials and methods : in this retrospective study carried at one trauma centre in india , a prospectively maintained neurotrauma registry was queried from may 2010 to february 2013 for patients who had severe traumatic brain injury ( gcs 8) at admission and had a motor response of m5 or lower on the gcs at discharge .
demographic and clinical data were analyzed , and outcome glasgow outcome scale ( gos ) assessed at 6 months using a telephonic questionnaire.observations and results : there were a total of 1525 patients with severe tbi in the study period .
of these 166 ( 10.9% ) were unconscious ( motor response m5 or lower on the gcs ) at discharge from the hospital .
139 were males and 27 females with a mean age of 33.9 years .
after a mean hospital stay of 24.31 days , the discharge motor score was m5 in 32 ( 19.3% ) , m4 in 44 ( 26.5% ) , m3 in 59 ( 35.5% ) , m2 in 44 ( 26.5% ) , and m1 in 9 ( 5.4% ) .
telephonic follow - up was available in 102 ( 61.4% ) of the patients .
54 ( 52.9% ) patients had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months .
only 16 patients ( 15.7% ) had a good outcome ( gos 12 ) at 6 months following an injury.conclusions:this is the only study of its kind on patients who remain unconscious at discharge following severe tbi and reveals that around 50% will die and another 30% remains vegetative at 6 months of discharge .
only a small percentage ( 15% in our study ) will become conscious and partially integrated in the society . |
their marbling has been increased over many decades to meet domestic consumer preferences . in both countries ,
highly marbled beef is greatly prized for traditional meat cooking methods such as sukiyaki for japanese and gogigui for korean . because of these demands , the use of heifers and steers instead of bulls , intensive feeding system , and genetic ability of wagyu and hanwoo cattle have resulted in greater fat deposition in these breeds compared to european breeds . as intramuscular fat ( imf ) improves beef quality at least in juiciness and flavor ( hornsterin and wasserman , 1987 ; wheeler et al . , 1994 )
it is assessed in abattoirs by meat graders in various countries , including usa , australia , japan , and korea . like other kinds of foods , meat has three functions : 1 ) it provides nutrition ; 2 ) it provides deliciousness ; and 3 ) it prevents disease .
although beef has these three functions , the main food in both japan and korea is boiled rice while beef is a side dish .
therefore , these two countries have developed the quality of beef rather than its quantity .
this is quite different from foreign countries where meat is consumed as a main dish . in japanese and korean cuisine , soft and delicious beef with imf and a good red color
in the past , fat was not given a good image in its role towards human health , although fat is an important energy resource for human .
recently , fat has been reported to have fewer adverse effects on health than carbohydrates , especially simple carbohydrates .
in fact , meat has played a crucial role in human evolution of a healthy and well balanced diet ( pereira and vicente , 2013 ) .
furthermore , meat plays a pivotal role in nutritious diets . high quality marbled beef not only has excellent eating quality , but also contains a lot of beneficial fatty acids ( troy et al . , 2016 ) . in this regard ,
the current paper reviews the characteristics and health benefit of highly marbled beef from wagyu and hanwoo cattle .
it has well known that wagyu cattle have high potential of accumulating imf and producing highly marbled beef .
hanwoo cattle are also known for their high imf for marbled beef similar to wagyu beef .
highly marbled wagyu loin contains more than 40% of imf , sometimes more than 60% ( horii et al . , 2009 ) , while quality grade 1 hanwoo , the highest quality grade , has approximately 28% of imf in longissimus thoracis muscle ( hwang and joo , 2016 ) .
wagyu cattle include four types of japanese cattle : the black , brown , short horn , and polled breeds .
numerous studies have investigated the meat quality , quantity , and muscle physiology of crossbreed wagyu ( japanese black cattle ) in foreign countries ( cafe et al . , 2006 ; cafe et al . , 2009 ; greenwood et al . , 2006 ; greenwood et al . , 2009 ; may et al . , 1993 ) .
they are identified by different coat color : brown ( major hanwoo ) , black face ( heukwoo ) , black ( jeju heukwoo ) , and tiger color ( chickso ) ( jo et al . , 2012 ) .
in this review , wagyu and hanwoo are used to describe the japanese black breed and the brown coat color hanwoo , respectively .
all four types of wagyu cattle have played important roles locally and in the history of mixed farming .
they also played important roles in the synergies between cattle and crops , especially rice .
farmers gradually began to replace the role of cattle as draft animals and started to use industrial fertilizers approximately 50 years ago . in recent years , japanese wagyu cattle have been raised more specifically for beef production .
the famous brand name wagyu not only includes the japanese black cattle produced in japan , but also includes animals or even cross - bred japanese black cattle produced in foreign countries such as australia and the united states .
similarly , the utilization of hanwoo cattle as an edible meat had been minimal for long time .
full - scale production of hanwoo as meat - type cattle has started since the 1970s . because hanwoo cattle have maintained stable traits through pure breeding , the current blood lineage is very valuable .
it is mainly spread in the korean peninsula ( kim and lee , 2000 ) .
recently , hanwoo beef has been reported to have highly marbled imf similar to wagyu beef .
especially , hanwoo beef has relatively thin muscle fiber and minimal content of connective tissues ( kim et al . , 1994 ) .
it has less subcutaneous fat depth with greater ossification scores and marbling scores than those of australian angus ( cho et al . , 2005 ) .
in 2013 , a total of 2.64 million heads of cattle were fed for beef production in japan .
approximately 1.71 million heads were japanese black cattle ( maff , 2013 ) , and approximately 873,400 were holstein cattle .
, the number of farmers producing beef was 613,000 , but 86.5% of these farmers fed less than 50 heads of cattle .
the mean body weight and carcass weight of beef at slaughter ( 26 - 30 mon of age ) were 725 kg and 470 kg , respectively .
high performance marbled beef production has caused japanese black cattle to comprise the greatest share of japan s wagyu cattle population ( albrecht et al . , 2011 ;
recently , the imf percentage of beef from japanese black cattle has an average value of greater than 30% ( albrecht et al . , 2011 ; horii et al . ,
the total number of slaughter cattle was 1,007,000 , including 883,593 hanwoo cattle , 66,485 holstein cows , and 56,923 holstein heifers and bulls ( kape , 2015 ) .
the number of cattle farming households was 99,858 , including 89,403 hanwoo farmers ( kape , 2015 ) . during the last decade ,
the number of households raising hanwoo cattle has drastically decreased from 186,000 households in 2006 to 89,403 households in 2015 ( kape , 2016 ) .
the average live and carcass weights of hanwoo cattle at slaughter ( 26 - 30 mon of age ) were 719 kg and 430 kg , respectively ( kape , 2015 ) .
wagyu carcasses are evaluated by accredited graders from the japan meat grading association ( jmga ) in accordance with beef carcass grading standards .
first established in 1988 , the present grading system assigns both yield grade ( a , b , and c ) and meat quality grade ( 1 , 2 , 3 , 4 , and 5 ) ( jmga , 2014 ) .
in korea , all cattle carcasses should be evaluated by korean carcass grading system . established in 1992 ,
the korean carcass grading system presently has three levels of yield grade ( yg ) for meat amount ( a , b , and c ) and five levels of meat quality grade ( qg ) ( 1 , 1 , 1 , 2 , and 3 ) ( kape , 2016 ) . for beef quality grading in japan ,
all cattle carcasses are graded at the 6 to 7 rib section at least one hour after ribbing .
the following four items are independently evaluated : beef marbling ; meat color and brightness ; meat firmness and texture ; and fat color , luster , and quality . meat quality grade of the carcass is then assigned according to the lowest grade of these four items .
korean beef quality grading is also estimated based on several factors , including marbling score , meat color , fat color , firmness and texture of lean meat , and maturity of the exposed loin muscle at the 13 rib interface .
this means that marbling score is the most dominating determinant in korea because korean consumers have an extraordinary preference for high marbled meats . in 1988 , wagyu marbling levels were assigned by the beef marbling standard ( bms ) using a plastic model made from silicone resin .
this standard was calculated based on the circumference and area percentage of marbling particles in the rib eye section ( longissimus thoracis ) . in october 2008 , a new marbling standard using carcass photographs replaced the 1988 standard . in march 2014 , an even newer marbling standard
1 ) . graders now determine the bms number ( 1 to 12 ) by comparing the actual carcass marbling to the standard photograph of marbling . during this process
, any larger inclusions of fat at the periphery of the rib eye are not considered as marbling according to the japanese grading system .
the bms of korean carcass grading system has been changed by the addition of marbling number . in 1992 , when the carcass grading system was established for the first time in korea , the bms had only 5 numbers ( 1 to 5 ) with 3 qg ( 1 , 2 , and 3 ) . however , in 1997 , new qg 1 was added with new bms no . 6 and no .
furthermore , in 2004 , another new qg 1 was added with new bms no . 8 and no .
hanwoo beef with qg 1 or 1 is considered as a premium class of beef in korea .
of hanwoo cattle slaughtered in 2015 , 10.0% were qg 1 , 26.4% were qg 1 , and 31.4% were qg 1 ( kape , 2016 ) .
the plentiful marbling of wagyu and hanwoo beef has attracted attention . in both japan and korea ,
the value of cattle carcasses is determined by a qg which considers marbling as a decisive determinant . since the liberalization of beef importation
, marbling has been greatly emphasized to differentiate domestic beef from imported beef ( hirooka , 2014 ; hwang et al . , 2010 ) .
the high content of imf can improve the texture and juiciness of hanwoo beef and thereby its acceptability ( jung et al . , 2016 ) .
korean consumers prefer qg 1 or 1 beef because of its high imf content ( kim et al . , 1999 ) .
2015 ) have demonstrated that an increase in crude fat content ( range 23.8 - 48.6% ) can increase the tenderness , juiciness , and fattiness .
however , they also reported that an increase in crude fat content can reduce the crude protein content and slightly reduce the content of umami components such as nucleic acid and glutamic acid .
it is well known that imf content varies depending on feeding time , finishing diet , and breed type . to produce high qg beef ,
great attention has been paid to more accumulation of imf in wagyu and hanwoo muscle .
one of good strategy to increase imf content in beef muscle is to extend slaughtering age . although the marbling score is increased and reached a plateau at about 24 mon of age ( choi et al . ,
2002 ) , the slaughtering age of hanwoo has been extended to increase the bms score ( jo et al . , 2012 ) .
in korea , the marketing age of hanwoo has been extended to an average of 31 mon with weight of 719 kg to fatten the cattle ( kape , 2015 ) .
however , average daily gain is decreased due to increased slaughtering weight ( paek et al . , 1993 ) .
recently , cattle in china are fed for unusually long periods of time before slaughter as wagyu and hanwoo .
this might have contributed to their high imf and oleic acid contents ( smith , 2016 ; tanaka , 1985 ) .
it is clear that imf increases with feeding time for grain - fed and pasture - fed cattle .
however , the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle ( smith et al . , 2009 ) .
it has been reported that wagyu fed on a high - concentration diet have higher expression of adipogenic transcription factors in the subcutaneous and intramuscular adipocytes than those fed on a high - rough - age diet ( yamada and nakanishi , 2012 ) .
the imf content and the numbers of preadipocytes and adipocytes are reported to be higher in wagyu than those in angus ( duarte et al . , 2013 ) .
( 2009 ) have reported that the imf contents in the longissimus muscle of wagyu , german angus , belgain blue , and holstein friesian are 23.3% , 4.4% .
the wagyu and european cattle breeds did not differ in their mechanisms of postnatal fat accretion .
however , they differed in their efficiency of accretion of imf ( gotoh et al . , 2009 ) .
for every 1% increase of imf in the longissimus muscle , the increase amounts of subcutaneous adipose tissue in wagyu , holstein friesian , german angus , and belgain blue were 3.0 , 4.3 , 7.9 , and 10.7 kg , respectively ( gotoh et al . , 2009 ) .
although imf content is the most dominating determinant of beef quality , the imf content is not the only parameter that decides the quality grade of beef carcass .
marbling is called shimo - furi in japanese and sang - gang in korean .
it literally means frosting . in japan , marbling with a fine appearance resembling frost is highly valued , but coarse marbling is not ( motoyama et al . ,
recently , korea also began to discriminate between fine and coarse marbling in hanwoo beef .
this marbling quality contributes to the tenderness of beef because imf deposits are found mainly between muscle fiber bundles , resulting in the disorganization of perimysium connective tissue ( nishimura , 2015 ; sasaki et al . , 2012 ) .
therefore , the sensory of tenderness could be qualitatively affected by histological difference in marbling due to difference in tissue disorganization extent .
there are several types of fatty acids : 1 ) monounsaturated fatty acids ( mufa ) , 2 ) polyunsaturated fatty acids ( pufa ) , and 3 ) saturated fatty acid ( sfa ) .
pufa such as linoleic acid , -linolenic acid ( n-3 ) , -linolenic acid ( n-6 ) , arachidonic acid , and so on contain many important compounds such as essential fatty acids .
the fatty acid that has the highest amount in beef is oleic acid ( c18:1n-9 ) .
it has been reported that fatty acid compositions are different depending on breeds ( smith et al . , 2006
the fatty acid compositions in highly marbled wagyu and hanwoo are considerably different from those in other cattle breeds .
highly marbled wagyu beef has a higher percentage of mufa within fat compared with other breeds ( yang et al . , 1999a ) .
( 2006 ) have investigated oleic acid concentrations in the subcutaneous adipose tissues of wagyu , hanwoo , australian crossbred , angus ( corn - fed ) , angus ( hay - fed ) , and angus ( weaned ) and found that they are 52.9% , 47.3% , 39.8% , 39.8% , 34.6% , and 32.9% , respectively .
a higher percentage of mufa will lead to a lower fat - melting point which contributes to the softness of beef fat and favorable beef flavor .
it may decrease the circulating concentration of ldl cholesterol in consumers ( melton et al .
, 1982 ; rudel et al . , 1995 ; smith , 1994 ) . therefore , fatty acid compositions of beef have recently become important in the beef industry , especially in highly marbled wagyu and hanwoo cattle
( 1995 ) have investigated the effect of breed type ( including japanese black ) and sex on fatty acid compositions of subcutaneous and intramuscular lipids in finishing steers and heifers of pure japanese black and holstein as well as crossbred japanese black , holstein , japanese brown , and charolais .
they have reported that the japanese black is genetically predisposed to producing carcass lipids containing higher concentrations of mufa than holstein , japanese brown , or charolais steers ( zembayashi et al . , 1995 ) .
1992 ) have also concluded that beef from purebred wagyu cattle raised in japan is rich in mufa .
( 2011 ) have compared intramuscular fatty acid composition of longissimus muscle in 26-month - old japanese black steers and holstein steers reared and fattened using a standard fattening system ( table 1 ) . in the longissimus muscle of japanese black steers ,
a higher percentage of unsaturated fatty acid was found than that in holstein steers ( gotoh et al . , 2014 )
2011 ) have also compared the imf content and fatty acid compositions of 21 major skeletal muscles using the same animals .
muscles from the japanese black cattle contained a greater proportion of numerous fatty acids , particularly mufa such as c16:1 , c18:1 , and c20:1 compared to fatty acids in holstein cattle .
in japanese black cattle , the proportion of sfa including c18:0 was much lower compared to that in holstein cattle .
sfa : saturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .
2005 ) have investigated the fatty acid compositions of hanwoo and australian angus beef and found a significant difference in fatty acid compositions between these two cattle breeds ( table 2 ) .
especially , angus beef had significantly higher n-3 pufa while hanwoo beef contained greater n-6 pufa in three different muscles ( cho et al . , 2005 ) .
the difference in fatty acid composition might be attributed to the influence of different diets , forage , and grain feeding , although fatty acid profile in ruminants is not a direct reflection of the dietary fatty acid composition due to hydrogenation by rumen microorganism ( enser et al . , 1998 ) .
sfa : saturated fatty acids , usfa : unsaturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .
f - ratio statistic : * if p<0.05 , * * if p<0.01 , * * * if p<0.001 .
therefore , it can be easily anticipated that hanwoo beef has a fatty acid profile similar to that of high concentratefed animals ( jo et al . , 2012 ) .
recently , hwang and joo ( 2016 ) have evaluated the fatty acid profile of ten muscles from high marbled ( qg 1 ) and low marbled ( qg 2 ) hanwoo carcass and found significant differences in fat content and fatty acid composition among 10 muscles and between high and low marbled hanwoo beef . in particular , high marbled hanwoo muscles had significantly higher proportion of mufa due to higher oleic acid ( c18:1 ) proportion , while low marbled hanwoo muscles had higher proportion of sfa due to higher proportion of stearic acid ( c18:0 ) ( hwang and joo , 2016 ) .
stearoyl - coa desaturase ( scd ) was first identified and reported as one of the genes associated with beef fatty acid composition ( taniguchi et al . ,
the composition of fatty acids stored in fat depots reflects the earlier action of scd on substrates such as stearic acid and palmitic acid ( kim and ntambi , 1999 ) .
1999b ) have reported interesting correlations between scd enzyme activity and fatty acid composition in bovine adipose tissue .
although the adipogenic mechanism is extremely complicated , several genes have been identified and confirmed as either associated with or responsible for the fatty acid composition in wagyu cattle ( gotoh et al . , 2014 ) .
it is generally accepted that the concentration of oleic acid in beef adipose tissue is dependent on scd expression and activity .
wagyu cattle are genetically disposed to produce more oleic acid ( smith et al . , 2006 ) .
very high heritability has been reported for oleic acid in wagyu cattle ( nogi et al . , 2011 ) .
higher levels of concentrated feed in the later fattening period can lead to higher mufa concentration in the subcutaneous adipose tissues of wagyu steer ( kimura et al . ,
interest in beef fat and fatty acids has been increasing , especially in highly marbled beef such as wagyu and hanwoo because fatty acids composition in the diet have impact on human health .
consumption of fat and cholesterol has been reported to be linked to cardiovascular disease , obesity , and cancer ( micha et al . , 2010 ;
consequently , reduction of total fatty acid intake and replacement of sfa with pufa have been recommended .
ulbricht and southgate ( 1991 ) have demonstrated that stearic acid has no effect on plasma cholesterol level and that oleic acid can lower serum cholesterol similar to pufa .
furthermore , pavan and duckett ( 2013 ) have suggested that a higher proportion of oleic acid in beef is desirable because the consumption of high - oleic acid ground beef can increase hdl - cholesterol concentration ( gilmore et al . , 2011 ) . according to smith ( 2016 ) ,
the amount of fat consumed in a typical portion of beef will not increase risk factors for cardiovascular disease .
clinical trials have demonstrated that ground beef containing elevated oleic acid can increase the concentration of hdl - cholesterol or at least has no negative effect on the concentration of hdl - cholesterol . in earlier research on oleic acid , the major mufa in beef , grundy et al .
( 1988 ) have found that it can lower ldl - cholesterol without affecting beneficial hdl - cholesterol .
( 2014 ) have reported that mufa can normalize or improve lipid metabolism and maintain the balance in cardiac muscle .
these have implied that mufa have little effect on total cholesterol and that they are heart - healthy dietary fat that can lower ldl - cholesterol and increase hdl - cholesterol ( lahey et al . , 2014 ) .
this effect is repeatable when natural foods are used to supplement diets with oleic acid . in this
regard , smith ( 2016 ) have concluded that beef cattle should be raised under production conditions to increase the concentration of oleic acid in their edible tissue , i.e. , by grain feeding over extended periods of time .
it is obvious that consumer in the world has an overwhelmingly negative attitude toward animal fats , especially saturated fat in meat for the last several decades ( ngapo and dransfield , 2006 ; williams and droulez , 2010 ) .
according to higgs ( 2000 ) , the per capita decline in beef consumption in the us and other western countries has been attributed in large part to animal fat phobia .
consumers have been warned to reduce saturated fat in their diet and to avoid meat cuts containing high fat content .
these health recommendations are obviously in conflict with the health of highly marbled wagyu and hanwoo beef .
many research studies have shown that the imf of wagyu and hanwoo beef contains a lot of mufa that could prevent arteriosclerosis .
researches have also demonstrated that high - oleic acid ground beef may reduce risk factors for cardiovascular disease ( adams et al .
thus , although some consumers in japan and korea consider highly marbled wagyu and hanwoo beef as being unhealthy , there is no scientific evidence to indicate that beef that is high in oleic acid will increase risk factors for diseases ( smith , 2016 ) . consequently , the role of animal fats in the diet should be re - evaluated because scientists around the globe increasingly doubt the validity of the so called diet - heart hypothesis
it is now generally accepted that diets with low fat , high carbohydrate failed to curb obesity ( drewnowski , 2015 ) . on the other hand ,
more recent functional medicine research studies have suggested that the intake of fat has positive effect on human health ( saito , 2016 ) .
it is essential to consume fats containing good quality fatty acids while reducing the consumption of food high in simple carbohydrates .
excessive intake of simple carbohydrates is detrimental to health because they have negative effects on the body ( yu et al . , 2013 ) .
in this regard , inclusion of high fat foods with superior sensory properties in a balanced diet such as highly marbled wagyu and hanwoo beef is likely to gain wider acceptance as a well - being food in the near future .
in japan and korea , highly marbled wagyu and hanwoo cattle are greatly prized for traditional meat cooking methods .
many researches have shown that wagyu and hanwoo cattle have high potential of accumulating imf and producing highly marbled beef .
the beef quality grading system in both countries is primarily determined by marbling score with bms and additionally adjusted by other carcass traits .
literature suggests that imf content varies on the basis of feeding time , finishing diet , and breed type .
great attention has been paid to more accumulation of imf to produce high quality grade beef .
the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .
highly marbled wagyu and hanwoo beef have higher proportions of mufa due to higher concentrations of oleic acid .
they are heart - healthy dietary fat because they can lower ldl - cholesterol while increasing hdl - cholesterol .
clinical trials have also indicated that highly marbled beef does not increase ldl - cholesterol and that beef high in oleic acid can consistently increase hdl - cholesterol . finally , literatures have concluded that high - oleic acid beef such as wagyu and hanwoo beef may reduce risk factors for cardiovascular diseases . | this review addresses the characteristics and health benefit of highly marbled wagyu and hanwoo beef .
marbling of wagyu and hanwoo beef has been increased in japan and korea to meet domestic consumer preferences .
wagyu and hanwoo cattle have high potential of accumulating intramuscular fat ( imf ) and producing highly marbled beef .
the imf content varies depending on the feeding of time , finishing diet , and breed type .
imf increases when feeding time is increased .
the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .
fatty acid composition are also different depending on breeds .
highly marbled wagyu and hanwoo beef have higher proportions of monounsaturated fatty acid ( mufa ) due to higher concentrations of oleic acid .
mufas have little effect on total cholesterol .
they are heart - healthy dietary fat because they can lower low - density lipoprotein ( ldl)-cholesterol while increasing high - density lipoprotein ( hdl)-cholesterol .
clinical trials have indicated that highly marbled beef does not increase ldl - cholesterol .
this review also emphasizes that high oleic acid beef such as wagyu and hanwoo beef might be able to reduce risk factors for cardiovascular disease . |
n - acetyl - l - cysteine ( nac ) is an endogenous aminothiol present both in human plasma and in urine .
n-(2-mercaptopropionyl)glycine ( mpg ) , also known as tiopronin , is a synthetic aminothiol antioxidant .
nac has been in clinical use for more than 40 years , primarily as a mucolytic agent in a variety of respiratory illness .
intravenous and oral administration of nac have been extensively used in the management of paracetamol ( acetaminophen ) poisoning .
mpg is primarily used in the treatment of cystinuria , but studies have shown that mpg can be used as a chelating , cardioprotecting , and radioprotecting agent , as well as an antidote to heavy metal poisoning . a number of electrochemical [ 59 ] , fluorometric [ 1012 ] , chemiluminescence [ 1315 ] , and liquid chromatographic [ 1618 ] methods have been developed for the determination of nac and mpg in biological samples and pharmaceuticals .
spectrophotometry is the most widely used technique in pharmaceutical analysis because it is simple , economic , and easily available to most quality control laboratories .
spectrophotometric methods have also been reported for the determination of nac and mpg in pharmaceutical formulations [ 1925 ] .
a coupled redox - complexation reaction has been reported for the spectrophotometric analysis of nac and mpg using 1,10-phenanthroline as the chromogenic reagent . in the present work ,
we report a simple and cost - effective spectrophotometric method for the reliable analysis of nac and mpg in pharmaceutical formulations .
the method is also based on the coupled redox - complexation reaction between nac or mpg and fe(iii ) but uses 2,4,6-tripyridyl - s - triazine ( tptz ) as the chromogenic reagent .
collins et al . have introduced tptz as chromogenic reagent for determination of fe(ii ) .
the fe(ii ) complex with tptz has a twice higher molar absorptivity coefficient ( 2.2 10 l mol cm ) than the fe(ii ) complex with 1,10-phenanthroline ( 1.1 10 l mol cm ) .
all spectrophotometric studies were carried out on an ultraviolet - visible , double - beam spectrophotometer ( uv-1601 shimadzu , kyoto , japan ) , and using 1 cm quartz cells .
measurements of ph were carried out with a mettler toledo sevenmulti potentiometer ( mettler toledo , schwerzenbach , switzerland ) equipped with a combined glass electrode mettler toledo in lab 413 .
a thermostated water bath ( mgw lauda , germany ) was used to keep a constant cuvette temperature of 25 0.5c .
all chemicals were of analytical - reagent grade , and solutions were prepared in milliq deionised water .
separate 10 mm stock solutions of nac and mpg were prepared by dissolving 163.2 mg ( 1 mmol ) of nac ( merck , darmstadt , germany ) or 163.2 mg ( 1 mmol ) of mpg ( sigma - aldrich , st .
louis , usa ) in deionised water up to 100.0 ml volume and stored in the dark bottle at 4c .
dilutions were prepared daily in deionised water . stock solution of fe(iii ) ( 10 mm ) was prepared by dissolving 270.3 mg ( 1 mmol ) of fecl3 6 h2o ( kemika , zagreb , croatia ) in 50.0 ml deionised water .
then 0.5 ml of concentrated hcl was added and the volume was adjusted to 100.0 ml with deionised water . a stock solution of 10 mm tptz ( merck , darmstadt , germany )
was prepared by dissolving 312.3 mg ( 1 mmol ) tptz in 2.0 ml of a 6.0 m hcl , followed by addition of deionised water up to a total volume of 100.0 ml .
m ) was used to cover the ph range 3.24.0 . for solutions of ph 1.0 and 2.0 ,
two different pharmaceutical formulations of nac were analysed by the present spectrophotometric method , that is , fluimukan 200 mg granules , and fluimukan akut 600 mg dispersible tablets ( lek , ljubljana , slovenia ) .
an accurately weighed portion of the powder containing about 200 mg of nac was transferred into a 500 ml volumetric flask , and nac was dissolved in and diluted to the nominal volume with deionised water .
ten tablets of the mpg - containing drug captimer ( mit gesundheit gmbh , germany ) were weighed and pulverised .
a powder quantity equivalent to 100 mg of mpg was dissolved in 300 ml of deionised water , filtered through filter paper ( blue ribbon , s&s , germany ) , and the filtrate collected in a 500 ml volumetric flask was diluted by deionised water to the nominal volume .
it is noteworthy that such solutions are not stable and should be analysed within 24 hours .
these solutions were further diluted quantitatively with water to obtain suitable concentrations for the analysis by the proposed spectrophotometric method .
acetate buffer ( 20.0 ml , ph 3.6 ) was pipetted into a 25.0 ml calibrated flask . then 1.25 ml of 10.0 mm fe(iii )
, 1.25 ml of 10.0 mm tptz , and 1.0 ml of nac or mpg solutions were added .
the flask with reaction solution was filled to the nominal volume with deionised water , mixed well , and kept at room temperature ( about 25c ) for 30 min ( mpg ) or 60 min ( nac ) .
the absorbance of the produced fe(ii)-tptz complex was measured at = 593 nm against a blank solution , prepared in the same manner with 1.0 ml water instead of 1.0 ml sample solution .
nac and mpg concentrations in pharmaceutical preparations were determined by using daily prepared calibration curves .
the eleven solution of every analyte were prepared for the concentration range from 1.0 m to 100.0 m .
the proposed method is based on the coupled redox - complexation reaction . in the first ( redox ) step of the reaction ( see ( 1 ) ) , rsh compound ( nac or mpg ) reduces fe(iii ) to fe(ii ) whereas rsh molecules themselves oxidize to thiyl radicals rs which combine to form the disulfide rssr . in the second step of the reaction ( see ( 2 ) ) , in situ formed fe(ii ) is immediately complexed by 2 molecules of tptz to form the deep - blue coloured , highly stable fe(tptz)2
complex which absorbs light at
max at 593 nm .
the net overall reaction can be expressed by reaction ( 3 ) :
( 1)fe3++rshfe2++h++rs
( 2)fe2++2 tptzfe(tptz)22 +
( 3)2 fe3++2 rsh+4 tptz2 fe(tptz)22++rssr+2 h+
krishnamurti and huang have reported that the complexation of tptz is specific for fe(ii ) so that this reaction can be performed in the presence of large amounts of fe(iii ) .
the results of the present study confirm these previous results for the drugs nac and mpg serving as the reducing agents . in the literature , we were not able to find the standard reduction potential for nac and mpg .
however , the calculated formal potential of the fe(iii)/fe(ii ) couple of 0.578 v , equations ( 4 ) and ( 5 ) indicate that its oxidizing power in solution with tptz is more negative than in solution with 1,10-phenanthroline ( 1.197 v ) .
this means that the proposed method with the tptz is selective for the determination of nac and mpg .
thiols or other reducing substances with standard ( formal ) potentials higher than 0.6 v would not interfere in the proposed method ( see ( 6 ) ) ;
( 4)e10=efe3+/fe2 + 00.05922log ( fe2+fe3+[tptz]2)2 ,
( 5)e10=0.771 v0.0592log 1.2651050.077(3104)2=0.578 v
( 6)e20=erssr / rsh00.0592ph .
in previous work
, we found that the initial redox - complexation reaction rate is higher with mpg compared to nac [ 28 , 29 ] . in the coupled redox - complexation reaction with mpg , the steady state value of the absorbance is reached after 30 min while in the reaction with nac the steady state value of the absorbance is reached after 60 min . with both thiols ,
. equation ( 6 ) indicates that the potential for the redox system rsh / rssr depends upon the ph value of the reaction mixture .
the effect of the ph was therefore investigated over the range 1.04.0 using 0.1 m hcl for ph 1 , 0.01 m hcl for ph 2 , and acetate buffer for the ph values 3.2 , 3.5 and 3.6 . in this experiment mpg
the absorbance at 593 nm increased with increasing ph up to the value of 3.6 . however , precipitation of iron hydroxide occurred at the ph above 3.8 .
therefore , a buffered reaction medium of ph 3.6 was chosen as a compromise for keeping fe(iii ) in solution and achieving quantitative formation of the fe(tptz)2
complex which is stable in the ph range 3.45.8 .
the influence of the fe(iii ) concentration on the determination of nac and mpg at the fixed concentration of 40 m each was studied in the concentration range from 20 m to 400 m , allowing a molar ratio fe(iii)/rsh from 0.5 to 10 . in figure 2 , the absorbance measured at 593 nm is plotted versus the molar fe(iii)/rsh ratio for nac and mpg .
figure 2 shows that the reaction can be forced to completion by increasing the fe(iii)/rsh ratio , for instance by increasing the fe(iii ) concentration .
the influence of the tptz concentration on the analysis of nac and mpg at the fixed concentration of 40 m rsh compound was studied in the range from 20 m to 400 m allowing a tptz / rsh molar ratio of 0.5 to 10 .
figure 3 shows that absorbance increased with increasing tptz / rsh molar ratio , that is , with increasing tptz concentration , to reach its maximal value at a molar excess of five .
the effect of the reaction temperature on the signal intensity was examined by varying the temperature from 25c to 40c using the thermostated water pump .
we found that the reaction rate increased by elevating reaction temperature ( see [ 28 , 29 ] ) .
since the proposed method is an equilibrium method , signal is recorded when the reaction reaches the state of equilibrium .
the signal intensity in the state of equilibrium is the same for all the examined temperatures .
the linearity of the method was investigated under the optimized conditions for nac and mpg in the concentration range from 1.0 to 100.0 m .
straight lines were obtained from linear regression analysis of the absorbance at 593 nm and the drug concentration ( table 1 ) .
the lowest quantifiable concentration of nac and mpg by this method was 1.0 m each .
the effect of some possible interfering cations and anions on the analysis of a fixed concentration of 40.0 m for nac and mpg was investigated for the maximum molar ratio of foreign ions .
the influence of excipients that can commonly accompany nac and mpg in pharmaceutical formulations was also studied .
the tolerance is defined as the foreign - ion / excipient concentration causing an error smaller than 5% for the determination of the analyte of interest .
the tolerable concentration of kno3 and na2so4 was 40.0 mm ( molar ratio , 1000 : 1 ) .
the tolerable concentration of glucose , fructose , sucrose , boric acid , and acetic acid was 20.0 mm ( molar ratio , 500 : 1 ) .
thus , the commonly excipients glucose , fructose , and sucrose do not interfere with the analysis of nac and mpg because they essentially do not react with the oxidizing agents .
it should be emphasized that the contaminant / analyte concentration ratios studied in the present work are much higher than those normally found in commercial pharmaceutical products .
the tolerable concentration of some other thiols , that is , d - penicillamine , l - glutathione , and l - cysteine , was 40.0 m ( molar ratio , 1 : 1 ) .
thiols or other reducing substances with standard ( formal ) potentials higher than 0.6 v would not interfere in the proposed method . in order to evaluate the potential of the proposed method to the analysis of real samples , the method was applied to three pharmaceutical formulations of the drugs nac and mpg .
the results of these analyses are presented in tables 2 and 3 . for comparison , the spectrophotometric method reported by raggi et al .
these authors used 1,10-phenantroline as an chromogenic reagent , instead of tptz which is used in the present work .
as shown in table 2 , there were no significant differences between the values obtained by the reported method and those obtained by the proposed method ( p > 0.1 , student t - test ) .
this actually suggests that the proposed method is accurate and precise as the earlier reported method .
the accuracy of the method was further ascertained through the recovery studies . to the drug solutions of the granules or the tablet powder , the standard solutions of the synthetic nac or mpg
these results indicate that the proposed method is accurate for the determination of nac and mpg in their commercially available pharmaceutical preparations without any significant interference by common pharmaceutical excipients which do not absorb light in the visible region .
performance characteristic of existing equilibrium spectrophotometric methods [ 19 , 2124 ] and the proposed method are compared in table 4 .
the proposed method is free from drastic experimental conditions such as heating unlike some of reported methods .
some other thiol compounds do not interfere in the proposed method at molar ratio 1 : 1 .
it is also worth mentioning that the proposed method was performed in the visible region ( = 593 nm ) away from the uv - absorbance of the uv - absorbing interfering excipient materials , which might be dissolved from pharmaceutical formulations .
undoubtedly , hplc is one of the most widely used techniques in routine analysis of pharmaceuticals , but it involves expensive instrumental set which many laboratories in developing and underdeveloped countries can not afford . the proposed equilibrium spectrophotometric method based on the coupled redox - complexation reaction of the thiol drug nac or mpg with fe(iii ) and tptz can be applied in every analytical laboratory as a reliable method for the determination of nac or mpg in pharmaceutical preparations . due to the use of tptz
the coloured fe(tptz)2
complex is stable in an extended period of time up to 24 hours .
regular excipients and additives present in the pharmaceutical preparations of nac and mpg do not interfere in this method . | a simple spectrophotometric method for the determination of
n - acetyl - l - cysteine ( nac ) and
n-(2-mercaptopropionyl)glycine ( mpg ) in pharmaceutical preparations was developed , validated , and used .
the proposed equilibrium method is based on a coupled two - step redox and complexation reaction . in the first step , fe(iii )
is reduced to fe(ii ) by nac or mpg .
subsequently , fe(ii ) is complexed with 2,4,6-tripyridyl - s - triazine ( tptz ) .
several analytical parameters of the method were optimized for nac and mpg analysis in the concentration range from 1.0 m to 100.0 m .
regression analysis of the calibration data showed a good correlation coefficient ( 0.9999 ) .
the detection limit of the method was 0.14 m for nac and 0.13 m for mpg .
the method was successfully applied to quantify nac and mpg in pharmaceutical preparations .
no interferences were observed from common pharmaceutical excipients . |
vascular malformation is known as one of the major therapeutic problem even to the most experienced clinicians .
these malformations grow proportionately with age , and may involve multiple anatomical spaces and encase critical neuromuscular structures , associated with nerve damage , massive bleeding , and deformity .
management of arteriovenous malformations ( avms ) remains challenging because of their unpredictable behavior and high recurrence rate if treated by curettage alone without inadequate ligation of the feeder vessels .
these lesions are treated by variety of techniques over the years , including surgical excision , irradiation , electrocoagulation , cryotherapy , intravascular magnesium or copper needles , systemic corticosteroids , interferon- , embolization , cryotherapy , lasers and sclerotherapy .
sclerotherapy is an effective , conservative and low cost technique for the treatment of benign vascular lesions , included vascular malformation .
the sclerosant agents used are 5% sodium morrhuate , sodium psylliate , quinine urethrone , 5% ethanolamine oleate , 1% polidocanol , sodium tetradecil sulfate , and hypertonic saline .
we present our experience in treating palatal vascular malformation using direct ethanol sclerotherapy as a cheep therapeutic modality for treatment of deeply seated vascular lesion .
twenty one - year old saudi male patient presented to the department of oral and maxillofacial surgery clinic at king abulaziz university complaining of swelling on the right side of the face ( figure 1a ) .
extra - oral clinical examination revealed swelling on the right side of the face , which is soft and compressible .
intraoral examination showed a bluish swelling at the junction of soft and hard palate that bleed easily on touch .
computerized tomography ( ct ) and magnetic resonance image ( mri ) studies revealed that it is a vascular malformation that extends to the infratemporal area ( figure 2 ) .
the patient was advised to have ethanol directly injected into the lesion as surgical excision was hazardous and difficult to achieve .
patient disagree with that option and disappear for 18 months and returned back with severe gingival inflammation around the whole right maxillary quadrant which appeared covered by heavy calculus as he could not brush his teeth because of bleeding that he described as extensive ( figure 1c ) .
advised was given to the patient that the only treatment option we can offer him is the ethanol injection .
the procedure and possible complications were explained to the patient and family and they agreed to that option . under general anesthesia using fibro optic intubations , as the lesion was approaching the soft palate and there was a fear that routine nasal intubations could be hazardous to the airway , contrast medium was injected toward the engorged vascular lesion using a no .
18 venipuncture catheter ( to determine the volume of the growths before treatment ) , followed by injection of one - third to one - quarter cavity volume of 95% ethyl alcohol into the tumor cavity according to lee and chen ( figure 3 ) .
the patient was kept intubated for 48 h to guard his airway , as there was edema .
intravenous dexamethasone ( 0.1 mg / kg ) was given for 3 days postoperatively to control inflammation .
patient was discharged from the hospital on the fifth post injection day and kept follow up every week for the first three months , then every 2 weeks for another 3 months .
the lesion remarkably shrinks and the hygienist was able to thoroughly scale his teeth without any bleeding ( figure 4 ) .
figure 1a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .
the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw .
a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .
the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw .
figure 2a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .
c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes .
a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .
c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes .
figure 3a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion .
a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion .
figure 4a ) postoperative picture of the lesion one and b ) three months following injection .
a ) postoperative picture of the lesion one and b ) three months following injection .
there were no complications reported in this case except some palatal ulceration which is improving with every visit .
treatment success was determined clinically by reduction in lesion size , ability of the patient to chew and to clean his teeth without bleeding , patient experience to pain and by patient satisfaction .
for example : surgical excision can lead to significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding and the complicated anatomy of the face and neck , may result in significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding if surgery is adopted .
laser therapy presents higher costs to the patient and may result in skin atrophy , transient or permanent hyperpigmentation , slight depression of the skin and scarring .
intralesional injection of corticosteroids may cause adrenal suppression , slowed weight gain , atrophy , and necrosis .
the use of sclerotherapy in the management of vascular anomalies has increased and replaced the traditional role of surgical therapy , especially for the venous lesions that are surgically difficult or at inaccessible areas .
it has the advantage of low cost , no external scaring , and few complications compared to surgical treatment .
varieties of sclerosing agents have been used for the management of various vascular malformations such as 5% sodium morrhuate , sodium tetradecyl sulphate , ethanolamine oleate , ok432 , bleomycin , ethanol , and hypertonic saline , alone or in various combinations .
absolute ethanol was adopted as a new scleroagent for this complex form of venous defects to improve overall treatment results with acceptable morbidity and recurrence rates .
ethanol is used most of often due to its low cost , antiseptic quality , wide availability and easy of use ; however , it requires general anesthesia because the procedure is very painful .
the aim of treatment is to eradicate the lesion nidus completely , which is the fundamental abnormality as even the smallest residual nidus may expand to form a recurrence . in the presented case it was difficult to achieve complete eradication of the lesion by surgical approach because of the possible hazards that are associated with the affected site .
it has been suggested that ethanol cause protoplasm precipitation which subsequently causes permanent obliteration of the vessel lumen . also do et al .
reported that ethanol has shown the ability to induce protein denaturation of the endothelial cells with subsequent vessel wall denudation and interruption , which results in the complete obliteration of the vessel lumen rather than just simple obstruction .
although various complications were reported to develop during the procedure such as local skin necrosis and fibrosis , haemoglobinuria , anaphylaxis , and some major complications were rarely reported including blood loss , acute pulmonary hypertension with cardio - pulmonary collapse , therefore , lee and chin used rubber bands to compress the patient 's forehead and chin to occlude facial venous return and minimize complications , also hammer et al . recommended to inject the ethanol slowly , over a period of several minutes and not exceeding 1 cc / kg of body weight while monitoring the pulmonary artery pressure to avoid such a catastrophic situation
also , ethanol should only be used in the head and neck region by individuals and centers with professional expertise and experiences including maxillofacial surgeon , general radiology , general medicine , cardiovascular medicine , physical medicine and rehabilitation , dermatology , psychiatry , nuclear medicine , and social services with maximum efficiency to handle or at least minimize various anticipated complications .
also , careful and routine monitoring of pulmonary arterial pressure are required by the interventional radiologist , and anesthesiologist .
vascular malformations were treated with direct percutaneous injection of absolute ethanol , which resulted in remission , and alleviation of their symptoms , with no major complications .
this technique provides a simple , low cost and reliable alternative treatment for venous malformation in the face and neck .
absolute ethanol sclerotherapy can deliver excellent results as an independent therapy to the infiltrating type of extra - truncular form of vm .
complications can occur but are rare . the morbidity involved should be clearly understood and accepted by the patient and family , and the risk of acute and chronic complications , either major or minor , | arteriovenous malformation is treated by variety of techniques over the years .
sclerotherapy is considered an effective and conservative technique for the treatment of benign vascular lesions and replaced the traditional role of surgical therapy , especially for the venous lesions that are surgically difficult or at inaccessible areas .
absolute ethanol was adopted as a new sclerosant agent for this complex form of venous defects to improve overall treatment results with acceptable morbidity and recurrence rates .
sclerotherapy has the advantage of no external scaring , low cost , and few complications in comparison to the surgical treatment . |
in the 1980s , the prevalence of rickets in extremely low birth weight ( elbw ) infants was around 50% and was 17% for fractures ( 1 , 2 ) .
after introduction of a high mineral containing diet in the late 1980s , the prevalence decreased to 30% or lower ( 3 , 4 ) .
there is little information , however , beyond 1985 , concerning the incidence of rickets in extreme prematurity and factors that relate to its occurrence .
in addition to inadequate mineral intake as a major contributing factor ( 5 ) , chronic co - morbidities , like chronic lung disease , short bowel syndrome , and parenteral nutrition associated cholestasis ( pnac ) could also be related factors ( 6 ) .
medications used in the neonatal intensive care unit ( nicu ) , such as steroids , methylxanthines , and diuretics could theoretically increase the risk of inadequate bone mineralization ( 7 - 9 ) .
other risk factors , such as immobilization from sepsis , cerebral pathology , muscular disorders and paralysis are associated with bone disease of prematurity ( 10 ) .
since mineral enriched formula for the preterms began 30 yr ago , it is the standard practice to provide minerals in an attempt to reach in utero mineral accretion rates . in theory , under ideal conditions , the adventage of high mineral intake formulas should be associated with a marked reduction of rickets .
however , it is often practically difficult to achieve ideal nutrition support in sick elbw infants , the current incidence of rickets in elbw infants is unknown , and the relationship of the severity of overall illness and degree of prematurity in this population is unclear .
the aim of this study was to characterize the risk factors for development of rickets in elbw infants fed with high mineral containing preterm formula .
as a retrospective case - control study , medical records of 55 elbw infants admitted into the severance children 's hospital nicu from 2004 to 2008 that survived until 1 month of age were reviewed .
all infants had their serum alkaline phosphatase ( alp ) , calcium ( ca ) and phosphate ( p ) levels reported weekly and bone screen radiography at 1 month of age . for nutritional care ,
fortified human milk ( ca 131 mg/100 kcal , p 71 mg/100 kcal ) or a mineral fortified preterm formula ( ca 112 - 141 mg / kcal , p 52 - 63 mg / kcal ) was used . during parenteral nutrition , ca , p and multivitamins with trace minerals were added to parenteral nutrition , appropriately .
grade 1 was defined as a loss of dense white line , increased lucency of bone shaft , and thinning of cortex ; grade 2 showed irregularity and fraying of a metaphysis with splaying and cupping ; and grade 3 presented evidence of fractures . as co - morbidities , hyaline membrane disease ( hmd ) , patent ductus arteriosus ( pda ) , necrotizing enterocolitis ( nec ; using the modified bell 's staging criteria ) , parenteral nutrition associated cholestasis ( pnac , defined as direct bilirubin [ db ] greater than 2.0 mg / dl ) , bronchopulmonary dysplasia ( bpd ; using nih classification ) , retinopathy of prematurity ( rop , included above stage 2 ) , and intraventricular hemorrhage ( ivh , above grade 3 ) were analyzed .
severe pnac was defined as elevation of db greater than 4.0 mg / dl for more than 1 month and aspartate aminotransferase ( ast ) and alanine aminotransterase ( alt ) greater than 60
moderate / severe bpd was defined in infants requiring either 28 days of supplemental oxygen therapy with oxygen , nasal continuous positive airway pressure ( cpap ) or positive pressure ventilation at 36 weeks postmenstrual age or discharge , whichever came first , whose gestational age was younger than 32 weeks , or at 56 days postnatal age or discharge , whichever came first , in infants whose gestational age was older than 32 weeks .
statistics were calculated with the student t - test and chi - square test for means and frequencies .
numeric data are presented as mean and standard deviation because the data were normally distributed .
calculations were performed with spss software version 17.0 ( spss , chicago , il , usa ) .
the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) .
the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) .
twenty - four elbw infants ( 26.3 1.9 weeks , 792 99 g ) were diagnosed as rickets of prematurity , and 31 infants without rickets ( 27.2 2.3 weeks , 871 93 g ) were the control group .
rickets of prematurity was diagnosed in 43.9% of elbw infants and fracture in 13% . for the rickets group , 7 infants ( 29% ) showed g1 , 14 infants ( 58% ) g2 and 3 infants ( 13% ) g3 .
rickets of prematurity was diagnosed at 48.2 16.1 days of life and improved by 85.3 25.3 days of life . in patient characteristics such as mean gestational ages , birth weight , gender , and frequency of intrauterine growth retardation ( iugr )
the cumulative amounts ( either enteral or parenteral ) of ca and p administration during hospitalization were not different between the groups . for the rickets group in comparison with the control group , mean days on oxygen and ventilator use were significantly longer ( p < 0.05 ) .
the duration of hospitalization and parenteral nutrition were significantly longer in the rickets group versus the control group ( table 1 ) . in univariate analysis , elbw infants with rickets had a significantly higher occurrence of pda ( p = 0.025 ) , pnac ( p = 0.041 ) , severe pnac ( p = 0.013 ) , bpd ( p = 0.019 ) , and moderate / severe bpd ( p = 0.012 ) ( table 2 ) .
common neonatal illnesses such as hmd , air leak , nec , rop , ivh and periventricular leucomalacia ( pvl ) were not different between the groups . in multiple regression analysis after adjustment of gestation and birth weight , rickets in elbw infants significantly correlated with severe pnac ( or 18.5 ; 95% ci , 1.1 - 285 ; p = 0.042 ) and moderate / severe bpd ( or 3.2 ; 95% ci , 1.2 - 26.5 ; p = 0.04 ) ( table 3 ) .
the mean level of peak alp was significantly higher in the rickets group than the control group ( 952.2 413.8 vs 524.7 158.2
iu / l ) , while the mean level of the lowest ca and p levels were not different between groups ( table 4 ) .
in the 1980s , fortified mineral formulas were introduced to reduce the rates of rickets and fractures in very low birth weight ( vlbw < 1,500 g ) infants ( 11 ) .
reports at that time indicated that up to 30% of vlbws had rickets with fractures ( 12 ) . with advances in preterm nutrition , especially with the introduction of mineral fortified formulas , theoretically
the incidence of rickets of prematurity should have dropped ( 3 , 4 ) . however , for increasing the survival of elbw infants , significant co - morbid conditions could affect actual mineral intake which might mitigate the effect of high mineral fortification ( 13 ) .
this is the first study to focus on the incidence of rickets in elbw infants after 2 decades of fortified formula use in korea .
it is also the first to characterize and compare risk factors and neonatal morbidities for rickets of prematurity in elbw infants .
we found that the incidence of radiologic rickets in elbw infants was extremely high at 44% ( 24/55 ) due to increased survival , and the incidence of significantly increased 18 times and 3 times for severe pnac and moderate to severe bpd , respectively .
factors commonly thought to relate to rickets of prematurity include mismatch of postnatal intake of minerals compared to intrauterine mineral transfer , low bone loading effects in the nicu , intrauterine growth restriction , chorioamnionitis , steroids , methylxanthines and diuretic usage and necrotizing enterocolitis ( 7 - 10 , 14 - 16 ) .
inadequate mineral nutrient ( calcium and phosphorous ) intake appears to play a major role in causing rickets of prematurity ( 10 , 17 ) . prolonged parenteral nutrition ( pn ) and delayed enteral nutrition complicate delivery of adequate mineral intake and would be particularly acute in elbw infants ( 10 ) .
it is standard practice in nicu to provide high mineral preterm formula and human milk fortifier as an attempt to reach in utero mineral accretion rate , including the use of minimal early enteral feeding , which appears to improve bone mass at term ( 3 , 17 - 19 ) .
however , in elbw infants , co - morbid illnesses often preclude full enteral tolerance and the actual delivery of mineral nutrients ( 13 ) .
further , when pn is needed , the provision of minerals in pn can be limited by solubility issues compounded by fluid restriction in sick infants ( 3 ) .
specific chronic co - morbidities , particularly chronic lung disease and short bowel syndrome which is generally associated with prolonged parenteral nutrition and pnac , are potentially serious risk factors for poor bone mineralization ( 6 , 20 , 21 ) .
thus our findings of markedly higher risk of rickets in elbw infants with pnac ( 18 times ) and bpd ( 3 times ) support these associations
. the limitations of this study are a small sample size of a single institution and a retrospective design . in conclusion
, there is a high incidence ( 44% ) of rickets in elbw infants , which shows an 18-fold increase in severe pnac and a 3-fold increase in moderate to severe bpd .
we suggest that in elbw infants with severe pnac or moderate / severe bpd , aggressive prevention or treatment for rickets and fractures can be instituted earlier in the course of management . | risk factors for rickets of prematurity have not been re - examined since introduction of high mineral formula , particularly in elbw infants .
we analyzed the incidence and the risk factors of rickets in extremely low birth weight ( elbw ) infants .
as a retrospective case - control study from 2004 to 2008 , risk factors were analyzed in 24 patients with rickets versus 31 patients without .
the frequency of rickets in elbw infants was 24/55 ( 44% ) .
infants with rickets were diagnosed at 48.2 16.1 days of age , and improved by 85.3 25.3 days . by radiologic evaluation ,
29% were grade 1 rickets , 58% grade 2 and 13% grade 3 . in univariate analysis , infants with rickets had significantly higher incidence of patent ductus arteriosus , parenteral nutrition associated cholestasis ( pnac ) , severe pnac and moderate / severe bronchopulmonary dysplasia ( bpd ) .
in multiple regression analysis , after adjustment for gestation and birth weight , rickets significantly correlated with severe pnac and with moderate / severe bpd .
serum peak alkaline phosphatase levels were significantly elevated in rickets ( p < 0.001 ) . in elbw infants ,
the incidence of rickets of prematurity remains high and the incidence of severe pnac and moderate / severe bpd was significantly increased 18 and 3 times , respectively . |
kyphosis is a progressive curvature of the thoracic spine , which results in rounding or bowing of the back .
a curved spine accompanied by restricted neck motion and poor lung reserve present challenges for an ophthalmic surgeon , who is used to patients neck and head resting flat on the operating table and the eye in the horizontal position .
intraoperative difficulties include poor view , inability to maneuver surgical instruments properly and constant threat of elevated vitreous pressure .
several innovative solutions have been published , including the maximal reverse trendelenburg position and pillows under the head and neck , use of a donut and wedge - shaped head positioner , allowing patients to remain in the upright sitting position while their head is positioned horizontally in a modified waiting room chair , performing surgery while standing [ 5 , 6 ] , using a 4-section orthopedic operating table , or laying the patients on their sides . however
a 49-year - old obese male with controlled secondary glaucoma needed cataract surgery in the right eye .
the past ocular history was positive for recurrent uveitis and glaucoma shunt surgery in the right eye .
cataract density was + 4 , and the best - corrected visual acuity was 20/200 .
the anterior chamber was shallow and the glaucoma shunt tube was visible at the 11 o'clock position .
the patient was planned for pupillary dilation using iris retraction hooks , lens capsule staining with trypan blue and phacoemulsification with intraocular implantation under local anesthesia .
the anesthesiologist was reluctant to use general anesthesia because of the patient 's disability and poor health . in the operating room ,
the patient could only recline to about 40 from the vertical axis despite maximal reverse trendelenburg position and a number of pillows under his back , neck , head and legs ( fig .
2 ) . while trying to focus the microscope , it became evident that by grasping the eye at the inferior limbus with a fine forceps and pulling it upwards , it could be moved to a more desirable horizontal position .
a single 7/0 vicryl ( polyglactin ) corneal retraction suture was placed at the limbus at the 6 o'clock position to achieve the desired view ( fig .
the assistant surgeon was able to rotate the eye to the desired positions during the whole procedure by relaxing or pulling the suture .
patients with kyphosis may present several unique challenges to an eye surgeon , the foremost being the inability to lay the head and neck in the ideal horizontal position .
the resulting poor view makes focusing and manipulation of tissues and instruments difficult . tilting the head end of the operating table down and raising the patient 's legs may compromise venous return from the orbit , causing venous engorgement and elevated posterior vitreous pressure during surgery .
in addition , the patient 's poor pulmonary reserve itself may further impede orbital / jugular venous return , resulting in increased posterior vitreous pressure . other features of kyphosis that may adversely affect intraocular surgery are tenderness and stiffness of the back and difficulty in breathing .
several practical solutions have been described by very experienced surgeons but complications may still occur .
a wedge - shaped pillow with a built - in donut for the positioning of the head has been used by some surgeons .
livingston and mackool recommended using more than one of these positioners in severe cases .
another approach suggested performing cataract surgery while standing , using loupes and making an inferotemporal scleral tunnel [ 5 , 6 ] .
the surgeons were unable to remove the subincisional cortex because of a shallow anterior chamber secondary to elevated posterior vitreous pressure .
prasad et al . recommended using a 4-section orthopedic operating table to enable an extreme reverse trendelenburg position for cataract and glaucoma surgery .
however , because of a blurred vision , these authors were unable to perform curvilinear capsulorhexis and had to resort to can - opener capsulotomy .
in short , there is no single solution for the different groups of difficult patients .
we have found that a single retraction suture at the inferior limbus can rotate the eye to any desired position .
this may allow both the patient and surgeon to be in a comfortable position without compromising good view .
it may be tried practically in all patients with kyphosis or other conditions that prevent them from reclining such as torticollis , chronic obstructive pulmonary disease , chronic congestive heart failure or morbid obesity .
the surgeon can still sit on the preferred temporal side of the patient and perform intraocular surgery of choice . with this maneuver
, it may be unnecessary to resort to extreme and uncomfortable tilting of the patient and avoid complications such as those described above .
the authors have no financial or proprietary interest in the materials described in the article . | we describe a 49-year - old man with advanced kyphosis and dense cataract , who could only recline to about 40 from the vertical axis despite a maximal reverse trendelenburg position and pillows under the head , neck , shoulders and knees . with a single corneal retraction suture at 6 o'clock , the eye could be rotated horizontally , which enabled the surgeon to perform a complex cataract surgery despite prior glaucoma shunt , posterior synechiae , a small pupil and the need to stain the capsule .
as the eye can be brought into any desired position with a retraction suture , patients with kyphosis or other conditions that prevent them from assuming a supine position can still have safe intraocular procedures .
this maneuver reduces the need to tilt patients to an uncomfortable position that may cause pain , increased breathing difficulty and elevated posterior vitreous pressure . |
using water as solvent in the organic reactions is one of the most important targets to organic chemists because of the easy availability , nontoxicity , and ecofriendly nature of the water [ 17 ] . in this endeavour
, a number of chemical reactions such as diels alder , hetero diels - alder , 1,3-dipolar cycloaddition , oxidations , reductions , and others are performed successfully in water [ 13 ] .
also , it is reported that in few cases addition of the water increases the rate and the yield of a reaction and also enhances the enantioselectivity in a chiral synthesis .
but the main problems associated with water as a solvent is its poor ability to solubilise organic reactants and incapability to create anhydrous condition for moisture sensitive organic compounds and catalysts . to overcome the solubility problem , generally a surfactant is introduced to the reaction mixture .
the surfactant , due to its hydrophobic and hydrophilic nature , forms micelles with water insoluble organic compounds and promote the desired reactions to occur inside the hydrophobic ambience of the micelle core [ 9 , 10 ] .
cleavage of the c = n bonds is a very important transformation in organic synthesis as the c = n functionality is widely used to protect both the carbonyl and amines .
there are a number of methods used for the cleavage of c = n bonds which include acidic reagents [ 1113 ] , oxidizing agents , metallic salts [ 15 , 16 ] , ( phseo)2o , nahso3 , and others .
most of these methodologies suffer from serious drawbacks like involvement of strong lewis and bronsted acids , use of toxic and costly transition metals ( i.e. , cr , pd , co ) , low temperature , longer reaction time , low yield , and difficulties in isolating the products
. therefore , development of efficient , mild and environment friendly reagents are always necessary . on the other hand
, bisindoles are recently emerging as extremely important class of compounds because of their novel antibacterial and anticancer activities [ 1921 ] .
that is why a number of methodologies have also been postulated for the synthesis of bisindoles [ 2229 ] . in our previous communications
, we reported that surfactant- ( sds- ) mediated cleavage of c = n bonds could be achieved with acetic anhydride and surfactant - i2-water can be used for the deprotection of imines to carbonyls .
we have also shown that bis- and tris ( indolyl)alkanes can be synthesized in presence of bronsted acid in water . in continuation of our research in hydrated media , herein , we wish to disclose the dual catalytic activity of the system i2-sds - h2o which behaves as a lewis acid for the cleavage of 2,3-diaza-1,3-butadiene , 1-aza-1,3-butadienes , and oximes to produce carbonyls and amines , and the resulting reaction mixture reacts with indoles to produce bis ( indolyl)alkanes in situ at room temperature under neutral conditions .
for an initial study , molecular iodine was added to a mixture of 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) ( 1 mmol ) and indole ( 2a ) ( 2 mmol ) in water .
it was presumed that the activated imine should produce carbonyl compound in the reaction mixture which might be trapped with indole .
but only a trace amount of 3,3-bis(indolyl)phenylmethane ( 3a ) was found to be formed in the reaction .
we envisioned that the poor yield of the product may be due to the insolubility of organic substrates in water .
accordingly , we added a surfactant ( sds ) to the reaction flask . to our delight ,
the reaction produced isolable amount of 3a as a brown solid but half of the 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) left unreacted in the reaction mixture .
so amount of indole ( 2a ) was doubled ( 4 mmol ) and the same reaction condition successfully produced quantitative amount of 3a .
the product was filtered out and the filtrate was treated with freshly distilled benzaldehyde ( 0.05 mmol ) in ethanol which furnished 1a ( m.p .
this infers the simultaneous involvement of two c = n bonds of bis - anils , leaving behind hydrazine in the reaction mixture .
also , no self - reaction of individual starting materials leading to indole dimer ( 4 ) or pyrrole formation ( 5 ) were observed under the same reaction conditions ( scheme 1 ) .
optimization of the reaction conditions was undertaken by employing different catalyst loadings under various surfactant conditions .
it was found that the best result was obtained by the application of 15 mol% of i2 containing sodium dodecyl sulphate ( sds ) and water at room temperature ( entry 1 , table 1 ) . in absence of the catalyst no formation of 3a
was observed even after stirring for 24 hours ( entry 5 , table 1 ) . to study the scope and limitations of the reaction ,
i2-sds - h2o system was applied to the reaction of indole ( 2 ) and 2,3-diaza-1,3-butadiene derivatives ( table 2 , entries a
the bisindoles were formed in excellent yields under the reaction condition . it was observed that the reaction was relatively faster when an electron withdrawing substituent , for example , no2 , was present in the phenyl ring of the 2,3-diaza-1,3-butadienes ( table 2 , entry e ) in comparison to the electron donating groups , for example , ome and oh ( table 2 , entries b and f ) .
identical results were obtained when 2-methylindole ( 2a ) was used in place of indole ( 2 ) ( table 2 , entries g and h ) .
all the products were characterized by their ir , h nmr , c nmr , and mass spectral data and also by comparison with the literature report ( scheme 2 ) [ 24 , 25 ] . in order to further explore the efficiency of the i2-sds - h2o system the reaction of the oximes 6 and indoles 2 was studied . when oximes ( 1 mmol ) and indole ( 2 mmol ) were allowed to react under the same reaction condition described earlier , bis - indolylalkanes formed ( table 3 ) . the product was filtered off and the filtrate was treated with benzaldehyde in ethanol .
the resulted product was benzaldoxime , which proved the liberation of hydroxylamine during the reaction .
it was found that no michael addition product was formed and only a trace amount of indole dimer 5 could be identified .
the system was also applied to the reaction of 1-aza-1,3-butadienes 7 with indole ( 2 ) which produced bis ( indolyl)alkanes 8 in very good yield eliminating aryl amine in the reaction mixture ( table 4 ) .
all the products were well characterized by comparison of their spectral and mass data with that of the reported value [ 24 , 25 ] .
in conclusion , we have shown the dual catalytic activity of i2-sds - h2o system which deprotects the azadienes , oximes , and azabutadienes and produces bis ( alkyl)indoles in situ when indole is present in the reaction medium .
the two - step reaction can be carried out without using acid , transition metals , and organic solvents . besides , the reaction condition is mild and can be done in water under neutral condition which contributes to the criteria of green chemistry .
h nmr spectra were recorded on avance dpx 300 mhz ft - nmr spectrometer .
chemical shifts are expressed in units relative to tetramethylsilane ( tms ) signal as internal reference .
ir spectra were recorded on ft - ir - system-2000 perkin elmer spectrometer on kbr pellets or in chcl3 .
the solvents for chromatography were distilled before use . in a 50 ml round bottom flask
, 15 mol% of i2 was first dissolved in water ( 10 ml ) .
2,3-diaza-1,3-butadiene ( 1 mmol ) and indole ( 4 mmol ) were added and stirred in the presence of sodium dodecyl sulphate ( sds ) ( 0.02 g ) for the stipulated time .
the product formed was filtered off and washed with water , dried , and recrystallized from ethanol .
identical reaction condition was followed when 1-aza-1,3-butadienes and oximes were used as reactants . in this case , 2 mmol of indoles were used to react with 1 mmol of imines .
colorless solid ; mp : 150152c ; ftir ( kbr ) : 3418 , 3058 , 1623 , 1611 , 1445 , 1093 cm ; h nmr ( 300 mhz , cdcl3 ) : 5.95 ( s , 1h , ar
ch ) , 6.73(s , 2h ) , 7.06 ( t , 2h , j = 6.8 hz ) , 7.187.27 ( m , 3h ) , 7.317.36 ( m , 2h ) , 7.367.42 ( m , 6h ) , 7.98 ( br , s , 2h , nh ) ; c nmr ( 75 mhz , cdcl3 ) : 40.7 , 111.2 , 119.1 , 119.5 , 120.4 , 122.1 , 123.8 , 126.9 , 126.9 , 128.2 , 129.1 , 136.8 , 144.8 ; hrms calcd for c23h18n2 ( m ) : 322.2851 , found 322.2832 ; anal.calcd . : c , 85.70 ; h , 5.59 ; n , 8.69 ; found c , 85.75 ; h , 5.56 ; n , 8.56 .
pink solid ; mp : 76 - 77c ; ftir ( kbr ) : 3415 , 3060 , 1491 , 1465 , 1095 cm ; h nmr ( 300 mhz , cdcl3 ) : 5.91 ( s , 1h , ar ch ) , 6.76 ( s , 2h ) , 7.08 ( t , 2h , j = 8.3 hz ) , 7.22 ( t , 2h , j = 7.9 hz ) , 7.287.42 ( m , 8h ) , 8.01 ( br , s , 2h , nh ) ; c nmr ( 75 mhz , cdcl3 ) : 39.8 , 111.5 , 122.6 , 123.8 , 127.1,128.4 , 129.8 , 130.1 , 130.9 , 131.0 , 131.6 , 137.2 , 143.5 ; hrms calcd for c23h17n2cl ( m ) : 356.7371 ; found 356.7324 ; anal.calcd .
: c , 77.52 ; h , 4.77 ; n , 7.86 ; found c , 77.48 ; h , 4.72 ; n , 7.80 .
brown solid ; mp : 323325c ; ftir ( kbr ) : 3420 , 1720 , 1455 , 1258 cm ; h nmr ( 300 mhz , cdcl3 ) : 5.98 ( s , 1h , ar - ch ) , 6.85 ( s , 2h ) , 7.107.55 ( m , 11h ) , 8.05 ( br , s , 2h , nh ) ,
c nmr ( 75 mhz , cdcl3 ) : 34.8 , 107.0 , 110.0 , 111.5 , 117.8 , 119.9 , 120.0 , 122.5 , 124.8 , 127.1 , 136.8 , 142.2 ; hrms calcd for c21h16n2o2 ( m ) : 312.2621 ; found 312.2611 ; anal.calcd .
c , 80.84 ; h , 5.12 ; n , 8.97 ; found c , 84.05 ; h , 5.15 ; n , 8.94 .
the spectral data of the compounds are available in the supplementary material available online at doi : 105402/2012/635835 . | a novel catalytic system consisting of i2-sds - h2o has been developed which cleaves 2,3-diaza-1,3-butadiene , 1-aza-1,3-butadienes , oximes and in presence of indoles in the medium uses the corresponding aldehyde products to produce bis(indolyl)alkanes in situ .
this one pot simple and mild dual catalytic system works in water at room temperature under neutral conditions . |
parental drug misuse and its effects on children and families are policy priorities in the uk and elsewhere ( flavin & paltrow , 2010 ; hm government , 2010 ) .
polydrug use is the norm among drug users in the uk , and benzodiazepine use is common ( bird & robertson , 2011 ; department of health ( england ) , 2007 ; hay , gannon , casey , & mckeganey , 2009 ; jones , mogali , & comer , 2012 ) . increased risks and harm , and a range of adverse effects are associated with long - term benzodiazepine use ( lader , 2011 , 2014 ; obrien , 2005 ) . however , existing research on drug use and parenting has tended to focus on opioids , opioid substitution therapy ( ost ) , or crack cocaine ( radcliffe , 2009 , 2011 ; rhodes et al . , 2010 ) , and in the case of pregnancy , also alcohol , nicotine , marijuana and amphetamines ( behnke & smith , 2013 ) . in this paper ,
we examine accounts provided by polydrug - dependent parents about their use of , and dependence on , benzodiazepines as compared to ost .
benzodiazepine use is highly prevalent among drug users worldwide ( jones et al . , 2012 ) .
opioid - dependent and ost patients use benzodiazepines to prolong and enhance the effects of opiates ( jaffe et al . , 2004 ) , and increased rates of dependence are associated with co - occurring psychosocial problems ( jones et al . , 2012 ) .
weizman , gelkopf , melamed , adelson , and bleich ( 2003 ) estimated a lifetime prevalence of benzodiazepine dependence in the opiate - dependent population ( in or out of treatment ) ranging from 6194% .
benzodiazepines were originally marketed in the 1960s for the relief of anxiety , stress and insomnia ( who , 1996 ) and remain one of the most widely used psychoactive drugs ( lader , 2011 ) .
their use within the general population is common , particularly among females ( obrien , 2005 ) .
the gendered cultural meanings of diazepam ( valium ) , a well - known benzodiazepine , was cemented in the 1966 rolling stones song mother s little helper .
benzodiazepines are recommended for the short - term treatment of anxiety and insomnia ( baldwin et al . , 2013 ) .
tolerance and withdrawal effects can develop after only a few weeks of treatment ( lader , 2011 ) .
adverse effects include sedation , impaired cognitive and psychomotor functioning , and increased likelihood of accidents and injuries ( lader , 2011 ; obrien , 2005 ) .
withdrawal effects vary in severity and duration , and include anxiety , insomnia , depression , impaired memory , muscle spasms and tension , and rarely , seizures and psychosis ( lader , 2011 ) .
combined benzodiazepine and opioid use is associated with increased sedation , decreased psychomotor performance ( lintzeris , mitchell , bond , nestor , & strang , 2007 ; lintzeris & nielsen , 2010 ) , poorer psychosocial adjustment and general health , heightened risk - taking behaviour , and increased risk of overdose ( bird & robertson , 2011 ) . however , evidence is limited ( jones et al . , 2012 ) , and
few studies have focused on the combined effects of opioid and benzodiazepine dependence on pregnancy or parenting ( acmd , 2003 ) , despite evidence to suggest that both drugs are problematic in these contexts . whilst there is evidence that parental opioid use can compromise parenting capacity and pregnancy outcomes ( acmd , 2003 ) , the health and wellbeing of children and families can also be negatively affected by social and structural factors such as poverty , poor housing , unemployment , parental stress , domestic violence and criminal justice involvement ( hepburn , 2004 ; klee , jackson , & lewis , 2002 ) .
however research indicates that despite this complexity , there is a tendency among service users and service providers to focus on drug use alone ( banwell & bammer , 2006 ; chandler et al . , 2013 ) , and in most cases the primary drug of misuse ( winklbaur et al . , 2008 ) .
while this focus may relate to concerns regarding the effects of specific drugs per se on parenting capacity and the immediate safety of children , it could also result in a limited view of drug dependence that neglects to consider polydrug use and the impact of socio - economic and structural inequalities . while the use of opioids and ost is stigmatising , especially in the context of pregnancy and parenthood ( holland , forrester , williams , & copello , 2013 ) , use of benzodiazepines in the context of mothering has historically been characterised in a less problematic ( though not entirely positive ) manner ( metzl , 2003 ) .
we found no published literature which compared benzodiazepine and opioid dependence , from the perspective of parents , or expectant parents .
therefore the aim of our study was to address this gap in the evidence and to undertake a comparative thematic analysis of the ways in which parents accounted for their use of opioids , ost and benzodiazepines during and after pregnancy .
this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .
the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .
the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .
ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .
there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .
notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .
participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .
inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .
all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .
five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .
all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .
eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .
this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,
semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .
this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .
the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .
accounts were compared and contrasted and analytic themes were generated and agreed by the research team .
participants gave informed consent and were provided with a 20 voucher for each interview completed .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just ,
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing .
accounts of substance use during pregnancy were for women especially more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .
as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal )
i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .
carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .
as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal )
i ve been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .
carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .
another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .
these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was controlled and unproblematic .
thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women
was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ?
where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and
i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines .
while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .
the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .
the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .
ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .
there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .
notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .
participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .
inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .
all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .
five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .
all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .
eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .
this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,
semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .
this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .
the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .
accounts were compared and contrasted and analytic themes were generated and agreed by the research team .
participants gave informed consent and were provided with a 20 voucher for each interview completed .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,
the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot
they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :
im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases
, women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,
the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot
they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :
im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases
, women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .
a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .
as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal )
i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .
carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .
as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal )
i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,
cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .
carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .
another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .
these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was
thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women
was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ?
where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like
, because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines .
while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
this longitudinal qualitative study explored accounts of poly - drug use within the context of pregnancy and parenting , highlighting differential understandings and practices surrounding benzodiazepine and opioid use .
our findings illuminate a number of issues with implications for theory , policy , prevention and education .
the accounts in our study resonate with others ( klee , 1998 ; radcliffe , 2011 ) in demonstrating the morally difficult nature of being a drug - using parent , and the extent to which stigma can play an important role in the day - to - day lives of parents .
our findings suggest that while drug use , in general , is seen as incompatible with parenting , there are clear indications that some drugs , and types of addiction , are more acceptable than others .
crucially , our analysis indicates important differences in the way opioid and benzodiazepine use are understood and narrated within the context of parenthood . unlike benzodiazepine use
, participants in this study often described ost in negative terms even when they acknowledged that ost was helpful in treating opioid dependence .
this dominant negative discourse on ost by drug users themselves , has been described elsewhere ( harris & mcelrath , 2012 ; lloyd , 2013 ; radcliffe & stevens , 2008 ) , and highlights the unique stigma attached to ost for the treatment of opioid dependence ( keane , 2013 ) .
participants described using benzodiazepines in constructive ways to enhance social and emotional functioning and engagement in normal life appealing to a self - medicating hypothesis of addiction to explain and justify continued use ( jones , et al . , 2012 ) .
these meanings served to legitimise benzodiazepine dependence , contrasting sharply with participants alignment of ost with
junkie identities , as documented elsewhere ( keane , 2013 ; radcliffe & stevens , 2008 ) . in the context of parenthood ,
parents were more likely to describe a desire to reduce opioid rather than benzodiazepine use , and there was evidence that illicit benzodiazepine use was considered less harmful than opioid use .
this is noteworthy , because alongside this complacency were occasional notes of concern , particularly in relation to benzodiazepine use by men , and its link with increased aggression and domestic abuse ( reported by some women participants ) . accounts from this study provide a tentative indication of the way gender may shape understandings of benzodiazepine use .
the long - term use of benzodiazepines by mothers was framed as being therapeutic and unproblematic .
in contrast , while men reported using benzodiazepine for similar reasons , their accounts indicated that practitioners did not legitimise this and prescriptions were denied .
these findings sit uneasily with the dominant recovery agenda in uk drug policy ( valentine & treloar , 2013 ; wardle , 2012 ) , where abstinence from all drug use is assumed to be both desirable and necessary for social reintegration .
it may be that in practice recovery refers to particular categories of drugs , with the hierarchy of acceptability identified among service users in this study being apparently mirrored in prescribing practices and approaches taken to treating addiction by clinicians ( anthierens , habraken , petrovic , & christiaens , 2007 ) .
thus , while opioid - dependent individuals may express a desire to reduce their opioid prescriptions , this is not necessarily reflected in their orientation towards other drug use , particularly benzodiazepines .
as such , the claim that opioid - dependent individuals would prefer abstinence - based treatment approaches may result from the unique stigma attached to opioids and may not apply to all drug use , or all drugs of dependence ( lloyd , 2013 ; mckeganey , morris , neale , & robertson , 2004 ; neale , nettleton , & pickering , 2011 ) .
our findings suggest a need for further preventative measures and targeted education to address benzodiazepine use and dependence within the context of pregnancy and parenting , especially in populations of parents engaged in ost programmes where stability on opioids may detract from issues associated with benzodiazepine use and dependence . reviews of benzodiazepine prescribing ( sirdifield et al . , 2013 ) ,
identify a variety of reasons for inconsistent practice , and conclude that improved education and training , provision of non - pharmacological interventions , and enhanced communication with patients is required .
our findings support these recommendations , especially the need for enhanced education and training of both clinicians and patients , and extra provision of non - pharmacological interventions to promote the psychological and social functioning of parents on ost .
this was an in - depth , qualitative exploration of the accounts of nineteen drug - dependent parents on ost in a specific geographical area .
as such , our findings and conclusions should be read as indicative and suggestive rather than definitive .
further qualitative exploration in different geographical areas and populations is needed , as it is likely that local or cultural contexts may shape the types of narratives expressed in respect of the range of substances consumed .
our findings may have limited applicability to opioid dependent parents who are not engaged in ost programmes .
however , the contrasting accounts depicted here underline the importance of qualitative research in illuminating understandings about the meanings that drugs of addiction have for those who use them ( neale , allen , & coombes , 2005 ) .
although long - term benzodiazepine use and dependence among opioid - dependent populations has been a topic of concern for some decades ( who , 1996 ) and is associated with increased risks and harm ( lader , 2011 , 2014 ) , research regarding the impact on pregnancy , parenting and family life has been limited ( acmd , 2003 ) .
our findings suggest that parents frame benzodiazepine use and dependence as largely unproblematic , less stigmatising and more legitimate than ost .
we suggest these divergent accounts can be understood as drawing on differing cultural meanings attached to ost and benzodiazepine use , which are reflected in policy and practice . as such , prevention ,
education and policy initiatives should pay greater attention to the kind of narratives and strategies that are employed in addressing benzodiazepine use and dependence within opioid dependent populations , especially in respect of the parenting and child welfare agenda . | aimsto explore the ways in which opioid - dependent parents accounted for their use of opioids and benzodiazepines during and after pregnancy.methodslongitudinal qualitative interviews [ n = 45 ] with 19 opioid - dependent adults recruited in scotland , uk , were held during the antenatal and post - natal period .
interviews focused on parenting and parenting support within the context of problem drug use and were analysed using a narrative informed , thematic analysis .
findingsthe majority of participants described using benzodiazepines in addition to opioids .
almost all indicated a desire to stop or reduce opioid use , whereas cessation or reduction of benzodiazepines was rarely prioritised .
in stark contrast to opioid dependence , benzodiazepine dependence was portrayed as unproblematic , therapeutic and acceptable in the context of family life . whereas opioid dependence was framed as stigmatising , benzodiazepine use and dependence was normalised .
an exception was benzodiazepine use by men which was occasionally associated with aggression and domestic abuse.conclusionsdrug-dependent parents attach different meanings to opioid and benzodiazepine use and dependence in the context of parenthood .
divergent meanings , and stigma , may impact on stated commitment to stability or recovery from dependent drug - use .
attention should be paid to the way in which policy and practice regarding ost and benzodiazepines reflects this divergence . |
in industrialized countries , cardiovascular disease ( cvd ) remains the predominant cause of morbidity and mortality .
it is current scientific opinion that cvd occurs as a part of a series of events that form a continuum .
the first stage of this continuum corresponds to the presence of risks factors that predispose to tissue injury , such as hypertension , dyslipidemia , and diabetes .
these risk factors usually activate a complex range of pathogenic pathways leading to the development of atherosclerosis and ischemic heart disease .
failure to effectively manage any stage of this continuum results in ventricular hypertrophy , heart failure , end - stage heart disease , and cardiovascular death .
one of the molecular pathways that could be activated in response to these risk factors and be involved in cvd development is that of osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) , as reviewed in .
opg and trail are both members of the tnf superfamily , and they are widely expressed in different tissues , including the heart and the vessels , in health and disease states [ 4 , 5 ] . although initially opg actions seemed to be limited to bone metabolism and those of trail to host defense against tumors , recent studies have suggested that both molecules might actually be involved in cvd development and progression .
epidemiological studies have shown that there is an association between circulating opg , trail , and cvd .
opg was found to be directly correlated [ 5 , 8 , 9 ] , while trail was inversely correlated , with cardiovascular morbidity and mortality [ 10 , 11 ] .
likewise , opg was significantly associated with the level of c - reactive protein , which is an independent predictor of acute vascular events and adverse outcomes , while trail was negatively correlated with it .
recently , secchiero and colleagues have shown that patients with coronary artery disease have an increased opg / trail ratio , which further increases in patients developing heart failure .
consistent with this epidemiological data , experimental studies suggest that opg and trail have opposite actions , as opg has proinflammatory and proatherogenic effects [ 14 , 15 ] , while trail seems to be anti - inflammatory [ 16 , 17 ] and antiatherogenic [ 1820 ] . based on these premises
, we hypothesized that dyslipidemia and diabetes , which are two well known risk factors for cvd , could modify the vascular and cardiac expression of opg and trail , leading to an increased opg / trail ratio at a tissue level .
the aim of this study was to explore the vascular and cardiac changes of opg and trail in experimental models of dyslipidemia and diabetes .
a total of 12 male c57bl/6j ( cnt ) mice and 24 male apolipoprotein e null ( apoe ) mice on a c57bl/6j background , aged 6 weeks , were studied for 14 weeks . at 6 weeks of age ,
apoe mice were further randomized to saline ( apoe ) or streptozotocin ( apoe + dm ) , which were delivered intraperitoneally in five consecutive daily doses .
the daily dose of streptozotocin ( stz ) ( sigma - aldrich ) was 55 mg / kg of body weight .
after one week , blood glucose was checked and only the mice with blood glucose levels greater than 15 mm were included in the apoe + dm group . during the study period , all the mice were fed with a standard diet .
the animals were kept in a temperature - controlled room ( 22 1c ) on a 12 h light/12 h dark cycle with free access to food and water and they were fed ad libitum for the length of the study . after 14 weeks , the animals were anesthetized with an intraperitoneal injection of thiobutabarbital sodium ( inactin , sigma - aldrich ) ( 60 mg / kg body weight ) and sacrificed by exsanguination via cardiac puncture .
all the animal experiments were approved by the animal ethics committee of the university of trieste ( i d 28.0.2008 ) and the italian ministry of health , conforming to the guide for the care and use of laboratory animals . at the end of the study , we measured body weight and systolic blood pressure ( sbp ) .
in particular , sbp was assessed by a computerized noninvasive tail cuff system in conscious mice .
glucose , total cholesterol , hdl cholesterol , and triglycerides were measured in the sera by autoanalyzer technique ( hitachi 917 , tokyo , japan ) .
circulating levels of opg and trail were measured in the sera by elisa ( r&d systems duoset # dy459 and # dy1121 ) . in half of the animals in each group ,
aortae were collected and placed in 10% neutral - buffered formalin for atherosclerosis quantification , before being processed for immunohistochemical analyses . in the other half ,
aortae were snap - frozen in liquid nitrogen and stored at 80c for rna extraction and gene expression analyses .
briefly , aortae were cleaned of excess fat and stained with sudan iv - herxheimer 's solution .
images were acquired with a dissecting microscope ( olympus bx-50 ) equipped with a high - resolution camera ( sony xc-77ce ) .
digitized images were then evaluated with an image analysis system ( image - pro plus 6.3 software , media cybernetics ) .
total plaque area was quantified as the percentage area of aorta stained red . as for the hearts
, they were divided and half was put in formalin and half was put in liquid nitrogen .
the part that was fixed in formalin was embedded in paraffin , cut in 4 m thick sections , and stained with hematoxylin and eosin ( h&e ) in order to evaluate cardiomyocyte hypertrophy .
cardiomyocyte hypertrophy was assessed by measuring the shortest diameter of 200 cells , which were selected when they showed the spindle - shaped transverse section including the elliptical nucleus .
the shortest transverse diameter was measured 3 times per cell , and the values were averaged , as reported by .
the other half that was snap - frozen in liquid nitrogen was used either for gene expression analyses or for immunohistochemical stainings . gene expression of opg and trail was determined by real - time quantitative rt - pcr ( reverse transcription - polymerase chain reaction ) in aortic and cardiac tissue . in order to isolate mrna from the aorta and the heart , tissue was homogenized and processed as previously reported .
then , mrna was treated with the dnase inactivation reagent ( ambion dna - free product # am-1906 ) , and 3 g of treated mrna was subsequently used to synthesize cdna with superscript first strand synthesis system for rt - pcr ( gibco brl ) .
the gene expression of opg and trail was analyzed by real - time quantitative rt - pcr using the taqman system ( life technologies ) for opg and the sybr green system ( life technologies ) for trail .
fluorescence for each cycle was quantitatively analyzed by an abi prism 7900ht sequence detection system ( applied biosystems ) .
gene expression of opg was normalized to 18s mrna , while that of trail was normalized to rps9 .
results were reported as ratio compared with the level of expression in untreated controls , which were given an arbitrary value of 1 .
the presence of opg and trail in the aortae was evaluated by immunostainings on 4 m thick paraffin sections . in particular , after antigen retrieval in citrate buffer ( ph6 ) endogenous peroxidase was quenched for 10 minutes using 3% h2o2 in pbs . to localize opg
, we used a biotinylated goat anti - opg antibody ( r&d systems , 1 : 10 dilution ) followed by a catalyzed signal amplification system ( dako ) . to localize trail
, we used a monoclonal mouse anti - trail antibody ( r&d systems , 1 : 50 dilution ) , applied overnight at 4c . biotinylated goat anti - mouse immunoglobulins ( vector laboratories , dilution 1 : 200 )
were then used as secondary antibody , followed by streptavidin - hrp ( dako ) .
the presence of opg and trail in the hearts was evaluated by immunostaining on 5 m thick frozen sections .
after neutralization of endogenous peroxidase , sections were incubated overnight with the goat anti - opg antibody ( r&d systems , 1 : 10 dilution ) and the monoclonal mouse anti - trail antibody ( r&d systems , 1 : 50 dilution ) .
biotinylated immunoglobulins , diluted 1 : 200 , were then applied as secondary antibodies , followed by streptavidin - hrp ( dako ) . in both tissues ,
after counterstaining with hematoxylin , all the sections were examined by light microscopy ( olympus bx50wi ) and digitized using a high - resolution camera ( q - imaging fast 1394 ) .
the proportional area of brown staining was measured by an image analysis system ( image - pro plus 6.3 software , media cybernetics ) in order to quantify opg and trail expression in aortae and hearts .
apoe mice had higher levels of total cholesterol and triglycerides as compared to the cnt mice . in line with the study protocol , the mice in the apoe + dm group were hyperglycemic . moreover , diabetes was associated with a significant reduction of body weight and a significant increase of total cholesterol as compared to nondiabetic apoe mice .
apoe mice had a significant increase in total plaque area , which increased further after the induction of diabetes ( figures 1(a ) and 1(b ) ) .
as for cardiac changes , dyslipidemia was associated with a significant increase in cardiomyocyte size ( figures 1(c ) and 1(d ) ) , indicating cardiomyocyte hypertrophy , with no further increase after the induction of diabetes . in the aorta , the induction of diabetes was associated with a significant increase in opg gene expression and in the opg / trail ratio , while dyslipidemia had no effect on opg / trail ratio as compared to controls ( figure 2(a ) ) . by contrast , in the heart , dyslipidemia was the major determinant of opg / trail tissue changes , not only by increasing opg but also by reducing trail gene expression .
so dyslipidemia increased significantly cardiac opg / trail ratio , with no further differences after the induction of diabetes ( figure 2(b ) ) .
immunostainings showed that diabetes significantly increased aortic opg expression as compared to the other groups , while trail was unchanged ( figure 3 ) . on the other hand , in the heart , it was dyslipidemia that significantly increased opg protein expression , with no further changes after the induction of diabetes .
as for cardiac trail , trail protein expression decreased in the apoe and apoe + dm groups ( data not shown ) . the levels of circulating
circulating opg was 1.91 0.21 ng / ml in the control group , 2.05 0.1 ng / ml in the apoe group , and 3.1 0.19 in the apoe + dm group , such that opg increased significantly in diabetic mice as compared to the other groups ( p < 0.05 , apoe + dm versus cnt and versus apoe group ) .
as for trail , we were unable to measure its circulating levels , as they were below the detection levels of the elisa we used .
it has already been argued that opg , which circulates at much higher levels than its ligands ( rankl and trail ) , may be a more stable overall measure of opg / trail activity than trail .
patients with coronary artery disease have an increased opg / trail ratio , which further increases in the group of patients who develop heart failure . dyslipidemia and diabetes
, we evaluated the effects of dyslipidemia , alone and in combination with diabetes , on aortic and cardiac opg and trail expression and found that while diabetes is the major determinant of opg / trail tissue changes in the vessels , dyslipidemia is it in the heart .
this data is in line with previous experimental works showing that diabetes increased opg expression and the opg / trail ratio in the aorta and that the onset of diabetes was associated with an increase of circulating opg .
consistent with these observations , in our study , we found that aortic and circulating opg increased significantly only in the group of mice with diabetes .
taken together , these findings seem to support the hypothesis that the vessels might be the source of increased circulating levels of opg in patients with diabetes and/or cvd .
it has to be noted that the increase of circulating and tissue opg could be not only a risk marker but also a risk factor for atherosclerosis and cvd development .
firstly , animal models point to a role for opg in glucose metabolism regulation , as opg injections increased significantly glucose levels [ 12 , 26 ] , which represent one of the risk factors for cvd .
this effect could be mediated by trail blockade , as trail lowers glucose levels . by offsetting trail effects ,
opg could also promote body weight gain and dyslipidemia , as well as atherosclerotic plaque development [ 18 , 20 ] .
secondly , several studies have shown that opg has proinflammatory and profibrotic effects on the vasculature .
consistent with these actions , our group has shown that opg increases leukocyte adhesion to endothelial cells , that it increases tgf - beta - mediated fibrogenesis and proliferation in vascular smooth muscle cells , and that it increases atherosclerosis extension in diabetic apoe mice .
by contrast , dyslipidemia alone did not affect opg / trail ratio in the vasculature .
this data is consistent with a few in vitro studies showing that oxidized ldl did not change opg expression in human coronary artery smooth muscle cells and lymphocytes .
nevertheless , in our study , we found that dyslipidemia significantly increased opg / trail ratio in the heart , while diabetes had no additional effect on it .
it has been shown that dyslipidemia is independently associated with left ventricle ( lv ) hypertrophy [ 5 , 30 , 31 ] .
this was confirmed by experimental studies on apoe mice , where dyslipidemia was associated with increased lv mass in the absence of hemodynamic stress .
consistent with previous reports , here we found that dyslipidemia was associated with cardiomyocyte hypertrophy .
it has been demonstrated that triacylglycerol overload and the increased availability of other lipid intermediates , such as ceramides , diacylglycerol , and oxidized phospholipids , can all activate inflammatory pathways and oxidative stress , leading to cellular apoptosis and myocardial scarring [ 33 , 34 ] .
it is possible that the increase in cardiac opg / trail ratio that was associated with dyslipidemia represents one of the mediators of lipid - induced cardiac remodeling . as for opg
, a recent study has demonstrated that opg delivery induced lv hypertrophy , while , in vitro , cardiomyocytes transfected with aav - opg increased dramatically the expression of hypertrophy - related proteins , such as anp , -mhc , and troponin i . in the cardiac fibroblast ,
by contrast , we showed that trail administration to mice with diabetic cardiomyopathy had cardioprotective effects , as it reduced cardiac fibrosis and apoptosis , which are generally contributing to cardiac remodeling . in conclusion , here we found that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system .
this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd .
most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling . | background . dyslipidemia and
diabetes are two of the most well established risk factors for the development of cardiovascular disease ( cvd ) . both of them usually activate a complex range of pathogenic pathways leading to organ damage . here
we hypothesized that dyslipidemia and diabetes could affect osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) expression in the vessels and the heart .
materials and methods . gene and protein expression of opg , trail , and opg / trail ratio were quantified in the aorta and the hearts of control mice , dyslipidemic mice , and diabetic mice .
results .
diabetes significantly increased opg and the opg / trail ratio expression in the aorta , while dyslipidemia was the major determinant of the changes observed in the heart , where it significantly increased opg and reduced trail expression , thus increasing cardiac opg / trail ratio . conclusions .
this work shows that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system .
this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd .
most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling . |
compounds 14 , 7 , 8 , and 11 were
synthesized following our previously reported synthetic routes .
saturated derivatives 5 and 6 , unique to this work , were synthesized via the hydrogenation
of diastereomers 1 and 2 , respectively .
palmyrolide alcohol ( 9 ) and acid ( 10 )
were
prepared exploiting the known acid - catalyzed ring opening of 1 , followed by treating the resultant
aldehyde with either nabh4 ( for 9 ) or naclo2 ( for 10 ) .
alcohol 12 could be accessed
in a similar manner from 14-epi - palmyrolide ( 2 ) . in order to detect an interaction of ( )-palmyrolide
a and
its analogues with voltage - gated sodium channels ( vgscs )
, we examined
the ability of these compounds to antagonize veratridine - stimulated
na influx in murine primary cerebrocortical neurons .
we
previously demonstrated that veratridine is a partial agonist at neurotoxin
site 2 on the vgsc subunit .
murine
cerebrocortical neurons were loaded with the na - binding
benzofuran isophthalate ( sbfi ) , and the ability of palmyrolide analogues
to block veratridine - induced elevation of neuronal [ na]i was determined .
sodium influx was monitored with sbfi ( 340/380 ) responses to veratridine
in the absence and presence of ( )-palmyrolide a and its analogues .
the compounds shown are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide a ( 5 ) ; ( c ) ( + ) -palmyrolide
a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide
a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f )
[ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) .
data shown are from a representative
experiment performed in 2 or 3 replicates for each concentration .
in total 47 experiments
nonlinear regression analysis of concentration response
data for active analogues of ( )-palmyrolide a. a three - parameter
logistic fit of the palmyrolide a analogue inhibition of the response
to veratridine is shown for each active compound .
the compounds shown
are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide
a ( 5 ) ; ( c ) ( + ) -palmyrolide a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) . data points
shown represent the mean sem of 25 experiments performed
with 210 replicates each ( a f ) .
values represent mean sem
of 25 experiments each performed with 210 replicates . naturally occurring ( )-palmyrolide
a is a neuroactive macrolide
that was previously shown to block veratridine - induced sodium influx
in neuro-2a cells ( ic50 = 5.2 m ) and to display significant inhibition of ca oscillation in murine cerebrocortical neurons , with an ic50 value of 3.70 m ( 2.295.98 m , 95% ci ) .
we have confirmed and extended these original
findings by demonstrating that synthetic ( )-palmyrolide a
( 1 , figures 2a and 3a ) and its enantiomer ( 3 , figures 2c and 3c ) produced concentration - dependent
antagonism of veratridine - induced increase in neuronal [ na]i .
the concentration response curves for these
compounds were best fit by a three - parameter logistic equation .
this
analysis yielded an ic50 value ( 95% confidence interval )
of 2.1 m ( 1.23.1 , 95% ci ) , which was in good agreement
with the ic50 value reported earlier for natural ( )-palmyrolide
a. we found that ( )-palmyrolide
a ( 1 ) and macrolide analogues 5 and 3 were approximately 39-fold more potent than the
other analogues tested ( figures 2a
the observation that saturated
congener 5 showed a comparable ic50 to synthetic
( )-palmyrolide a suggests that the enamide double bond is
not essential for activity .
it is moreover noteworthy that acyclic
analogues of ( )-palmyrolide a with the natural absolute configuration
were inactive as blockers of veratridine - stimulated na influx .
these data indicate that the three - dimensional molecular
shape of ( )-palmyrolide a was crucial for the interaction
with vgscs . in order to support these observations and to better
understand
how structure relates to biological function , an exhaustive nmr and
computational analysis was next conducted on 1 and three
diastereomers ( i.e. , 2 , 7 , and 8) .
saturated derivatives 5 and 6 exist
as a mixture of rotational isomers and precluded precise nmr assignments
from being made .
chemical shift assignments of ( )-palmyrolide
a ( 1 ) and synthetic diastereomers 2 , 7 , and 8 were made using dqf - cosy , hsqc , hmbc ,
and h2bc spectra .
the chemical shifts of these four diastereomeric compounds follow
the same general trends but differ slightly , indicating that the conformations
of the macrolide ring are different . for example , in each diastereomer
a different methylene group exhibited degenerate chemical shifts for
its two protons .
stereospecific assignments were made for most of
the prochiral atoms that possessed nondegenerate shifts .
the noesy
spectra provided 74 nonredundant distance restraints for 1 , 83 for 2 , 84 for 7 , and 77 for 8 .
h coupling in the h nmr spectra
and from the heteronuclear couplings measured by the hetloc experiments
( see supporting information for all restraints ) .
applying the distance and torsion restraints during simulated annealing
calculations produced fairly well - defined ensembles of structures .
table 2 summarizes the inputs
and results of the structure calculations . to test the specificity
of the restraints and their ability to discriminate between the four
diastereomers ,
restrained simulated annealing calculations were performed
for all 16 combinations of starting structure and restraint set . if
the restraints correctly discriminated between conformations , then
the correct combination should have the least restraint violations
and the lowest energy .
for all four structures we found the best restraint
set for each starting structure was the correct one , namely , the restraints
derived from nmr spectra of that compound ( table 3 ) .
this confirms that the restraint sets were able to correctly
discriminate between the diastereomers .
the four ensembles generally
have the same overall conformation ,
with the major differences being due to the orientations of the substitutents .
all four diastereomers have a cisoid configuration
of the n - methyl group ( n - ch3 ) and the
adjacent carbonyl despite the starting structures having a transoid configuration .
examining the trajectories reveals
that the configuration converts early in the simulated annealing as
the restraints take effect .
the driving force in these simulations
appears to be noes between h-18 and the h-2 methylene group . to quantify differences between the ensembles , we calculated root - mean - square
deviations ( rmsd s ) between the representative structures of
each ensemble ( table 4 ) .
the rmsd s
were calculated using the differences in the positions of the carbon ,
nitrogen , and oxygen atoms of the macrolide ring after aligning the
structures to minimize these differences .
representative structures
were selected as the structure having the smallest rmsd among all
other structures in its ensemble .
the rmsd s between all four
representative structures were similar ( table 4 ) , with 2 having the lowest values overall , suggesting
that this ensemble is the most central in comparison
to the other diastereomers .
visual inspection of the ensembles
shows that the macrolide ring
is relatively flat , but the orientation of the tert - butyl and methyl groups alters the global shape ( figure 4 ) . in 1
the tert - butyl
and methyl groups lie close to the plane of the ring , but in 7 the tert - butyl group projects almost perpendicular
to the plane of the ring , and in 8 the two methyl groups
project in opposite directions above and below the ring . examining
the surface electrostatic potential of the representative structures
( figure 5 ) we find that 8 is the
most similar to the active compound 1 . however , the c-14
methyl group in 1 projects to the side , whereas in 8 it is situated above the macrolide ring . taken together ,
these differences between diastereomers may account , at least in part ,
for the observed differences in biological activity .
in addition ,
due to the highly flexible nature of the macrolide , as evidenced through
computational modeling , the minimized ligand ensembles reported here
may not necessarily reflect the structure these molecules adopt when
bound to their molecular target .
further consideration and investigations
into structure activity relationships are ongoing and will
be reported in due course .
number of diastereotopic hydrogens
for which stereospecific assignments were obtained , and total number
of protons with nondegenerate chemical shifts .
the three numbers reported for each
combination of starting structure and restraint set are number of
selected structures , average amber energy , and average violation energy .
the italic cells indicate the best set of restraints for each starting
structure , with respect to amber energy and violation energy , and
also correspond to the correct pairing .
the final row is the sum of the
three rmsd s above , a measure of the total difference from
the other structures .
the four structures in the top row are shown in
a similar orientation to figure 4 . in the lower
row
the structures have been rotated 180 about the horizontal
axis to show the rear face .
the electrostatic potential ranges from
red ( 2 kcal / mole ) through white ( 0 ) to blue ( + 2 kcal / mole ) .
these
findings suggest that both synthetic ( )-palmyrolide
a and its enantiomer function with equal potency as vgsc antagonists
to block veratridine - induced sodium influx . with regard to identification
of the pharmacophore of this natural product
, it appears that the
intact macrolide is required since acyclic versions were inactive
as inhibitors of vgscs .
moreover , analogues lacking the enamide double
bond were of high potency in this assay , and thus this functionality
appears unnecessary for blocking vgscs . finally ,
using detailed nmr
and computational approaches with the natural product and three diastereomeric
analogues , it was found that the c-5c-7 anti / c-7c14 syn arrangement of stereocenters
produces a unique combination of three - dimensional shape and electrostatic
potential that is responsible for the potent biological activity of
the natural product . in an effort to understand
the similar biological
activity found for the natural stereoisomer and its enantiomer , continued
investigations in this area will focus on uncovering the specific
molecular target and associated binding site , which may also assist
in future analogue development of novel sodium channel blocking analgesics
derived from palmyrolide a.
unless otherwise noted ,
reactions were performed in flame - dried glassware under an atmosphere
of dry nitrogen .
reaction solvents ( ch2cl2 ,
thf , and et2o ) were purified before use in a solvent purification
system under a flow of dry nitrogen .
all other solvents and reagents
were purchased from commercial suppliers and used as received , unless
otherwise specified .
thin - layer chromatography ( tlc ) was performed
using plates precoated with silica gel 60 f-254 ( 250 m )
and visualized by uv light , kmno4 , or anisaldehyde stains ,
followed by heating .
ir samples were prepared by evaporation
from chcl3 or ch2cl2 on nacl plates
and run on a perkinelmer spectrum one ft - ir spectrometer . for the
synthetic studies ,
h and c nmr spectra were
recorded at 300 and 75 mhz ( oxford magnet with a varian 300 console ) ,
at 400 and 100 mhz ( oxford magnet with a varian unity 400 console ) ,
and at 600 and 150 mhz ( magnex magnet with a bruker avance
iii 600
console ) , respectively , and are reported relative to residual solvent
peak ( h 7.26 and c 77.0 for h and c in cdcl3 ) .
high - resolution
mass spectra were obtained using positive electrospray ionization
on a bruker 12 t apex - qe fticr - ms with an apollo ii ion source at
the cosmic laboratory facility at old dominion university , va . to a solution of ( s , e)-6-iodo-2-methylhex-5-enoic
acid ( 0.096 g , 0.37 mmol ) in thf ( 3.7 ml )
was added diisopropylethylamine
( 0.100 ml , 0.60 mmol ) followed by the yamaguchi reagent ( 0.071 ml ,
0.45 mmol ) .
the mixture was allowed to stir at room temperature for
3 h before being concentrated in vacuo .
the resultant
mixed anhydride was dissolved in toluene ( 7.4 ml ) and then added ,
via cannula , to a flask containing ( 5r,7r)-7-hydroxy-5,8,8-trimethylnonanamide ( 0.051 g , 0.23 mmol ) and dmap
( 0.046 g , 0.37 mmol ) . after stirring at room temperature ( rt ) for
12 h
, the reaction mixture was diluted with ch2cl2 , washed with a saturated aqueous solution of nahco3 ,
dried over mgso4 , and concentrated .
purification via flash
column chromatography ( 1:1 hexanes / etoac ) afforded 0.088 g ( 81% ) of
the coupled product as a colorless oil : [
]d22.5 + 13.3 ( c 1.98 ,
chcl3 ) ; ir ( neat , thin film ) 3429 , 3351 , 3203 ,
2962 , 2934 , 2868 , 1725 , 1665 , 1607 , 1461 , 1380 , 1366 , 1259 , 1181 ,
1119 , 1067 , 957 , 935 cm ; h nmr ( 300
mhz , cdcl3 ) 6.49 ( dt , j = 7.2 ,
14.4 hz , 1 h ) , 6.02 ( d , j = 14.4 hz , 1h ) , 5.88 ( bs ,
1 h ) , 5.46 ( bs , 1 h ) , 4.79 ( dd , j = 4.5 , 6.3 hz ,
1 h ) , 2.45 ( sextet , j = 6.9 hz , 1 h ) , 2.232.15
( m , 2 h ) , 2.122.05 ( m , 2 h ) , 1.861.69 ( m , 2 h ) , 1.611.25
( m , 6 h ) , 1.17 ( d , j = 7.2 hz , 3 h ) , 1.110.99
( m , 1 h ) , 0.910.87 ( overlapping doublet / singlet , 12 h ) ; c nmr ( 75 mhz , cdcl3 ) 176.5 , 175.8 , 145.6 ,
79.0 , 75.5 , 39.3 , 37.7 , 35.7 , 34.79 , 34.77 , 33.9 , 32.3 , 29.2 , 26.1 ,
22.8 , 21.1 , 17.5 ; hresims m / z 474.1471
[ m + na ]
( calcd for c19h34ino3na , 474.1476 ) . to the coupled product ( 0.088 g , 0.19
mmol ) in thf ( 19.0 ml )
was added cui ( 0.005 g , 0.03 mmol ) and cs2co3 ( 0.100 g , 0.30 mmol ) .
the mixture was first
degassed using nitrogen ( 10 min ) before n , n-dimethylethylenediamine ( 0.050 ml , 0.46 mmol )
was added .
the rubber septum was quickly replaced with a glass stopper ,
and the reaction mixture heated to 60 c for 19 h. after cooling
to rt , the reaction mixture was diluted with etoac , filtered through
a short plug of silica , and concentrated .
the crude product was purified
via flash column chromatography ( 8:2 7:3 1:1 hexanes / etoac )
to yield the n - h enamide intermediate ( 0.022 g ) ,
which was immediately dissolved in thf ( 0.7 ml ) and cooled to 0 c .
to this
was added nah ( 0.014 g , 0.34 mmol , 60% in mineral oil ) in
a single portion .
the reaction mixture was allowed to stir for 20
min before mei ( 0.100 ml ) was added dropwise , followed by removal
of the ice water bath . after being allowed to stir at rt for 20 min ,
the reaction mixture was again cooled to 0 c before being quenched
with h2o , extracted using etoac , dried over mgso4 , and concentrated .
the crude product was purified via flash column
chromatography ( 9:1 hexanes / etoac ) to yield 14-epi - palmyrolide a ( 0.016 g , 25% over two steps ) as a colorless oil :
[ ]d24.5 11.4 ( c 0.58 , chcl3 ) ; ir ( neat ,
thin film ) 2959 , 2927 , 2873 , 1728 , 1675 , 1646 , 1466 , 1413 ,
1384 , 1366 , 1333 , 1298 , 1240 , 1205 , 1193 , 1171,1121 , 933 cm ; h nmr ( 400 mhz , cdcl3 ) 6.63 ( d , j = 13.6 hz , 1 h ) , 4.92 ( ddd , j = 5.2 ,
8.8 , 13.6 hz , 1 h ) , 4.85 ( dd , j = 2.0 , 9.6 , 1 h ) ,
3.05 ( s , 3 h ) , 2.602.42 ( m , 3 h ) , 2.32 ( dt , j = 7.2 , 13.6 hz , 1 h ) , 2.242.17 ( m , 1 h ) , 2.031.95
( m , 1 h ) ,
1.841.74 ( m , 1 h ) , 1.681.54 ( m , 2 h ) , 1.491.30
( m , 4 h ) , 1.26 ( d , j = 7.2 hz , 3 h ) , 1.030.93
( m , 1 h ) , 0.89 ( d , j = 6.4 hz , 3 h ) , 0.86 ( s , 9 h ) ; c nmr ( 100 mhz , cdcl3 ) 175.4 , 172.8 , 129.8 ,
110.2 , 76.5 , 37.2 , 36.6 , 35.4 , 34.3 , 33.6 , 31.0 , 29.8 , 29.5 , 27.3 ,
25.9 , 24.9 , 20.0 , 19.2 ; hresims m / z 360.2504 [ m + na ] ( calcd for c20h35no3na , 360.2509 ) .
to a solution
of ( )-palmyrolidea ( 0.008 g , 0.022 mmol ) in anhydrous meoh
( 1 ml ) was added 10% palladium on carbon ( 0.005 g ) .
the atmosphere
in the flask was replaced with hydrogen , and the reaction mixture
was allowed to stir at rt for 18 h before being filtered through a
short plug of silica .
after rinsing several times with ether , the
filtrate was concentrated and purified via column chromatography ( 8:2
hexanes / etoac ) to afford compound 5 ( 0.006 g , 77% ) as
a mixture of rotational isomers ( 2:1 ratio ) : [ ]d23 + 35.6 ( c 0.48 , chcl3 ) ; ir ( neat , thin film )
2954 , 2931 , 2871 , 1720 , 1651 , 1269 , 1250 , 1073 cm ; h nmr ( 300 mhz , cdcl3 , mixture of rotational
isomers with mostly overlapping peaks , making the assignment of major
and minor components difficult ) ( major isomer ) 4.82 ( d , j = 10.0 hz , 1 h ) , 3.30 ( t , j = 5.0 hz ,
1 h ) , 2.98 ( s , 3 h ) , 2.532.65 ( m , 2 h ) , 2.212.33 ( m ,
2 h ) , 1.211.73 ( m , 14 h ) , 1.18 ( d , j = 6.9
hz , 3 h ) , 0.94 ( d , j = 5.9 hz , 3 h ) , 0.86 ( s , 9 h ) ,
( minor isomer , partial ) 4.92 ( d , j = 10.8 hz , 0.6
h ) , 2.89 ( s , 1.5 h ) , 0.91 ( d , j = 6.1 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , mixture of rotational isomers )
175.5 , 174.8 , 173.5 , 77.8 , 77.2 , 48.9 , 46.3 , 40.4 , 40.0 , 38.3 ,
37.2 , 35.2 , 35.0 , 33.6 , 33.2 , 32.98 , 32.96 , 31.2 , 39.7 , 27.9 , 27.6 ,
27.0 , 26.0 , 25.9 , 25.4 , 23.5 , 23.2 , 22.2 , 20.4 , 20.1 , 18.0 , 16.5 ;
hresims m / z 362.2660 [ m + na ] ( calcd for c20h37no3na ,
362.2665 ) .
the crude product was purified via column
chromatography ( 8:2 hexanes / etoac ) to afford compound 6 ( 0.006 g , 96% ) as a mixture of rotational isomers ( 2:1 ratio ) :
[ ]d23 + 42.1 ( c 0.5 , chcl3 ) ; ir ( neat , thin
film ) 2956 , 2930 , 2872 , 1727 , 1647 , 1459 , 1192 , 1156 cm ; h nmr ( 300 mhz , cdcl3 , mixture
of rotational isomers with mostly overlapping peaks , making the assignment
of major and minor components difficult ) ( major isomer ) 4.86
( d , j = 10.5 hz , 1 h ) , 3.513.42 ( m , 1 h ) ,
3.233.14 ( m , 1 hz ) , 2.88 ( s , 3 h ) , 2.312.48 ( m , 2
h ) , 2.182.31 ( m , 2 h ) , 1.971.79 ( m , 2 h ) , 1.231.66
( m , 15 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.92 ( d , j = 5.8 hz , 3 h ) , 0.86 ( s , 11 h ) , ( minor isomer , partial )
4.83 ( d , j = 11.6 hz , 0.6 h ) , 2.91 ( s , 1 h ) , 1.15
( d , j = 7.2 hz , 1.5 h ) , 0.96 ( d , j = 5.7 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 ,
mixture of rotational isomers ) 176.8 , 176.6 , 173.3 , 173.0 ,
77.2 , 49.1 , 46.0 , 40.6 , 40.1 , 38.0 , 37.3 , 35.2 , 34.0 , 33.9 , 33.2 ,
33.1 , 33.0 , 31.3 , 29.7 , 28.9 , 27.2 , 25.9 , 24.8 , 23.9 , 22.8 , 19.9 ,
19.8 , 19.2 , 19.1 ; hresims m / z 362.2659
[ m + na ]
( calcd for c20h37no3na , 362.2665 ) . to a solution
of 1 ( 0.007 g , 0.021 mmol ) in chcl3 ( 2 ml )
was added a 6 n aqueous hcl solution dropwise until tlc analysis showed
complete consumption of the starting material .
the reaction mixture
was then quenched with a saturated aqueous solution of nahco3 , followed by extraction using etoac .
the
resultant crude aldehyde was dissolved in anhydrous meoh ( 1 ml ) and
treated with nabh4 ( 0.015 g , 0.39 mmol ) .
the reaction mixture
was allowed to stir at rt for 1 h before being quenched with h2o , extracted using etoac , dried over mgso4 , filtered ,
and concentrated .
the crude alcohol was purified by column chromatography
( 100% etoac ) to afford alcohol 9 ( 0.006 g , 73% over two
steps ) as a colorless oil : [ ]d24.3 + 26.8 ( c 0.20 , chcl3 ) ; ir ( neat , thin film ) 3296 , 2956 , 1728 , 1651 , 1562 ,
1462 , 1366 , 1164 cm ; h nmr ( 300 mhz ,
cdcl3 ) 6.10 ( bs , 1 h ) , 4.76 ( dd , j = 2.2 , 9.5 hz , 1 h ) , 3.63 ( t , j = 6.4 hz , 2 h ) ,
2.79 ( d , j = 4.8 hz , 3 h ) , 2.392.51 ( m , 1
h ) , 2.002.23 ( m , 2 h ) , 1.91 ( bs , 1 h ) , 1.631.80 ( m ,
2 h ) , 1.221.60 ( m , 10 h ) , 1.16 ( d , j = 7.0
hz , 3 h ) , 0.87 ( d , overlapped , 3 h ) , 0.86 ( s , 9 h ) ; c
nmr ( 75 mhz , cdcl3 ) 176.8 , 174.0 , 78.6 , 62.5 , 40.2 ,
37.6 , 36.2 , 34.5 , 33.3 , 32.5 , 28.8 , 26.2 , 25.9 , 25.9 , 23.5 , 23.0 ,
20.8 , 17.4 ; hresims m / z 380.2764
[ m + na ] ( calcd for c20h39no4na , 380.2771 ) . approximately 0.3
ml of an oxidant solution prepared via the mixing of naclo2 ( 0.060 g ) and nahpo4 ( 0.040 g ) in h2o ( 1 ml )
was added to a solution of palmyrolide aldehyde ( prepared as above ,
0.006 g , 0.016 mmol ) in tert - butanol ( 0.25 ml ) and
2-methyl-2-butene ( 0.1 ml ) . the reaction mixture was allowed to stir
for 30 min before being quenched with water , extracted with etoac ,
dried over mgso4 , filtered , and concentrated .
the crude
acid was purified by column chromatography ( 100% etoac ) to afford
acid 10 ( 0.002 g , 25% ) as a colorless oil : [ ]d23 + 10.8 ( c 0.10 , chcl3 ) ; ir ( neat , thin film )
3306 , 2960 , 1727 , 1633 , 1164 cm ; h nmr ( 300 mhz , cdcl3 ) 6.04 ( bs , 1 h ) , 4.78 ( dd , j = 10.2 , 1.9 hz , 1 h ) , 2.82 ( d , j = 4.8
hz , 3 h ) , 2.48 ( q , j = 6.9 hz , 1 h ) , 2.312.41
( m , 2 h ) , 2.022.23 ( m , 3 h ) , 1.601.82 ( m , 4 h ) , 1.211.59
( m , 6 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.921.05
( m , 1 h ) , 0.88 ( d , overlapped , 3 h ) , 0.87 ( s , 9 h ) ; c
nmr ( 75 mhz , cdcl3 ) 176.4 , 78.5 , 40.1 , 37.6 , 36.2 ,
34.6 , 34.4 , 33.0 , 29.7 , 28.7 , 26.3 , 26.0 , 23.0 , 22.7 , 20.7 , 17.2 ;
hresims m / z 394.2558 [ m + na ] ( calcd for c20h37no5na ,
394.2563 ) .
the crude product was purified via column chromatography
( 100% etoac ) to afford compound 12 ( 0.004 g , 94% ) : [ ]d25.5 + 30.8 ( c 0.24 , chcl3 ) ; ir ( neat , thin film )
3304 , 2950 , 1729 , 1649 , 1560 , 1462 , 1366 , 1163 , 1074 cm ; h nmr ( 300 mhz , cdcl3 ) 6.25 ( bs ,
1 h ) , 4.76 ( dd , j = 9.9 , 1.6 hz , 1 h ) , 3.553.70
( m , 2 h ) , 2.77 ( d , j = 4.8 hz , 3h ) , 2.372.50
( m , 1 h ) , 2.27 ( bs , 1 h ) , 2.002.20 ( m , 2 h ) , 1.631.83
( m , 2 h ) , 1.211.60 ( m , 10 h ) , 1.16 ( d , j =
7.0 hz , 3 h ) , 0.921.04 ( m , 1 h ) , 0.86 ( d , overlapped , 3 h ) ,
0.85 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )
177.0 , 174.1 , 78.4 , 62.2 , 40.0 , 37.5 , 36.2 , 34.6 , 34.5 , 33.1 , 32.4 ,
28.8 , 26.1 , 25.9 , 23.6 , 23.1 , 20.7 , 17.7 ; hresims m / z 380.2764 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) .
primary cultures of
cerebrocortical neurons were obtained from embryonic day 17 swiss - webster
mice as described elsewhere .
briefly ,
pregnant mice were euthanized by co2 asphyxiation , and
embryos were removed under sterile conditions .
cerebral cortices were
collected , stripped of meninges , minced by trituration with a pasteur
pipet , and treated with trypsin for 25 min at 37 c .
the cells
were then dissociated by two successive trituration and sedimentation
steps in soybean trypsin inhibitor and dnase - containing isolation
buffer , centrifuged , and resuspended in eagle s minimal essential
medium with earle s salt ( mem ) supplemented with 1 mm l - glutamine , 10% fetal bovine serum , 10% horse serum , 100 iu / ml penicillin ,
and 0.10 mg / ml streptomycin ( ph 7.4 ) .
cells were plated onto poly - l - lysine - coated 96-well ( 9 mm ) , clear - bottomed , black - well culture
plates ( midsci ) at a density of 1.5 10 cells / well .
cells were then incubated at 37 c in a 5% co2 and
95% humidity atmosphere .
the culture media was changed every other
day , starting from day 5 in vitro using a serum - free
growth medium containing neurobasal medium supplemented with b-27 ,
100 iu / ml penicillin , 0.10 mg / ml streptomycin , and 0.2 mm l - glutamine .
all animal use protocols were
approved by the institutional animal care and use committee ( iacuc )
of creighton university .
[ na]i measurement and full in situ calibration of sodium - binding benzofuran isophthalate fluorescence
ratio were performed as described previously .
cells were grown in 96-well plates and washed four times with locke s
buffer ( 8.6 mm hepes , 5.6 mm kcl , 154 mm nacl , 5.6 mm glucose , 1.0
mm mgcl2 , 2.3 mm cacl2 , 0.1 mm glycine , ph 7.4 )
using an automated microplate washer ( biotek instruments ) . after measuring
the background fluorescence of each well , cells were incubated for
1 h at 37 c with dye - loading buffer ( 100 l / well ) containing
10 m sbfi - am ( invitrogen ) and 0.02% pluronic f-127 ( invitrogen ) .
cells were washed five times with locke s buffer , leaving a
final volume of 100 l in each well .
the plate was then transferred
back to the incubator for 15 min to allow the cells to equilibrate
after washing and placed in a flexstation ii ( molecular devices ) chamber
to detect na - bound sbfi emission at 505 nm ( cells were
excited at 340 and 380 nm ) .
fluorescence readings were taken once
every 5 s for 60 s to establish the baseline , and then 50 l
of different concentrations of ( )-palmyrolide a and/or veratridine
was added to each well from the compound plate at a rate of 26 l / s ,
yielding a final volume of 200 l / well . after correcting for
background fluorescence , sbfi fluorescence ratios ( 340/380 ) and concentration
each palmyrolide a analogue was dissolved in
dmso at a stock concentration of 50 mm and stored at 20 c
until used . on the day of an experiment ,
this dilution in locke s
buffer resulted in palmyrolide a analogue solutions that were 4 times
the desired final concentration .
( )-palmyrolide
a ( 1 ) and analogues ( 2 , 7 ,
and 8) were dissolved in cdcl3 at a concentration
of 2.5 mg in 50 l and transferred to a 1.7 mm nmr tube .
the noesy mixing time was 500 ms , the hmbc delay was set to 6.25 ms
( 8 hz ) , and the hetloc used a dispi-2 spinlock of 60 ms duration .
chemical shift assignments were based on analysis of the dqf - cosy ,
hsqc , and hmbc 2d experiments using ucsf sparky .
topspin ( bruker biospin ) was used to analyze coupling constants
in the h nmr and hetloc spectra .
noesy peak heights were
converted to upper distance restraints of 3 , 4 , 5 , or 6 using
in - house perl scripts . where restraints involved a group of atoms ,
pseudoatom corrections were applied by the amber restraint generation
tools .
force field parameters were estimated using
the gaff force field and the antechamber
program in the amber 14 package .
molecular
dynamics simulations were nonperiodic , vacuum simulations with a distance - dependent
dielectric term .
using sander in the amber 14 package each starting
structure was energy minimized , then subjected to 20 ps of simulated
annealing with the nmr - derived restraints in which the temperature
was raised from 10 k to 600 k then cooled to near 0 k. the simulated
annealing was run 20 times for each structure with a different random
number seed each time .
each collection of 20 structures was sorted
by restraint violation energy , and outliers were discarded . to align
structures ,
the selected ensemble was superimposed on c17 ,
o12 , c1318 , and n19 using cpptraj . to stereospecifically assign pairs of diastereotopic hydrogens ,
pairs of noes that involved the diastereotopic atoms and had differential
intensities
were identified , then compared with the distances between
these atoms in the ensemble of selected structures .
if the ensemble
mean of one distance was more than one standard deviation from the
alternative , then the diastereotopic hydrogens were stereospecifically
assigned . | a small library of synthetic ( )-palmyrolide
a diastereomers ,
analogues , and acyclic precursors have been examined with respect
to their interaction with voltage - gated sodium channels ( vgscs ) . toward
this goal ,
the ability of ( )-palmyrolide a and analogues to
antagonize veratridine - stimulated na+ influx in primary
cultures of mouse cerebrocortical neurons was assessed .
we found that
synthetic ( )-palmyrolide a and its enantiomer functioned as
vgsc antagonists to block veratridine - induced sodium influx . a detailed
nmr and computational analysis of four diastereomers revealed that
none had the same combination of shape and electrostatic potential
as exhibited by natural ( )-palmyrolide a. these data indicate
that the relative configuration about the tert - butyl
and methyl substituents appears to be a prerequisite for biological
function . additional testing revealed that the enamide double bond
was not necessary for blocking veratridine - induced sodium influx ,
whereas the acyclic analogues and other macrolide diastereomers tested
were inactive as inhibitors of vgscs , suggesting that the intact macrolide
was required . |
the online version of this article ( doi:10.1007/s00414 - 009 - 0352 - 9 ) contains supplementary material , which is available to authorized users .
microbes can negatively interfere with the postmortem assessment of alcohol abuse and in this way pose problems for forensic investigators .
however , microbial forensics is often chiefly associated with the detection of highly pathogenic microbes to which humans are deliberately exposed in cases of biological terrorism [ 2 , 3 ] . however , human fingertip microflora left behind on touched objects at crime scenes may potentially contain forensically relevant information that may be useful for human host inferences accessible via microbial dna fingerprinting of physical fingerprints .
for example , if endogenous microbial skin species / strains with a geographically restricted distribution could be retrieved from touched objects via microbial dna analysis , the geographic origin of the human host individual could be determined indirectly .
information about the geographic region of origin can be relevant in suspect - less forensic cases where the evidence dna sample does not match either a suspect s dna profile or any in a criminal dna database .
in such cases , geographic information derived from crime scene samples is expected to reduce the potential pool of suspects by allowing police investigations to concentrate on specific groups of people , i.e. , those from a restricted geographic region .
numerous human genetic markers have been suggested for inferring human genetic ancestry mostly to the continental level [ 47 ] , and a recent study indicated that inferring the subregion of origin of an unknown european may be feasible from autosomal genetic data .
however , direct ancestry inference based on human genetic markers is currently far from perfect , initiating the question whether microbial dna may be used to supplement human dna markers in reliable ancestry reconstruction of unknown persons .
recently , it has been shown that the gastric pathogenic bacteria helicobacter pylori has intimately coevolved with its human host [ 9 , 10 ] .
although this example may be of limited direct relevance for forensics , because samples containing h. pylori are usually not found at crime scenes ( with the exception of bodies in cases of missing persons ) , it shows that in principle human geographic signatures are inferable from microbial genomes .
the human skin is a complex microbial ecosystem consisting of multiple niches , which can differ drastically from each other .
interactions between skin microbes and the human host , as well as between the microbial occupants , are still poorly understood .
the current knowledge on skin microbiota primarily derives from cultivation - based studies [ 12 , 13 ] , although molecular fingerprinting techniques have been employed more recently [ 14 , 15 ] .
if a comparable relationship exists between humans and their skin microbiota , as has been observed for h. pylori , new methods for human geographic origin determination could be developed based on dna analysis of fingertip microflora , with interesting new applications to molecular analyses of physical fingerprints left at crime scenes .
volunteers and microbiological procedures five dutch european subjects from rotterdam and three volunteers from dhaka , bangladesh , placed their right index finger on a colombia iii blood agar plate ( biomerieux , marcy - letoile , france ) .
fingerprint sampling for dutch volunteers was performed in rotterdam whereas that of bangladeshi volunteers was performed in dhaka . after washing this finger with hospital soap and air drying , another fingerprint , from the same finger ,
was placed next to the first . whereas the first fingerprint was expected to provide a mixture of transient and resident microbiota
, the second one was assumed to provide resident microbiota only as soap removes the upper dead - skin layers including their transient microflora .
after overnight incubation at 37c , colonies with different morphologies were isolated , with 20 per fingerprint ( two each ) from the five dutch individuals ( n = 200 strains ) .
three weeks later , this procedure was repeated for assessment of bacterial persistence in the dutch volunteers .
agar plates from bangladeshi volunteers were shipped by courier to rotterdam after overnight incubation at 37c for further analyses . given the diversity in genotypes encountered in the time - wise resampling experiments using dutch volunteers ( see below ) , this procedure was not repeated for the volunteers from bangladesh .
gram staining and the determination of catalase activity were applied for the identification of staphylococci using commercially available test kits ( biomrieux , marcy - letoile , france ) .
staphylococcus epidermidis sensu stricto was identified among the isolates by a species - specific polymerase chain reaction assay as described elsewhere .
a coagulase test with slidex staph plus ( biomrieux , marcy - letoile , france ) was performed to detect staphylococcus aureus isolates .
were classified with the i d 32 staph api system ( biomrieux , marcy - letoile , france ) .
genotyping bacterial isolates in order to generate dna fingerprints for the most relevant bacterial isolates , all staphylococcal isolates were subjected to pulsed - field gel electrophoresis ( pfge ) .
pfge can be used to electrophoretically separate dna restriction fragments ranging in length between 50 and 1,000 kb .
after lysostaphin and proteinase treatment and smai macrorestriction , pfge was performed in a 1% incert agarose ( fmc ) gel in 0.5 tbe at 14c , using a constant electric field of 6 v cm with pulse ramping from 0.1 to 30 s at a 60/60 angle for 18 h. pfge patterns were analyzed for levels of similarity with bionumerics 4.5 ( applied - maths , sint martens latem , belgium ) .
developing forensic dna markers for human host inferences from physical fingerprints relies on the examination of the transient exogenous microflora left behind together with the physical fingerprint .
however , any human host inference from fingertip microflora using physical fingerprints would require that the transient microflora is representative of the resident endogenous microflora of the same individual and is not simply reflecting microbial species / strains picked up from the environment . to test this prerequisite , we investigated and compared by means of dna fingerprinting techniques the transient superficial skin microbiota and the resident endogenous microflora of human fingertips within several individuals .
in addition , we collected data at two time points to investigate time - wise microflora persistency within individuals . to this end , we used samples from five dutch volunteers and gram staining as well as catalase activity testing revealed that all bacterial skin isolates that were selected were gram - positive cocci .
most isolates were staphylococcus spp . , a small number of colonies represented micrococci ( ~5% of total isolates ) , and the vast majority ( 97% ) of staphylococci were coagulase negative .
healthy skin is known to harbor gram - positive pleomorphic bacilli , including corynebacterium , brevibacteria , and propionibacteria sp . , but these were not detected here .
pfge is the most discriminatory typing method that is currently available for typing of staphylococci .
transient microflora ( tm ) was collected from physical fingerprints before washing with soap and endogenous microflora ( rm ) after soap washing ( see materials and methods
the tm showed higher numbers of pfge types than those seen in the rm in three of the five dutch subjects ( table 1 ) .
this can be explained by nonendogenous species picked up from the environment and being transferred .
significant variability between the subjects was seen for both the tm and rm , as has been previously reported for other skin areas [ 10 , 13 ] .
notably , the rm types encountered after washing were very similar intraindividually but very different interindividually .
this suggests that certain types of bacteria are able to efficiently and persistently colonize the skin of a given individual . within the rm , all dutch subjects had one or two dominant types , mostly staphylococcus warneri species .
these types were also present within the tm of three of the five subjects , while s. epidermidis was present at various time points in all of the subjects tm . only one subject ( subject 1 ) showed the major rm type as the major tm type . in several subjects , the dominant rm type did not appear at all in the tm , although there was considerable overlap in nondominant species between the tm and rm in many subjects . in general , pfge types within the rm and within the tm for the same subject appeared consistent over time .
although the degree of variability changed in the 3-week period for every subject , in some subjects , it changed more drastically than in others . at time point 1 , overlapping pfge types were seen in four of the five subjects in the tm and rm .
however , by time point 2 , only one subject ( subject 4 ) showed any overlap .
in one subject ( subject 4 ) , the major rm type did not occur in the tm of the same sampling time point 2 but was present in considerable frequency in the tm of the earlier time point 1 .
thus , although we see some resemblance between rm and tm as well as some time - wise consistency , the pattern detected is far from perfect . by viewing our results only qualitatively ( ignoring type frequencies ) ,
rm signals were detectable from the tm analyses in only 50% of the dutch experiments ( combining individuals and time points ) , which appears discouraging as basis for developing molecular markers for forensic applications to infer individual host information .
table 1staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) we chose geographic inference as a test example for the type of human host information that would be interesting to retrieve from physical fingerprints by means of microbial dna analysis for forensic applications .
a prerequisite for developing microbial skin markers for inferring human geographic origins would be that the endogenous fingertip microflora of humans from different parts of the world shows differences in their genetic diversity and that retrievable microbial genotypes cluster according to geographic origin of the host individuals . to test this , we obtained fingerprint microflora from native inhabitants of bangladesh collected in dhaka , bangladesh , and compared their microbiological profiles with those of the dutch volunteers .
fingertip microflora of three bangladeshi volunteers were isolated and processed in the same manner as for the dutch volunteers , although only at one time point ( three individuals , two fingerprints each and 20 bacterial isolates per fingerprint , hence 3 2 20 = 120 strains ; table 2 ) . correspondence between rm and tm
was observed in two of the three subjects for one pfge type , although the persistent pfge type found differed between the subjects .
notably , subject 3 from bangladesh showed an unexpectedly higher variety of staphylococci in the rm compared to tm . comparing pfge types between dutch and bangladeshi subjects revealed some differences , e.g. , bangladeshi skin microbiota contained less s. warneri and much less s. epidermidis types compared with the dutch ones .
were found among the tm in bangladeshi ( 45% ) than among the dutch ( 7% ) .
furthermore , none of the pfge types from the bangladesh fingerprints were seen in the dutch collection , indicating extensive geographic heterogeneity within the staphylococcus species . however , when each pfge type obtained during this study was subjected to dice - based cluster analysis for visualizing geographic structure in the skin staphylococci ( see supplementary material fig . 1 ) , the bangladeshi pfge patterns efficiently mixed with the dutch ones . hence , information about geographic origin of the dutch and bangladeshi donors was not obvious among genotype data of the staphylococci inhabiting the human fingertip skin .
although in general a limited number of five and three persons tested would not allow for cluster analysis , the full absence of clusters as identified in the present study already shows that individual geographic inferences for forensics purposes is not possible using the approach employed here .
table 2staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) to conclude , we see only limited geographic differentiation between microbial dna fingerprints from dutch europeans and bangladeshi south asians , indicating that geographic inferences of human hosts from fingertip microbial dna analysis is not feasible .
furthermore , our results from dna profiling of transient and resident fingertip microflora show that human fingertip microflora is too dynamic and thus does not fulfill the criteria required for forensic marker developments to infer any human host information from physical fingerprints .
| human fingertip microflora is transferred to touched objects and may provide forensically relevant information on individual hosts , such as on geographic origins , if endogenous microbial skin species / strains would be retrievable from physical fingerprints and would carry geographically restricted dna diversity .
we tested the suitability of physical fingerprints for revealing human host information , with geographic inference as example , via microbial dna fingerprinting .
we showed that the transient exogenous fingertip microflora is frequently different from the resident endogenous bacteria of the same individuals . in only 54% of the experiments , the dna analysis of the transient fingertip microflora allowed the detection of defined , but often not the major , elements of the resident microflora .
although we found microbial persistency in certain individuals , time - wise variation of transient and resident microflora within individuals was also observed when resampling fingerprints after 3 weeks . while microbial species differed considerably in their frequency spectrum between fingerprint samples from volunteers in europe and southern asia
, there was no clear geographic distinction between staphylococcus strains in a cluster analysis , although bacterial genotypes did not overlap between both continental regions .
our results , though limited in quantity , clearly demonstrate that the dynamic fingerprint microflora challenges human host inferences for forensic purposes including geographic ones .
overall , our results suggest that human fingerprint microflora is too dynamic to allow for forensic marker developments for retrieving human information.electronic supplementary materialthe online version of this article ( doi:10.1007/s00414 - 009 - 0352 - 9 ) contains supplementary material , which is available to authorized users . |
assisted / supported mechanical ventilation is designed to interact with patient muscle activity and ' share ' the work of breathing . if properly done , assisted /
, the ventilatory 's flow and pressure delivery must synchronize with patient effort during all three phases of breath delivery : breath initiation , flow delivery , and breath termination ( cycling ) .
dys - synchronous interactions can overload ventilatory muscles ( ' imposed ' loads ) , compromise alveolar ventilation , overdistend alveolar units , disrupt sleep patterns , and cause patient discomfort , prompting additional sedation .
carlucci and colleagues have recently studied patient - ventilator interactions in 69 acutely ill patients receiving non - invasive ventilation ( niv ) for a variety of reasons .
they grouped these patients according to obstructive or restrictive pathophysiologies and carefully measured respiratory system mechanics ( including esophageal pressure - a surrogate for pleural pressure ) .
the patients were then initiated on niv according to a standard protocol and were assessed for triggering dys - synchronies defined as missed triggers , delayed triggers , or double triggers .
importantly , they found that these dys - synchronies were common ( 58% of patients experienced them with missed triggers being the most frequent ) .
trigger dys - synchronies were associated with niv intolerance and their prevalence was similar among the different patho - physiologic patterns .
this study is important as it underscores that patient - ventilator trigger dys - synchrony is common and can be a major barrier to niv effectiveness .
this study also underscores the fact that the causes of trigger dys - synchrony are complex and affect all types of patients requiring assisted / supported mechanical ventilation [ 3 - 5 ] . like invasive mechanical ventilation , niv trigger dys - synchrony can stem from insensitive / poorly responsive breath triggering mechanisms . however , other factors also involved likely include ventilatory muscle loading patterns and their effects on the ventilatory controller , ventilatory muscle function , lung volumes at end - expiration ( that is , intrinsic positive end - expiratory pressure ( peep ) ) , gas exchange effectiveness , and cortical inputs to the patient 's ventilatory control center such as pain [ 3 - 5 ] . in addition , the triggering process with niv can be further hampered by the mask system with its inherent leaks and claustrophobia exaggerating dyspnea and neural drive .
importantly , this study did not address issues of flow synchrony or cycle synchrony , two other forms of patient ventilator interactions likely to impact niv tolerance but not well studied in the current literature .
flow synchrony is defined as the ability of the ventilator to provide flow in accordance with patient demand as manifest by the ventilatory muscle contraction pattern .
if flow is synchronous with that contraction pattern , the inspiratory muscle pressure time profile conceptually should resemble a near normal pattern .
instead it means providing flow to ' re - shape ' the inspiratory muscle 's pressure - time or pressure - volume profile to a more physiologic shape . in general
, the variable flow features of pressure targeted breaths tend to flow synchronize easier with patient effort than clinician set flow - volume targeted breaths .
an assisted / supported mechanical breath termination shorter than the neural inspiratory time ( machine ti < neural ti ) can lead to muscle activity beyond the machine 's flow delivery phase , which can lead to high muscle loading , excessive tidal volumes and/or triggering of a second breath .
in contrast , when mechanical breath cycling terminates after the inspiratory effort has ended ( machine ti > neural ti ) , dyspnea and expiratory muscle recruitment may occur in an effort to terminate the breath .
importantly , it is worth noting that dys - synchronous interactions often result in anxiety and dyspnea , which can stimulate overall ventilatory drive .
thus , improving synchrony in one area ( for example , triggering ) can help facilitate achieving synchrony in other areas ( for example , flow demand ) . in summary ,
patient - ventilator interactions are complex and are important to recognize and manage during both invasive and non - invasive mechanical ventilation .
carlucci and colleagues have demonstrated how common the trigger dys - synchronies are during niv and that understanding them requires assessment not only of respiratory system mechanics , but also the complex interactions of neural drive , gas exchange , muscle function , and muscle loading .
much research is needed in this area as dys - synchronies of all types can lead to overloaded muscles and dyspneic patients requiring sedation - two factors that clearly can lead to prolonged mechanical ventilation days and poor outcomes .
| patient - ventilator trigger dys - synchronies are common with the use of assisted forms of mechanical ventilatory support , including non - invasive mechanical ventilatory support ( niv ) .
future system designs need to address this in order to improve the effectiveness of niv . |
postanginal septicaemia in the early 20 century ; originally associated with a mortality reaching 90% it involves a classic triad of oropahryngeal sepsis , internal jugular vein ( ijv ) thrombophlebitis and metastatic abscess formation ( 1 ) .
clinical findings can therefore vary considerably from neck pain and swelling associated with thrombophlebitis to pleuritic chest pain , haemoptysis , dyspnoea or arthralgia as a result of septic thromboemboli .
we report a case of predominantly external jugular vein ( ejv ) involvement in an adolescent male with negative blood culture results .
a twelve year old boy presented to the children s assessment unit with a 24 hour history of unilateral face and neck swelling on a background of seven days progressive sore throat and dysphagia .
minor systemic upset was also noted including headache , vomiting and non specific abdominal pain .
a provisional diagnosis of tonsillitis had been made five days earlier by the local gp and a course of erythromycin and penicillinv commenced .
examination revealed tender swellings over the left temporal area , angle of the mandible and upper third of the sternocleidomastoid extending into the submandibular region .
examination of the oral cavity was complicated by trismus , however no obvious tonsillar enlargement , erythema or exudate was recorded .
initial blood results showed an elevated c - reactive protein of 155mg / l and white cell count of 16.6(10 * 9l ) .
initial imaging of the neck comprised an ultrasound scan ; this demonstrated normal right sided vasculature but abnormal left ejv and retromandibular veins .
these veins appeared dilated with increased wall vascularity and showed no recordable signal on doppler imaging , an appearance in keeping with an inflammatory thrombophlebitis . to note
an mri brain with venography ( mrv ) sequences provided greater anatomical delineation ( figure 1 ) . despite a coincidental finding of a smaller calibre left venous sinus ; thrombus was not seen to extend intracranially .
lastly an echocardiogram was sought confirming a normal heart with no septal defects , vegetations or thrombus .
mrv brain images demonstrate bilateral sagittal , straight , transverse and sigmoid sinuses with no evidence of thrombus extension . both right and left ijv as well as right ejv are clearly patent . the left ejv could not be demonstrated .
treatment was started promptly on clinical suspicion of ls and consisted of intravenous ( iv ) metronidazole , benzyl penicillin and dexamethasone ; anticoagulation was not given .
the patient remained in hospital on iv antibiotics for a total of 14 days , the noted swellings slowly decreased in size , blood results normalized and the trismus resolved .
he remained afebrile throughout , developed no subsequent complications and was discharged home to complete seven weeks of oral antibiotics .
despite ijv thrombophlebitis forming a main diagnostic criterion for ls , we report a case in which the primary vascular involvement is that of the ejv , with a corresponding normal ijv .
studies have previously described ls with thrombosis of the carotid artery , intracranial venous sinus and abdominopelvic vessels ( 1 ) ; only three have ever described involvement of the ejv .
this unusual finding helps to highlight the variable nature of this condition and the diagnostic confusion it may herald . to note there are reported cases of ls in which there was no demonstrable vascular involvement .
numerous theories have tried to explain the spread of organisms from oropharynx to the surrounding vein ; haematogenous via the tonsillar vein , secondary to lymphangitis or via direct spread through deep neck spaces .
( 3 ) the incidence of ls within developed countries is now thought to lie around one per million per year , significantly reduced from the pre antibiotic era , however a resurgence over the last decade has recently been proposed .
mortality has been recorded at 6.9% however morbidity and complications remain high as the early diagnosis can often be missed and treatment delayed .
( 3 ) although classically described as following an oropharyngeal infection ls has been reported developing from other primary infective sites including the middle ear , sinuses , mastoid and teeth ( 1 ) .
the development of sepsis in ls is often delayed , presenting three to seven days from signs of the primary infection , which may have all but resolved ( 4 ) , making accurate history taking and a high index of clinical suspicion ever more important .
ls is classically but not exclusively associated with an obligate oropharyngeal gram negative bacillus ; fusobacterium necrophorum .
other causative pathogens described in the literature include streptococcus , staphylococci and bacteroides species ( 5 ) .
it has however been reported that in almost 13% of cases no pathogen is isolated on culture ( 6 ) .
ls can also be associated with thrombophillia ( thought to be endotoxin induced ) ( 5 ) .
the role of anticoagulation however remains controversial with no conclusive literature evidence ; the decision should therefore be made on clinical grounds ; assessing severity of symptoms , poor treatment response and degree of thrombus extension .
a recent history of a sore throat in conjunction with sepsis and or a suggestive and often declining clinical picture should always prompt early antibiotic therapy whilst a diagnosis of ls is sought ; hopefully preventing the development of late embolic complications .
differential diagnoses to consider include infectious mononucleosis , parapharyngeal abscess , lymphoma , pneumonia ( including atypical forms ) and other embolic sources ( e.g. infective endocarditis ) . | oropharyngeal infections are routinely encountered within general practice and accident and emergency departments .
most settle with simple analgesia and antibiotics ; occasionally such patients may develop lemierre s syndrome ( ls ) a rare and potentially fatal sequela that can be easily overlooked .
we aim to highlight the main symptoms , pathology , investigations and management . |
helicobacter pylori is a gram - negative , spiral and microaerophilic microorganism that was first isolated from the stomach of patients afflicted with peptic ulcer ( 1 , 2 ) .
this bacterium can cause gastritis and peptic ulcer in the short - term and carcinoma of the stomach in the long - term ( 3 , 4 ) .
polymerase chain reaction ( pcr ) is the desired method of proving the presence of h. pylori in tissues due to its high sensitivity and specificity ( 5 , 6 ) .
it was previously thought that the human stomach was the sole source of h. pylori ; however , it was later found that they are present in human dental plaques , mouth , saliva , tonsils , adenoids , middle ear , nasal polyps and sinus mucosa ( 7 ) .
it is believed that gastroesophageal reflux is involved in many cases of upper respiratory tract disorders including pharyngoesophageal disorders , croup , oropharyngeal dysphagia , rhinosinusitis , otalgia and different forms of otitis media ( 8 , 9 ) .
samples from tonsil and adenoid tissues showed that they might be sources of h. pylori ( 10 , 11 ) .
researchers have proved that there is a connection between chronic tubotympanal disorders and gastroesophageal reflux , but the mechanism is unknown ( 12 , 13 ) .
it is suspected that gastroesophageal reflux is involved in the pathogenesis of chronic otitis media .
evidence showed that stomach pepsin is present in the middle ear cavity of patients afflicted with chronic otitis media , as compared to the control group , supports this suspicion ( 14 , 15 ) .
gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments reduce frequency of infection in the middle ear ( 16 ) .
many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 - 19 ) .
however , the correlation between chronic suppurative otitis media and its various aspects , such as tympanosclerosis in which deposition of acellular hyaline and calcified deposits accumulate in the lamina propria of the middle ear has not been sufficiently studied ( 20 , 21 ) .
so , we decided to use the pcr technique to determine the role of h. pylori in tympanosclerosis .
this case - control and cross - sectional study was conducted on patients who were diagnosed with chronic infection of the middle ear and were candidates to undergo surgery in the imam khomeini or apadana hospitals of ahvaz in 2013 .
patients who had operations for chronic infections of the middle ear and those who had ventilation tube inserted into their ears in the past were excluded from the study .
the middle ear mucosa of the patients was examined during the surgical operations and based on this clinical evaluation , they were placed in one of the four types of normal , edema and hypertrophy , hyalinized and sclerotic and polyploid with or without cholesteatoma and this classification was recorded in the questionnaire . during the operations
, samples were taken of the middle ear mucosa of the patients . in patients with sclerotic plaques ,
the samples were kept under sterilized conditions at 20 degrees centigrade , and were eventually sent to the laboratory of jundishapur university of medical sciences , ahvaz , iran . in the laboratory , dna was extracted using the roche high - pure template pcr kit ( roche , germany ) according to the manufacture protocol .
primers used to amplify the typing region were va1-f ( a3436 ) 5-atggaaatacaacaaacacac-313 and va1-r ( c1226 ) 5-ctgcttgaatgcgccaaac-313 for amplified 286-bp band and vag - f 5-caatctgtcaatcaagcgag-14 and vag - r 5-ctagcgtcaaaataattccaagg-314 for amplified 630-bp .
the extracted dna was amplified using 5 l dna template , 0.3 l taq polymerase , 1 l dntp , 0.25 l each forward and reveres primers , 2 l mgcl2 and 5 l 10x pcr buffer , all mixed in a micro - tube and the ultimate volume reach to 50 l using nuclease - free distilled water .
after activation of reaction in 95c for 5 minutes , amplification performed for 35 cycles .
cycle program for first pair primers was as : 94c for 60 , 52c for 60 and 72c for 90 seconds . a final extension 72c for 5 minutes was the ultimate step of cycles .
for the second pair primers , cycle program was as : 94c for 30 seconds , 56c for 60 seconds and 72c for 90 seconds .
the chi - squared and the independent - samples t - test were also used to analyze the data . the selected level of significance was p < 0.05 .
fifty - six patients participated in this study , of whom 29 ( 51.8% ) were men and 27 ( 48.2% ) women .
the biopsy samples of the middle ear mucosa of 31 ( 55.4% ) patients were h. pylori - positive . of the 29 male patients , 15 cases were ( 51.7% ) h. pylori - positive , while of the 27 female patients 16 cases ( 59.3% ) were h. pylori - positive .
this difference was not significant in pearson s chi - squared test ( p = 0.571 ) .
the average age of h. pylori - positive patients was 30.3 years old ( standard deviation = 7.6 ) , while the average age of h. pylori - negative patients was 33.4 years ( standard deviation = 9.7 ) .
this difference was not significant in the independent samples t - test ( p = 0.571 ) .
therefore , there was no significant correlation between the presence of h. pylori and the age and gender of the patients .
the most common pathological type of middle ear mucosa was hyalinized and sclerotic type in 19 cases ( 33.9% ) .
the polyploid type with or without cholesteatoma type was reported in 15 ( 26% ) cases and the edematous and proliferative type with 12 ( 21% ) cases .
examination of the middle ear mucosa of the 19 patients of the hyalinized and sclerotic type revealed that 16 ( 84.2% ) were h. pylori - positive ( figure 1 ) and 3 ( 15.8% ) were h. pylori - negative ; while of the 37 patients whose middle ear mucosa was not sclerotic,15 cases ( 40.7% ) were h. pylori - positive ( figure 2 ) 22 ( 59.3% ) h. pylori - negative .
this was a significant difference ( p = 0.002 ) , which means there was a correlation between the presence of h. pylori and tympanosclerosis of the middle ear ( table 1 ) .
as for other types of middle ear mucosa ( normal , polypoid with or without cholesteatoma and edema with hypertrophy ) , there was no significant correlation between them and the presence of h. pylori .
l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 2 : case h. pylori - positive ( 286 bp ) , case h. pylori - negative .
l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 3 : case h. pylori - positive ( 630 bp ) , negative control , case h. pylori - negative .
results showed that 26 ( 46.4% ) of 56 patients had dry ears for at least three months before surgery .
of the 30 patients who did not have dry ears , 13 cases ( 34.3% ) were h. pylori - negative and 17(56.7% ) were h. pylori - positive .
this was not a significant difference in pearson s chi - squared test ( p = 0.832 ) .
40 ( 45.2% ) of the 31 patients with perforations of more than 50% were h. pylori - negative and 17 ( 54.8% ) were h. pylori - positive .
h. pylori was not significant in pearson s chi - squared test ( p = 0.931 ) .
therefore , there was no correlation between the presence of h. pylori and the size of the perforation or dryness of the middle ear .
zollner ( 22 ) was the first to use the term tympanosclerosis in 1956 to describe a common complication resulting from middle ear infections .
tympanosclerosis is an abnormal condition in the middle ear cavity in which lime particles accumulate in the tympanic membrane , tympanic cavity and ossicular chain ( 23 , 24 ) .
it is a long - term complication of infections of the middle ear ( 4 , 25 - 27 ) . in available reports ,
the prevalence of tympanosclerosis varies from 14.1% in bhaya s report ( 28 ) to 35.64% in wu s study(21 ) . despite the extensive available information on the clinical , histologic and pathologic aspects of tympanosclerosis ,
tympanosclerosis is observed as irreversible changes in the temporal bone ( 13 , 24 ) .
it is thought that gastroesophageal reflux may be involved in the pathogenesis of otitis media and evidence indicating the presence of stomach pepsin in the middle ear cavity of patients with otitis media , as compared to the control group , confirms it ( 14 , 29 ) .
gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments make frequent middle ear infections go away ( 16 ) .
many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 , 18 ) ; however , the correlation between chronic suppurative otitis media and its various aspects such as tympanosclerosis has not been sufficiently investigated . in tympanosclerosis ,
the process of deposition of acellular hyaline and calcium deposits in the lamina propria of the middle ear takes place , following which it is even possible for fixation of ossicles to occur leading to conductive hearing loss ( 20 ) . the exact etiology and pathogenesis of tympanosclerosis
are not completely known ( 9 ) . in some studies , tympanosclerosis has been observed also as a complication of frequent otitis ( 21 , 29 ) . in a recent research
, it has been suggested that h. pylori is the pathologic factor in tympanosclerosis ( 21 ) . in our research ,
designed to study the correlation between h. pylori and tympanosclerosis , we found that h. pylori has a prevalence of 84.2% among patients with suppurative infection of the middle ear , while its prevalence was 40.5% in patients without tympanosclerosis .
this difference ( p = 0.002 ) indicates that h. pylori plays a role in the pathogenesis of tympanosclerosis .
( 26 ) showed that there are similar risk factors , such as increased homocysteine levels , between coronary artery atherosclerosis and tympanosclerosis .
although tympanosclerosis and atherosclerosis are two separate pathologies , they have similar histological and pathophysiologic elements and the same genetic reason has been proposed for both ( 2 ) .
( 30 ) studied evidence concerning the development of inflammation caused by h. pylori in 38 patients with atherosclerotic plaques who had undergone endarterectomy and found that in most of these patients h. pylori was correlated to the inflammation , which indicated the correlation between h. pylori and atherosclerosis .
( 31 ) show that contact with h. pylori increases the risk of cardiovascular diseases even in the absence of other risk factors .
our findings suggest that h. pylori probably plays a role in the development of tympanosclerosis .
( 26 ) carried out a case - control study and found that 100% of the patients with tympanosclerosis were h. pylori - positive while only 26.9% of the patients not suffering from tympanosclerosis were h. pylori - positive ( p < 0.01 ) ( 21 ) . results of our study , while confirming those of iriz et al .
( 26 ) used the campylobacter - like organism ( clo ) test in h. pylori identification , while we employed the pcr method . since the pcr method is more accurate than the clo test , our results are probably more acceptable than those iriz et al .
( 26 ) , both show that there is a correlation between the presence of h. pylori and tympanosclerosis , and suggest the presence of h. pylori as the etiology for this disease ; however , neither study can consider h. pylori as the definite pathogenesis for tympanosclerosis . to prove this correlation
, a study should be designed to evaluate the correlation between tympanosclerosis and h. pylori by considering those pathological changes found in the examination of the middle ear mucosa that attribute the presence of inflammatory symptoms to the activity of h. pylori in the middle ear .
this study offers a better understanding of the etiological and pathogenic aspects of h. pylori in tympanosclerosis that can be used in designing new treatment methods for this disease . | background : tympanosclerosis is a condition caused by calcification of tissues in the middle ear mucosa that sometimes results hearing loss .
helicobacter pylori is one of the pathological and etiologic factors in the development of tympanosclerosis.objectives:the purpose of this study was to show the role of h. pylori in the different aspects of chronic suppurative otitis media using the polymerase chain reaction ( pcr ) technique.patients and methods : this case - control and cross - sectional study was performed on all patients with chronic otitis media , candidates for surgical operations , in 2013 .
they were allocated into the case group with tympanosclerosis and the control group without tympanosclerosis . during the surgical operation
, biopsy was done from middle ear and the samples were studied to see if they contained h. pylori using the pcr method.results:from a total of 19 patients with tympanosclerosis , 16 cases ( 84.2% ) were h. pylori positive , while in the control group 15 ( 45.4% ) cases out of the 37 cases were h. pylori positive , which showed a significant difference ( p = 0.002 ) .
age and gender of the patients , ear dryness and perforation size were not correlated with the presence or absence of h. pylori.conclusions:there is a significant correlation between tympanosclerosis and h. pylori ( p = 0.002 ) .
this correlation can single out h. pylori as a pathological factor in the development of tympanosclerosis ; however , further studies are needed to prove this correlation . |
trigeminal neuralgia ( tn ) is a disorder of cranial nerve ( cn ) v that results in severe episodes of shock - like or lancinating pain in one or more of its three divisions ( v1v3 ) .
idiopathic tn and cases due to vascular compression of cn v are categorized as classical tn .
patients diagnosed with symptomatic tn experience trigeminal - related facial pain secondary to a brain tumor , skull deformity , or multiple sclerosis ( ms ) .
evidence suggests that the majority of cases of tn are the consequence of focal compression of the entry zone of the root of the trigeminal nerve , while only 2% of cases are observed in patients diagnosed with ms .
other than excruciating facial pain , there are no other direct medical symptoms associated with tn , and the condition does not decrease life expectancy .
however , many patients with tn struggle with accomplishing tasks that affect quality of life , which is how this disorder elicits a negative impact on the social and mental wellness of the patients who suffer from this illness . following the diagnosis of tn
however , many patients experience only limited relief from medication or are unable to endure the side effects of the prescribed drugs , and in turn seek neurosurgical intervention .
currently , surgical approaches include microvascular decompression ( mvd ) or a number of techniques that target the trigeminal ganglion or root which involve the destruction or blockage of portions of those anatomical structures [ 1 , 2 ] .
although the neurosurgical modalities are preferred in many clinical situations and have proven to be effective in achieving initial pain control , they are known to come with a variety of complications , and facial pain recurrence is likely .
stereotactic radiosurgery ( srs ) has proven to be an effective management approach for patients with medically and surgically refractory tn as a primary and repeat treatment modality .
the use of radiosurgery in the treatment of tn dates back to sweden in the 1950 's , where professor lars leksell performed radiogangliotomies directed at the gasserian ganglion .
since the time of leksell , advancements in radiosurgery and imaging technologies has led to the increasing popularity of srs as a treatment option for patients with tn .
one form of srs that can be delivered to a patient is through a machine called the gamma knife ( gk ) .
the gk device is a cobalt-60-based machine , with 201 separate 4 to 18 mm collimator openings , that emits multiple gamma rays that converge on a target specified by computer planning . for specific medication intolerable patient subsets , gamma knife radiosurgery ( gkrs )
can be used as an initial management approach , or as a secondary management approach following radiosurgery or one or more of the various surgical modalities . as the evidence examining the role of gkrs in the management of patients with tn is increasing ,
it is of utmost importance for physicians to understand the criteria associated with gkrs , so that the optimal course of treatment for their patients can be prescribed . an evidence - based review on the evaluation and treatment of tn by gronseth et al . found level c evidence indicating that gasserian ganglion percutaneous techniques , gkrs , and mvd may be considered for facial pain management for medically refractory patients .
however , questions remain regarding optimal treatment modalities in specific patient subsets . for this reason ,
the goal of this paper is to provide a modern review of the literature thoroughly analyzing the efficacy of gkrs in the treatment of patients with tn , as well as evaluating the treatment planning and methods associated with this evolving modality .
to identify contemporary studies assessing the clinical outcomes of patients treated with gkrs for tn , a pubmed search from 2006 to april 2011 was performed .
keywords for search included gamma knife or gamma knife radiosurgery or stereotactic radiosurgery trigeminal neuralgia or tic douloureux .
studies analyzed in this review included retrospective cohort studies and prospective cohort studies with 5 evaluated patients .
studies published only in abstract form and studies published in a language other than english were excluded from our analysis . due to our broad search strategy and the vast amount of world literature ,
we reviewed a total of 19 studies [ 4 , 5 , 925 ] analyzing the efficacy of patients with tn who were treated once with gkrs ( table 1 ) .
thirteen of the 19 evaluated studies [ 4 , 5 , 919 ] utilized the barrow neurological institute ( bni ) pain intensity scale as a measurement of response to treatment ( see section 3 ) .
of these 13 studies , only two [ 9 , 13 ] analyzed patients treated with gkrs as an initial management approach . with a median followup of 31 months , sheehan et al .
classified 87% of patients in bni class i - iiib , while chen et al .
classified 91% of patients in bni class i - iiib ( median followup = 15 months ) .
chen et al . also reported that five of the 44 patients ( 11% ) treated with gkrs developed hypoesthesia following the procedure .
the other 11 bni pain intensity scale studies we reviewed included patients where previous surgical procedures were performed in a fraction of patients [ 4 , 5 , 1012 , 1519 ] or all patients . of the 10 studies where previous surgical procedures were performed in a fraction of patients , nine reported outcomes in terms of categorizing patients in bni class i - iiib [ 4 , 5 , 1012 , 1518 ] .
classified 58.6% of patients in bni class i - iiib ( median followup = 48 months ) , kondziolka et al .
classified 71% of patients in bni class i - iiib at three years , dhople et al .
classified 72% of patients in bni class i - iiib ( median followup = 29 months ) , han et al
. classified 76.7% of patients in bni class i - iiib ( mean followup = 58 months ) , dhople et al .
classified 81% of patients in bni class i - iiib ( median followup = 5.6 years ) , matsuda et al .
classified 82% of patients in bni class i - iiib ( median followup = 37 months ) , little et al .
classified 83% of patients in bni class i - iiib ( median followup = 6.3 years ) , dellaretti et al .
classified 89.5% of patients in bni class i - iiib ( mean followup = 20.3 months ) , and park and hwang classified 94% of patients in bni class i - iiib with a minimum followup of 3 years .
reported clinical outcomes with respect to bni class i , which contained only 5.7% of patients .
the study that evaluated gkrs where previous surgical procedures were performed in 100% of patients classified 85% of patients in bni class i - iiib , with a median followup of 36 months .
we also reviewed two studies that used the excellent - good - fair - poor ( egfp ) categorical scale to assess patient outcomes [ 20 , 21 ] ( see section 3 ) .
treated 30 patients with tn with gkrs at iran gamma knife center between 2006 and 2007 .
the authors reported that 40% of patients had an excellent outcome , 10% of patients had a good outcome , 33% of patients had a fair outcome , and 17% of patients had a poor outcome following the procedure .
after surgery , 15 patients ( 54% ) reported an excellent outcome , one patient ( 4% ) reported a good outcome , two patients ( 7% ) reported a fair outcome , and 10 patients ( 36% ) reported a poor outcome .
the complications from mvd included facial numbness in six patients ( 21% ) , dysesthesias in three patients ( 11% ) , and delayed facial palsy in one patient ( 4% ) .
four of the 19 studies we evaluated [ 2225 ] used other methodologies in determining the effectiveness of gkrs . in a prospective controlled trial ,
rgis et al . analyzed 100 patients with tn treated with gkrs and reported that 83 patients ( 83% ) were completely pain free , 58 of which ( 58% ) discontinued all medication following the procedure ( minimum followup = 12 months ) .
ten patients ( 10% ) experienced radiation - induced complications , which included facial paresthesia or hypesthesia .
performed a study investigating the short - term efficacy of gkrs in 67 patients with medically refractory tn .
overall , 77.6% of patients witnessed some degree of pain relief , with 32.6% of those patients becoming completely pain free .
of the 67 patients , 10 ( 14.9% ) experienced complications from the procedure , which included hypoesthesia and paresthesia .
sixty eight patients ( 42.5% ) underwent prior invasive treatments . in clinical analysis , it was found that 61% of patients were pain free without medication , 29% of patients were pain free with medication , and 10% of patients did not respond to gkrs .
thirty eight patients ( 49.4% ) exhibited some level of pain improvement following gk treatment , with 23 of those patients ( 29.9% ) reporting a pain - free outcome .
twelve patients ( 15.6% ) experienced complications , which were reported to be mild facial sensory changes and mild facial nerve dysfunction .
as gkrs has proven to be an effective initial treatment for tn , numerous reports have been published analyzing patients treated on multiple occasions ( > 1 ) with gkrs .
we reviewed six studies evaluating patients treated more than once with gkrs [ 2732 ] ( table 2 ) .
of these six articles , two [ 29 , 32 ] utilized the bni pain intensity scale .
sixteen patients ( 76.2% ) exhibited compelling improvements and were placed in bni class i - ii .
huang et al . analyzed 65 medically refractory patients with tn who were treated with gkrs as a second treatment modality .
of these 65 patients , 30 ( 46% ) had undergone gkrs as an initial management approach .
the authors placed 22 patients ( 34% ) in bni class i , 11 patients ( 17% ) in bni class ii , four patients ( 6% ) in bni class iiia , and five patients ( 8% ) in bni class iiib .
overall , with a median followup of 64 months , 65% of patients reported successful results in terms of pain control rates . a total of three of the six reviewed studies evaluated patients using the egfp categorical scale [ 28 , 30 , 31 ] .
analyzed 37 patients treated a second time with gkrs for recurrent tn and reported that 17 patients ( 46% ) achieved excellent pain relief , nine patients ( 24% ) achieved good pain relief , five patients ( 14% ) achieved fair pain relief , and six patients ( 16% ) achieved poor pain relief .
however , the authors concluded that 57% of patients experienced some form of trigeminal dysfunction following repeat radiosurgery .
, huang et al . treated 28 patients with repeat gkrs and reported that 12 patients ( 43% ) exhibited excellent pain relief , five patients ( 18% ) exhibited good pain relief , and two patients ( 7% ) exhibited fair pain relief .
in addition , the authors found a statistically significant ( p = 0.047 ) correlation between cumulative radiation doses > 115 gy and facial numbness . in a separate study , huang et al .
specifically , a total of eight patients underwent mvd a mean of 7.6 months following repeat gkrs .
of the eight patients , seven ( 87.5% ) were completely pain free at a mean of 21 months following neurosurgery .
this data supports the use of mvd if multiple gk procedures are deemed ineffective .
kimball et al . treated 53 patients with repeat gkrs and analyzed the patients not lost during followup using the marseille scale , which categorizes patients into one of five classes , with a higher class statistically indicating a worse prognosis for the patient . with a mean
followup of 42 months , 20 patients ( 43.5% ) were categorized in marseille class i - ii , six patients ( 13% ) were categorized in marseille class iii - iv , and 20 patients ( 43.5% ) were categorized in marseille class v. the authors also reported a statistically significant ( p = 0.047 ) correlation between facial numbness and superior long - term pain relief .
a total of 22 patients ( 48% ) experienced trigeminal dysfunction of any kind , while 21 patients ( 46% ) experienced numbness in the face .
since gkrs can be performed as both initial and salvage treatment options for patients who suffer from tn , its efficacy has been compared in patients who undergo one versus multiple radiosurgery procedures .
we reviewed eight studies to further examine this matter [ 3 , 3339 ] ( table 3 ) .
four of the eight studies utilized the bni pain intensity scale to evaluate patient outcomes [ 3 , 3335 ] .
, it was reported that 75% , 60% , and 58% of patients with idiopathic tn had bni scores of i iiib at 1 , 3 , and 5 years , respectively .
the 1- , 3- , and 5-year - bni scores of i iiib in patients with ms - related tn were 56% , 30% , and 20% , respectively .
the authors concluded that repeat gkrs exhibited similar success rates when compared to the initial procedure .
similar to verheul et al . , park et al . did not find differences in terms of time to initial response , time to pain recurrence , and overall pain relief when comparing patients who underwent one versus two gk treatments .
however , it was observed that patients who received two gk treatments were more likely to have facial sensory changes when compared to patients treated a single time with radiosurgery .
little et al . performed a study where 79 patients with typical tn were treated with gkrs as a salvage procedure .
approximately five years following salvage gkrs , the authors reported that 50% of patients experienced pain relief and 20% of those patients were completely pain free .
in addition , a statistically significant ( p = 0.029 ) correlation between gkrs failure and prior mvd was found .
treated 37 patients ( 78% had failed prior surgery ) with ms - related tn with gkrs .
nine patients ( 24% ) underwent gkrs as their first procedure . the reported 1 , 3 , and 5 year bni scores of i iiib were 82.6% , 73.9% , and 54% , respectively . the other four studies we reviewed utilized the egfp categorical scale as a measurement of response to treatment [ 3639 ] .
two of the evaluated studies [ 36 , 37 ] were conducted by fountas et al . and analyzed patients treated with gkrs for idiopathic tn based on whether or not they had undergone previous surgical or radiosurgical procedures for facial pain control .
one of the studies evaluated 106 patients ( 19 previous radiosurgery procedures ) and concluded that the treatment group without a previous history of surgical or radiosurgical procedures exhibited superior clinical outcomes , with 1-year and 2-year complete pain relief rates of 82.5% and 78% , respectively .
the 1-year and 2-year complete pain relief rate in the patient group with a history of surgical or radiosurgical procedures was 69.4% and 63.5% , respectively .
as expected , similar results were found in the other study by fountas et al .
; however , no prior radiosurgical procedures were performed in the patient group with a history of prior procedures .
huang et al . conducted a study where 89 patients with idiopathic tn were treated with gkrs as an initial management approach , 20 of which underwent a subsequent gkrs procedure for facial pain recurrence . following the initial radiosurgical procedure ,
50 patients ( 56% ) had an excellent response , 12 patients ( 13.5% ) had a good response , and 7 patients ( 7.9% ) had a fair response .
following the second radiosurgical procedure , 11 patients ( 55% ) had an excellent response and one patient ( 5% ) had a good response . in a separate study , huang et al .
assessed 21 patients with benign tumor - related tn who were treated with gkrs as an initial or repeat procedure .
following the initial gk procedure to the tumor , 12 patients ( 57% ) had an excellent response and 1 patient ( 5% ) had a good response .
a total of eight patients were treated with a subsequent gkrs procedure targeted at the ipsilateral trigeminal root or ganglion due to facial pain recurrence . following the second radiosurgical procedure , the authors reported four patients ( 50% ) with an excellent response .
we identified six studies comparing patients treated with gkrs with patients treated with one of the various surgical modalities [ 2 , 4044 ] ( table 4 ) .
the authors of this review acknowledge the importance of percutaneous techniques in the management of tn ; however , our modern literature search predominantly yielded comparison studies analyzing the efficacy of mvd when compared to gkrs .
specifically , four of the six studies [ 2 , 4042 ] analyzed patients treated with gkrs against patients treated with mvd .
specifically , 36 were treated with mvd ( 45% ) , while 44 were treated with gkrs ( 55% ) .
the mvd treatment arm statistically differed from the gkrs treatment arm with respect to age ( median of 54 versus 74 years ) , preoperative symptom duration ( median of 2.6 versus 7.5 years ) , and the presence of major comorbidities ( 2.8 versus 58.3% ) .
the authors reported that patients treated with mvd exhibited superior levels of initial ( 100% ) , 2 year ( 88% ) , and 5 year ( 80% ) actuarial pain - free rates when compared to the patients treated with gkrs ( 78 , 50 , and 33% , resp . ) , with a p value of 0.0002 .
in addition to increased levels of patient satisfaction , as reported by required patient surveys , the mvd treatment group also had a decreased level of permanent mild ( 5.6% ) and severe sensory loss ( 0% ) when compared to the gkrs treatment group ( 6.8% and 2.3% , resp . ) .
two patients ( 5.6% ) in the mvd group experienced permanent mild paresthesias or numbness , one ( 2.8% ) patient experienced a cerebrospinal fluid leak from the wound , and one patient ( 2.8% ) experienced hearing loss and diplopia .
three patients ( 6.8% ) in the gkrs group experienced permanent mild paresthesias or numbness , one patient ( 2.3% ) experienced a more permanent sensory numbness , and one patient ( 2.3% ) experienced a transient headache and nausea following the gk procedure .
all patients were diagnosed with typical tn and did not undergo previous gk or mvd procedures . it was reported that patients treated with mvd exhibited superior levels of complete pain relief at 12 ( 68% ) and 18 months ( 68% ) when compared to the gkrs group , who 's complete pain relief rate was 58% at 12 months and 24% at 18 months ( p = 0.089 ) .
the treatment arms did not statistically differ in terms of 90% pain relief at 12 and 18 months .
this study could be criticized due to the large difference in the number of patients constituting the two treatment arms .
oh et al . evaluated a total of 45 elderly patients ( > 65 years of age ) diagnosed with idiopathic tn who were treated with either mvd ( 27 patients ) or gkrs ( 18 patients ) .
it was reported that three mvd patients ( 11% ) and three gk patients ( 17% ) underwent previous percutaneous procedures .
the mean followup period was reported to be 35.9 months for the mvd group and 33.1 months for the gkrs group . according to the bni pain intensity scale ,
the mvd group had a superior prognosis , with 17 patients ( 63% ) classified in bni class i - ii compared with the 10 patients ( 56% ) in the gkrs group classified in bni class i - ii .
the observed complications following mvd included constant headache in 11 patients ( 40.7% ) , facial paresthesia in five patients ( 18.5% ) , paresthesia of the tongue in two patients ( 7.4% ) , infection at the site of incision in one patient ( 3.7% ) , an acute subdural hemorrhage in one patient ( 3.7% ) , temporary hearing loss in one patient ( 3.7% ) , and otitis media with cerebrospinal leakage in one patient ( 3.7% ) . two patients ( 11% ) in the gkrs group experienced paresthesia .
compared the clinical outcomes of 19 patients treated with mvd with 15 patients treated with gkrs .
nine gk patients ( 60% ) and four mvd patients ( 21% ) underwent previous surgical procedures . the treatment
arms statistically differed ( p = 0.0005 ) with respect to mean patient age , with the mean age of the gkrs group exceeding the mvd group by 13 years ( 74 versus 61 years ) .
in addition , patient satisfaction was graded on a scale of 1 ( unsatisfied ) to 10 ( completely satisfied ) .
it was reported that the mean tn complexity grade was statistically different ( p < 0.001 ) between the treatment arms ( gk = 5.8 ; mvd = 3 ) .
the average response following the procedure was determined to be 3.4 for the mvd group and 2.4 for the gkrs group ( p = 0.017 ) . also , it was found that the satisfaction score for the mvd group was superior to the gkrs group ( 8.7 versus 6.4 ) , with a p value of 0.02 .
the authors reported a statistically significant correlation between tn complexity grade and clinical response ( p < 0.001 ) , as well as tn complexity grade and patient satisfaction ( p < 0.001 ) . to date
, no randomized trials have been conducted analyzing the outcomes of patients with tn who are treated with mvd compared to gkrs . in a large review on tn management ,
zakrzewska and linskey found evidence that mvd is an effective treatment for long - term facial pain relief but comes with an increased risk of ipsilateral hearing loss .
in addition , the authors concluded that single - dose srs is an effective treatment for long - term facial pain relief but puts patients at risk for facial numbness or facial paresthesias .
investigation into this matter in the form of a randomized controlled trial would provide the best evidence in terms of facial pain relief and procedure - related complications .
in addition , we reviewed two studies comparing patients treated with gkrs with patients treated with posterior fossa exploration ( pfe ) [ 43 , 44 ] , both of which were conducted by pollock and colleagues at the mayo clinic college of medicine .
one of the studies was a specific prospective comparison of 91 patients treated with pfe and 49 patients treated with gkrs for idiopathic tn as an initial management approach .
the treatment arms statistically differed in terms of age ( gkrs = 67.1 years ; pfe = 58.2 years ) , with a p value < 0.001 .
it was reported that patients treated with pfe were more likely to be pain free and off medications at 1 year ( 84% ) and 4 years ( 77% ) when compared to the gkrs group ( 66 and 56% , resp . )
retreatment for recurrent facial pain was performed in 15% of the patients in the pfe treatment arm and 35% of patients in the gkrs treatment arm ( p = 0.009 ) .
also , it was found that nonbothersome facial numbness occurred more frequently in the gkrs group ( p = 0.04 ) .
an additional study from the mayo clinic evaluated patients with recurrent tn who underwent 3 or more surgical procedures .
the authors reported that patients treated with pfe exhibited superior levels of complete pain relief at 3 years of followup when compared to patients treated with gkrs , balloon compression , and glycerol rhizotomy ( p < 0.01 ) and underwent additional surgery for recurrent facial pain less often when compared to patients treated with the other modalities ( p = 0.02 ) .
clinical 006futcomes did not differ between patients treated with gkrs and patients treated with the percutaneous techniques .
srs can be performed by a variety of tools , which include gkrs , cyberknife technology , and linear accelerator ( linac)-based treatment .
our analysis yielded one study whose primary endpoint was to devise a method using cyberknife treatment planning that would mimic the dosimetric characteristics of the gk treatment plan in five patients undergoing radiosurgery for tn .
both the isodose lines and critical structures were identified using the gkrs treatment plan and were transferred to the cyberknife treatment planning system .
it was reported that the average length of the trigeminal nerve receiving a dose of 60 gy was 4.5 mm for the gk , 4.5 mm for the nonisocentric cyberknife , and 4.4 mm for the isocentric cyberknife .
the authors found it more difficult to minimize the dose to critical structures when using cyberknife technology .
also , the dose falloff of gkrs was found to be steeper when compared to cyberknife technology due to , what the authors hypothesized , the large number of gamma rays produced which converge on the focal point with precision .
as previously mentioned , the gk machine 's primary functional unit is cobalt-60 , which is used to emit photon energy through 201 separate 4 to 18 mm collimator openings that converge on a target specified by a treatment planning system .
balamucki et al . performed a study examining if the half life of cobalt ( 5.26 years ) relates to the outcomes for patients being treated for tn with gkrs .
the authors collected data on 239 gkrs procedures performed at their institution between 1999 and 2004 .
patient surveys were used to measure responses to radiosurgery . with the followup time ranging from one to six months , it was reported that 80% of patients experienced some degree of pain relief and that 56% of those patients were pain free .
the authors concluded that clinical outcomes remained consistent during the first half life of cobalt-60 .
an area of controversy in the treatment of patients with tn is defining the optimal maximum radiosurgery dose that can be delivered to specific patient subsets .
we analyzed five studies whose primary endpoint was to assess gkrs - dosing efficacy [ 4852 ] .
kim et al . utilized the bni pain intensity scale to assess 66 tn patients treated with a gk maximum dose of 80 gy and 44 tn patients treated with a gk dose of 85 gy . although the two groups did not statistically differ in terms of facial rain relief and procedure - related complications , the authors did report that patients treated with a gk dose of 85 gy experienced a more rapid response to treatment when compared to the patients treated with a gk dose of 80 gy .
arai et al . analyzed 165 patients with tn treated with a gkrs dose of 80 gy .
specifically , the authors divided the patients into two groups , which differed in the radiation dose rate received ( low - dose rate = 1.212.05 gy / min ; high - dose rate = 2.063.74 gy / min ) . using the bni pain intensity scale as a clinical evaluation method
, it was reported that the low - dose - rate group and the high - dose - rate group did not statistically differ in terms of initial pain relief , maintenance of pain relief , and clinical complications .
patients in group one were treated with a gk dose < 90 gy with no beam channel plugging , patients in group two were treated with a gk dose equal to 90 gy with no beam channel plugging , and patients in group three were treated with a gk dose equal to 90 gy with beam channel plugging . although the trend did not reach full statistical significance ( p = 0.054 ) , patients in group three exhibited the highest level of pain relief , while patients in group one exhibited the lowest level of pain relief .
the authors also observed that the three groups statistically differed ( p < 0.0001 ) in terms of trigeminal nerve dysfunction , with patients in group three experiencing the highest rate of mild and bothersome complications and patients in group one experiencing the lowest rate of mild and bothersome complications .
similar to the results of massager et al . , morbidini - gaffney et al . reported positive outcomes in patients treated with gk doses > 85 gy .
the authors also found that patients treated with two isocenters were more likely to have superior bni pain intensity scale scores during their course of followup when compared to patients treated with a single isocenter .
the median initial dose was 80 gy , and the median retreatment dose was 45 gy . although the authors did not report any predictors in terms of facial pain control and patient morbidity , they did compare the results of their study with seven published retreatment articles and found that successful levels of pain control ( > 50% ) were significantly correlated with cumulative gkrs doses > 130 gy , as well as new trigeminal nerve dysfunction ( > 20% ) . in addition to dose selection efficacy in select patient cohorts , the radiosurgical target of cn v is another subject matter that requires further clinical investigation .
we reviewed three studies [ 5355 ] analyzing specific gkrs targeting methods in the treatment of tn and one study that examined the accuracy of gkrs to its image - guided target .
compared patients treated with gkrs targeted at the dorsal root entry zone ( 59 patients ) with patients whose radiosurgical target was the retrogasserian zone of the trigeminal nerve ( 41 patients ) . with a median followup of 30 months , the dorsal root entry target group exhibited statistically superior levels of initial complete pain remission ( p = 0.003 ) and experienced less complications than the retrogasserian zone group ( p = 0.009 ) .
chen et al . also reported positive results with the dorsal root entry zone - targeting approach , with a success rate of 82.8% and a complication rate of 15% .
park et al . compared the dorsal root entry zone and retrogasserian zone - targeting methods in the treatment of 39 patients with medically refractory tn .
the authors reported that the two treatment arms did not statistically differ in treatment success ( bni class i - iiib ) with respect to the bni pain intensity scale .
however , patients treated with the retrogasserian zone - targeting method experienced a substantially shorter time of response to gkrs than patients treated with the dorsal root entry zone - targeting method ( p = 0.044 ) .
although the two groups did not statistically differ with regard to treatment - related morbidities , it was found that the patients whose targeting approach was the dorsal root entry zone experienced a greater amount of bothersome complications than the retrogasserian zone group .
massager et al . analyzed the targeting accuracy of gkrs in 65 patients treated for tn whose six month followup mri showed focal contrast enhancement of the trigeminal nerve .
the authors found that the median deviation of the coordinates between the intended radiosurgical target and the center of contrast enhancement was 0.91 mm in euclidean space .
the median radiation dose fitting into the contrast enhancement region was determined to be 77 8.7 gy .
this small deviation from the gkrs target explains the high accuracy and precise nature of the machine .
the two most common methods of measuring patient outcomes from gkrs in the management of tn are the barrow neurological institute pain intensity scale ( table 5 ) and the excellent - good - fair - poor ( egfp ) categorical scale ( table 6 ) .
the bni pain intensity scale divides patients into one of five classes , with a higher class indicating a worse prognosis for the patient .
patients in bni class iiia do not experience trigeminal pain but require the use of medication .
patients in bni class iiib experience some trigeminal pain that can be satisfactorily managed with medication . patients in bni class iv experience some trigeminal pain that is not satisfactorily managed with medication . patients in bni class
excellent outcomes are defined as complete pain relief without the need of medication .
good outcomes are defined as complete pain relief with the need of medication .
poor outcomes are defined as a < 50% pain relief rate or treatment failure .
for patients with medically refractory forms of tn , gkrs has proven to be an effective initial and repeat treatment option .
cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .
however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .
although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .
further evidence in the form of a phase iii - randomized trial is needed to confirm the clinical outcomes of patients treated with either modality .
questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter . | since its introduction by leksell , gamma knife radiosurgery ( gkrs ) has become increasingly popular as a management approach for patients diagnosed with trigeminal neuralgia ( tn ) .
for this reason , we performed a modern review of the literature analyzing the efficacy of gkrs in the treatment of patients who suffer from tn . for patients with medically refractory forms of the condition ,
gkrs has proven to be an effective initial and repeat treatment option .
cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .
however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .
although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .
questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter . |
a 55-year - old caucasian female was undergoing elective coronary artery bypass surgery following a recent myocardial infarction .
intravenous midazolam was given to the patient in the holding area , and the right radial artery was cannulated . in the operating room , intraoperative monitors , included five - lead electrocardiography , pulse oximetry , a radial artery catheter , capnography ( etco2 ) , and bladder temperature .
the patient was intubated without any difficulty after routine intravenous induction with etomidate , sufentanil , and rocuronium .
the right internal jugular vein was cannulated with the patient in the trendelenburg position , and a pulmonary artery catheter was advanced to the wedge position without difficulty .
additional monitors included a pulmonary artery catheter with venous saturation and continuous cardiac output monitoring as well as central venous pressure .
an adult multiplane 6.0 mhz transesophageal echocardiography ( tee ) probe ( acuson , siemens , washington , dc , usa ) was placed .
systematic tee images were obtained according to the american society of echocardiography - society of cardiovascular anesthesiologists guidelines .
as the cardiac surgeon harvested the left internal mammary artery , the first assistant harvested the saphenous vein from the left leg via an endoscopic approach ( stryker endoscopy , san jose , ca , usa ) using co2 insufflation at 4 l / min and a pressure of 14 mmhg .
approximately 20 minutes after endoscopic vein harvesting started , we noticed the patient 's end - tidal co2 ( etco2 ) had increased from 36 to 44 mmhg followed by increasing pulmonary artery pressure from 42/20 to 60/39 mmhg within 1 minute .
subsequently , systemic blood pressure dropped from 101/60 mmhg to 65/30 mmhg while o2 saturation remained at 100% .
the cardiac index dropped approximately 15% from 2.2 to 1.9 l / min / m , but heart rate and central venous pressure were only slightly elevated .
the inspiratory oxygen concentration fraction was adjusted from 50% to 100% , and vasopressors were administered to support the pressure .
mid - esophageal four - chamber and mid - esophageal aortic valve long - axis views were examined , but no new mitral or aortic regurgitation was noted .
however , the mid - esophageal right ventricular inflow - outflow view revealed a massive snowflake appearance suggestive of gas bubbles from the right atrium to the right ventricle ( fig .
however , mild dilatation of the right ventricle was noted . at that time , all intravenous fluids through the central line were stopped , and all intravenous connections were checked to make sure no intravenous fluids were running . despite these maneuvers , the snowflake appearance suggestive of gas bubbles was still present .
we concluded that the gas bubbles were not caused from turbulence due to intravenous fluids or from a disconnected intravenous line , so a co2 embolism was suspected .
after this observation , we kept the patient in the trendelenburg position and increased the ventilation rate to expire more co2 . a nitroglycerine drip was started to lower pulmonary artery pressure , and phenylephrine boluses were administered to maintain systemic blood pressure within an acceptable range .
further examination of tee in the mid - esophageal bicaval view revealed that the gas bubbles were originating from the inferior vena cava ( fig .
, we concluded that the source of the gas embolism was most likely from co2 insufflation .
the surgical team was informed of these findings and they reassured us that the proximal saphenous vein was completely ligated and confirmed that most branches were adequately cauterized .
the surgical team requested 5 additional minutes to complete vein harvesting based on patient stability .
our hemodynamic assessment revealed that the patient was stable , and the tee examination revealed a significant reduction in air bubbles , so the decision was made to proceed with the endoscopic vein harvesting .
over that course , the patient remained on 100% fio2 , and a higher ventilation rate .
the nitroglycerin drip was discontinued and intermittent small epinephrine boluses were administered for inotropic support , and phenylephrine or ephedrine were administered to maintain systemic blood pressure > 90/50 mmhg .
the patient 's hemodynamic status improved shortly after vein harvesting was completed , and co2 insufflation was discontinued .
we noted the patient 's etco2 had returned back to 35 mmhg , pulmonary artery pressure had decreased steadily back to a baseline level of 40/20 mmhg , and continued down to 30/18 mmhg , whereas systemic blood pressure stabilized at 100/60 mmhg without vasopressor support .
a tee examination in the mid - esophageal right ventricular inflow - outflow view and mid - esophageal bicaval view revealed no further evidence of gas embolism .
coronary artery bypass grafting was completed without further events , and the patient recovered without any adverse neurological sequelae .
our case not only confirms the vital role of tee in the quick and accurate diagnosis of a co2 embolism during endoscopic vein harvesting but also allowed us to focus on the mid - esophageal bicaval view , a view not commonly used to identify a co2 embolism .
further review of the mid - esophageal right ventricular inflow - outflow view revealed that gas was entering the right side of the heart , but we could not determine whether the bubbles originated from the superior vena cava or the inferior vena cava . using the mid - esophageal bicaval view , we were able to confirm that the gas embolism originated from the inferior vena cava , and that the lower extremity endoscopic site was the most likely suspect .
the era of minimally invasive surgery started two decades ago and has extended its influence into cardiac surgery .
the first endoscopic vein harvesting was described by lumsden et al . in 1994 .
the endoscopic technique is associated with a shorter time ( 51.07 vs. 75.94 min ) , better leg wound healing , and a lower incidence of wound infections ( 12.0% vs. 8.8% ) compared to those of conventional open vein harvesting .
other advantages of endoscopic vein harvesting compared to conventional open vein harvesting include a smaller incision , less post - operative pain , a shorter hospital stay , and better cosmetic appearance .
the endoscopic vein harvesting procedure requires co2 insufflation at a flow of 4 l / min and pressure of approximately 14 mmhg to create a potential subcutaneous space .
in contrast , there have been reports of co2 embolism , pneumothorax , subcutaneous emphysema , and hypercarbia .
the reported incidence rate of co2 embolism ranges from 1 - 200 cases in every one million laparoscopic surgeries .
the incidence of a co2 embolism during endoscopic saphenous vein harvesting with co2 insufflation procedures decreases as the severity of clinical symptoms and the volume of embolized gas increases .
the incidence of venous co2 embolism of any size is 17.1% , whereas the incidence of massive gas emboli is only 0.5% .
the incidence of a co2 embolism is even lower when an endoscopic approach is used to harvest arterial conduits . in our hospital
, the endoscopic vein harvesting technique has been used in approximately 1,500 cases since 2005 , with no reported incidence of a co2 embolism .
manifestations of a co2 embolism include hypotension , reduced cardiac output , and decreased mixed venous oxygen saturation , along with elevated pulmonary artery and central venous pressures .
however , this sign may not be as dependable as was the case in our patient , as we observed an initial rise in etco2 instead of a reduction .
a drop in etco2 is an indication of increased dead space and insufficient pulmonary perfusion .
this condition does not generally occur unless the trapped gas in the pulmonary vascular system or the right ventricular outflow prevents blood from flowing into the lung , a condition that is generally observed with a massive embolism . whenever co2 is used for endoscopic insufflation , co2
can directly enter the injured saphenous vein or its branches , resulting in elevated etco2 .
furthermore , a smaller amount of co2 embolized to the right heart could be released acutely , resulting in elevated etco2 . as was seen in our case ,
treatment for a co2 embolism includes immediate discontinuation of co2 insufflation , placing the patient in the trendelenburg position , increasing fio2 to 100% , and aspirating the central line .
the latter intervention is less successful with a pulmonary artery catheter and may divert the attention of the anesthesia team from other rescue efforts . furthermore , inotropic agents should be started to support cardiac output to push the co2 embolism into the lungs .
cardiac massage and even urgent cardio - pulmonary bypass or extracorporeal membrane oxygenation have also been described if the patient does not respond to initial resuscitation efforts . in our case
, we choose to allow continued co2 insufflation to complete saphenous vein harvesting based on the patients stabile hemodynamics and on the observation that a reduction in gas entry to the right heart occurred shortly after detecting the co2 embolism .
this finding was interesting , as the surgical team did not reduce insufflation flow or pressure .
we believe that the source of co2 absorption was eliminated at this point , as the surgical team cauterized the open vascular structures .
in summary , the use of endoscopic vein harvesting for the saphenous vein has become increasingly more common and awareness and prevention of the potential for a co2 embolism is important . as seen in our case , tee played a vital role in detection , and we believe that the mid - esophageal bicaval view should be used as the standard view during endoscopic vein harvesting .
it has been suggested that the surgical team should pay special attention when cauterizing or ligating all vein branches to reduce the potential for co2 entry into the venous system to prevent further co2 embolisms .
although we allowed endoscopic vein harvesting to continue after confirmation of the co2 embolism with direct inspection of the saphenous - femoral vein junction and meticulous closure of the tears were confirmed , further investigation into the safety of this practice is warranted . | a carbon dioxide ( co2 ) embolism during endoscopic vein harvesting is a rare but potentially fatal complication .
early and accurate diagnosis is crucial for limiting the extent of the embolism and stabilizing the resulting cardiovascular compromise .
we report a case of co2 embolization during endoscopic vein harvesting .
transesophageal echocardiography was instrumental in the diagnosis and management of this patient by further improving the decision making process , which resulted in the best outcome .
mid - esophageal bicaval view is the best view to determine whether a co2 embolism is coming from the upper or lower extremities . |
drywood termites cause significant damage to wood in structures in the united states , with two species , incisitermes minor ( hagen ) and cryptotermes brevis ( walker ) , being responsible for the majority of damage ( su and scheffrahn , 1990 ; grace , 2009 ) .
the economic cost of control and repair of damage is second only to that for subterranean termites ( su and scheffrahn , 1990 ) .
remedial control of drywood termites in the united states relies primarily on either ( 1 ) fumigation of the entire structure with a toxic gas or heated air , or ( 2 ) localized chemical or physical treatments designed to eradicate small , localized colonies ( lewis and haverty , 1996 ; lewis , 2008 ; lewis and rust , 2009 ) .
fumigation treatments are likely to kill all of the termites in a structure , while localized treatments can destroy all termites in a colony , provided the treatment is delivered to the entire gallery system .
the size , number , and dispersion of drywood termite colonies in a structure can vary greatly , depending on the age of the infestation and the success of preventative or remedial treatments .
drywood termite colonies also are considered single piece nesters , i.e. , they nest within their food source and do not forage from the nesting site to a food source ( abe , 1987 ) .
in fact , large , dispersed colonies ( or even small colonies ) can live in a single piece of wood or in multiple pieces of wood that are joined together ( grace et al . ,
drywood termites are cryptic , seldom leaving obvious or visible external signs of their presence in wood . a commonly used sign for determining the presence of drywood termites is the occurrence of fecal pellets , ejected through a kick - out hole in the external surface of wood from the internal galleries ( ebeling , 1975 ) .
these pellets often are found as conical piles or are scattered on horizontal surfaces below infested wood .
these hexagonally sided pellets are diagnostic for drywood termites , and can be used to distinguish damage from that by other wood - destroying insects ( ebeling , 1975 ; moore , 1992 ) .
grace and yamamoto ( 2009 ) demonstrated the relationship between the cellulose and lignin content of the food utilized by small groups of c. brevis and incisitermes immigrans ( light ) , and the quantity of fecal pellets produced over time .
they also discussed the use of the size and number of fecal pellets for estimating both size and age of drywood termite colonies .
drywood termites excrete feces in the form of hard , even - shaped fecal pellets .
these fecal pellets contain the same mixture of hydrocarbons as the insects that produced them , albeit in slightly different proportions ( haverty et al .
, 2005 ) . because cuticular hydrocarbons are species specific in termites ( page et al . , 2002 )
rather than simply signaling the general presence of termites or providing a diagnosis of the species of termite inhabiting the wood , we postulated that these pellets could be chemically characterized so as to determine the status of a colony as active ( alive ) or inactive ( dead ) . here , we report quantification of the hydrocarbons in pellets of i. minor aged for up to 1 year after they were produced .
we document the changes in proportions of selected hydrocarbons as an indication of the length of time since the pellets were excreted .
collection of termites and preparation of termite containment unit termites , i. minor , were removed from one naturally infested board ( 98.9 13.3 271.8 cm ) collected on 19 july 2006 from lakeview , california , and stored at the university of california richmond field station .
the board was cut across the grain into pieces , 58 cm thick , and stored at room temperature .
a wood chisel was used to separate pieces of wood into smaller pieces , 0.51.0 cm thick .
all remaining live termites , including soldiers and primary reproductives ( alates ) , were placed in a termite containment unit ( tcu ) .
termites thus collected were likely from a large mixed colony ( booth et al . , 2010 ) or from multiple colonies .
we were not able to assign the various galleries in the wood to any particular colony.fifteen birch tongue depressors were bundled together , and three equally spaced holes drilled across the length of the bundle using a 2.8-mm bit .
bamboo skewers , 1013 cm in length , were inserted into each of the three holes in the bundle of tongue depressors .
spaces , 36 mm , were left between each tongue depressor allowing termites access to all tongue depressors .
two sets of skewered tongue depressors were placed into a clean plastic container ( 17.5 12.5 6 cm ) , one on top of the other , such that the tongue depressors on the top layer fit into the gaps created by the tongue depressors on the bottom layer .
tongue depressors were lightly misted with water , and then 6,662 drywood termites ( a mixture of pseudergates or workers , alates , and soldiers ) were placed in the tcu .
tcus , with termites , were maintained in a dark cabinet in the laboratory under ambient conditions prior to collection of fecal pellets .
a single colony or a mixture of two or more colonies , representing a single location within california , was prepared in this manner . pellet collection and aging process
after all the tcus had acclimated to laboratory conditions over several weeks , new holding chambers were prepared .
all wood debris , pellets , dead termites , and damaged tongue depressors were discarded .
the termites in the new holding chambers were maintained in the laboratory under ambient conditions for 2 weeks . at the end of this period
, all pellets were removed , and sub - samples collected and readied for the aging study .
concurrently , three samples of 20 workers , three samples of 20 alates , and one sample of 20 soldiers were also collected for hydrocarbon analysis .
live termites were placed in a 20-ml scintillation vial and frozen until extraction.fecal pellets were separated from debris by sequentially sifting them through successively smaller sieves ( haverty et al . , 2005 ) .
thus , all substances such as hand lotion , lip balm , parafilm , and waxed paper were kept away from the work area .
gloves were worn when handling pellets , and implements that came in contact with pellets were wiped with a paper towel dampened with absolute ethanol to remove any oils or waxes .
pellets were separated from fine debris by removing individual pellets with forceps or with an aspirator , until a sample weighing approximately 200 mg was obtained.in developing this protocol we made one estimate and one critical assumption .
the estimate was that at least 1,000 pellets were needed for each hydrocarbon analysis ( based on preliminary analyses of pellets collected from drywood termites maintained in our laboratory ) .
( 1997 ) using c. brevis and i. snyderi ( light ) from southern florida . in that study , 40 i. snyderi ( pseudergates or workers ) produced 573 pellets over 4 weeks , slightly less than 150 pellets / wk or about 3.5 pellets / individual / wk . in a study with small groups of c. brevis and i. immigrans , grace and yamamoto ( 2009 ) found that these species produced 4.9 to 7.0 pellets / individual / wk . for our study , we assumed that i. minor would produce pellets at roughly the same rate and , therefore , we needed about 3,000 to 5,000 individuals in a tcu for this study .
we prepared one tcu to collect the requisite quantity of fecal pellets.four sub - sampling intervals , each replicated three times , were used : 0 , 30 , 90 , and 365 days from the initial collection date .
sub - samples were stored in clean , 20-ml scintillation vials , sealed with 1.5 1.5 mm mesh screen .
vials were stored in a dark cabinet at the university of california richmond field station at ambient temperature.voucher samples of i. minor pseudergates or workers and soldiers from this study ( fresh , not dried ) were preserved in 85% ethanol and deposited in the essig museum , university of california at berkeley ( haverty et al . ,
extraction procedure and characterization of hydrocarbons hydrocarbons from workers , alates , soldiers , and fecal pellets of i. minor were extracted , characterized , and quantified as previously reported ( haverty et al . , 2005 ) .
frozen termite samples were thawed and dried at 70c for approximately 1 h before extraction . each sub - sample of fecal pellets or termites
was placed in a 20-ml scintillation vial , and immersed in 10 ml of n - hexane for 10 min . after extraction
, hydrocarbons were separated from other compounds through 4 cm of activated sigma silica gel ( 70230 mesh ) in pasteur pipette mini - columns .
the resulting hydrocarbon fractions were evaporated to dryness under a stream of nitrogen and redissolved in 60 l of n - hexane for gas chromatography - mass spectrometry ( gc - ms ) analysis .
a 3-l aliquot was injected into the gc-ms.gc-ms analyses were performed on an agilent 6890 gas chromatograph interfaced with an agilent 5973 mass selective detector , using agilent chemstation data analysis software ( g1701ca version c.00.00 ) .
the gc - ms was equipped with an hp-1ms , fused silica capillary column ( 30 m 0.25 mm i.d , 0.25 m film thickness ) , and was operated in split mode ( split ratio of 30:1 ) , using helium as carrier gas .
the column oven was programmed from 200320c at 3c min , with a final hold of 11 min .
electron impact ( ei ) mass spectra were obtained at 70 ev.all chemicals of interest were identified by their retention times and mass spectra .
mass spectra of methyl - branched alkanes were interpreted as described by blomquist et al .
olefins were identified by their mass spectra , although double bond positions were not determined ( haverty et al . , 2005).in the text , tables , and figures , we use shorthand nomenclature to identify individual hydrocarbons or mixtures of hydrocarbons .
this shorthand uses a descriptor for the location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) .
thus , pentacosane is represented as n - c25 , 11-methylnonacosane as 11-mec29 , 13,17-dimethylhentriacontane as 13,17-dimec31 , and heptatriacontatriene as c37:3 .
hydrocarbons are presented in the tables for each caste and worker fecal pellets in the order of elution from our gc - ms system .
statistical analysis gc - ms peak ( some peaks contained more than one compound or a mixture of positional isomers ) areas were converted to percentages of total hydrocarbon fraction , enabling mean ( sd ) relative amounts of each hydrocarbon peak for workers and alates , and overall mean ( sd ) of each hydrocarbon peak for the fecal pellets , to be calculated.the percentage of each hydrocarbon peak for pellets of each aging period was transformed to the log of the percentage ( dependent variable y ) and regressed against the days of aging ( independent variable x ) .
hydrocarbons with slopes different from 0 ( = 0.05 ) were separated into two groups : those with a significant positive slope and those with a significant negative slope . for each aging period ( 0 , 30 , 90 , and 365 day ) , an index of age ( iage ) was created by subtracting the sum of the percentages of the hydrocarbons with a negative slope over time from the sum of the percentages of the hydrocarbons with a positive slope over time [ iage = ( peakpositive ) minus ( peaknegative ) , for each aging period ] .
the iage for each aging period then was regressed against the aging period ( r development core team , 2004 ) .
the cuticular hydrocarbons for i. minor pseudergates and fecal pellets were characterized previously ( haverty et al . , 2000 , 2005 ) .
the composition of the hydrocarbon mixture for this species and the hydrocarbons from their fecal pellets in the present study are displayed in fig .
1total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colontable 1relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbonspseudergates n = 3alates n = 3soldiers n = 1fecal pellets n = 122-mec220.06 ( 0.02)0.08 ( 0.02)0.090.11 ( 0.02)n - c231.30 ( 0.06)1.34 ( 0.05)1.481.35 ( 0.18)2-mec231.45 ( 0.40)1.72 ( 0.35)2.112.13 ( 0.28)3-mec231.31 ( 0.38)1.38 ( 0.30)1.712.06 ( 0.29)n - c240.46 ( 0.01)0.43 ( 0.02)0.480.32 ( 0.05)2-mec240.13 ( 0.03)0.19 ( 0.03)0.190.15 ( 0.02)3-mec240.08 ( 0.02)0.10 ( 0.02)0.120.11 ( 0.02)n - c2516.35 ( 0.16)11.75 ( 0.40)12.648.65 ( 1.05)2-mec250.18 ( 0.01)0.27 ( 0.01)0.240.14 ( 0.02)3-mec250.23 ( 0.04)0.36 ( 0.04)0.340.26 ( 0.03)n - c262.25 ( 0.15)1.17 ( 0.07)1.510.87 ( 0.12)2-mec260.14 ( 0.01)0.13 ( 0.01)0.140.07 ( 0.01)n - c2714.04 ( 0.55)6.81 ( 0.31)10.055.91 ( 0.65)2-mec270.13 ( 0.01)0.09 ( 0.01)0.130.06 ( 0.01)3-mec270.20 ( 0.01)0.19 ( 0.01)0.250.12 ( 0.01)n - c280.43 ( 0.05)0.17 ( 0.01)0.350.15 ( 0.03)n - c293.38 ( 0.33)1.27 ( 0.07)2.971.38 ( 0.18)11-mec290.06 ( 0.01)0.15 ( 0.01)0.100.09 ( 0.01)9,13-dimec290.70 ( 0.02)0.82 ( 0.01)0.910.63 ( 0.06)9,13,17-trimec290.11 ( 0.02)0.27 ( 0.00)0.160.22 ( 0.03)n - c300.04 ( 0.00)0.07 ( 0.00)0.100.0013-mec300.01 ( 0.02)0.08 ( 0.00)0.040.0010,14-dimec300.64 ( 0.06)1.45 ( 0.02)0.880.90 ( 0.03)10,14,18-trimec300.18 ( 0.02)0.43 ( 0.01)0.240.28 ( 0.02)n - c310.14 ( 0.01)0.10 ( 0.01)0.200.09 ( 0.01)15- ; 13- ; 11- ; 9-mec310.36 ( 0.03)0.82 ( 0.01)0.420.49 ( 0.02)13,17- ; 11,15-dimec317.45 ( 0.68)13.74 ( 0.52)8.5510.67 ( 1.80)9,13,17-trimec310.57 ( 0.05)1.19 ( 0.02)0.720.89 ( 0.06)7,11,15-trimec310.54 ( 0.04)0.99 ( 0.01)0.720.81 ( 0.09)14- ; 12-mec320.17 ( 0.01)0.33 ( 0.02)0.190.27 ( 0.03)12,16-dimec322.01 ( 0.14)3.57 ( 0.05)2.222.97 ( 0.28)10,14,18- ; 8,12,16-trimec320.46 ( 0.03)0.80 ( 0.02)0.610.82 ( 0.06)15- ; 13-mec330.93 ( 0.05)1.57 ( 0.03)0.921.30 ( 0.08)13,17- ; 11,15- ; 9,13-dimec334.63 ( 0.31)6.87 ( 0.11)5.117.41 ( 1.03)9,13,17-trimec331.30 ( 0.08)2.02 ( 0.02)1.863.47 ( 0.33)7,13,17-trimec330.21 ( 0.02)0.32 ( 0.01)0.320.47 ( 0.07)c35:31.01 ( 0.03)2.93 ( 0.17)1.641.76 ( 0.17)c35:22.31 ( 0.06)4.46 ( 0.14)2.713.55 ( 0.15)12,16-dimec34 ; c35:10.68 ( 0.02)1.16 ( 0.03)0.851.40 ( 0.10)10,14,18- ; 8,12,16-trimec340.28 ( 0.00)0.50 ( 0.04)0.450.89 ( 0.14)15- ; 13- ; 11-mec350.56 ( 0.02)0.85 ( 0.05)0.671.11 ( 0.09)13,17-dimec351.27 ( 0.09)1.96 ( 0.02)1.833.95 ( 0.46)9,13,17-trimec350.14 ( 0.01)0.28 ( 0.03)0.270.93 ( 0.14)c37:31.74 ( 0.06)3.33 ( 0.16)2.723.28 ( 0.34)c37:21.67 ( 0.06)2.55 ( 0.08)2.182.90 ( 0.10)12,16-dimec360.17 ( 0.01)0.34 ( 0.02)0.280.71 ( 0.08)c37:10.78 ( 0.02)0.76 ( 0.03)0.810.83 ( 0.06)10,14,18-trimec36 ; c37:10.44 ( 0.03)0.35 ( 0.05)0.430.58 ( 0.10)13 ; 11-mec370.71 ( 0.01)0.60 ( 0.04)0.770.89 ( 0.14)13,17- ; 11,15-dimec370.87 ( 0.05)1.27 ( 0.01)1.252.13 ( 0.22)9,13,17-trimec370.09 ( 0.01)0.13 ( 0.01)0.130.30 ( 0.06)c39:30.09 ( 0.02)0.18 ( 0.02)0.290.49 ( 0.16)c39:3 ; 12-mec380.49 ( 0.04)0.56 ( 0.03)0.761.03 ( 0.18)14,18 ; 12,16-dimec380.02 ( 0.03)0.14 ( 0.01)0.140.24 ( 0.04)c39:1 ; 10,14,18-trimec382.73 ( 0.19)1.73 ( 0.04)2.531.74 ( 0.23)13- ; 11-mec391.75 ( 0.09)1.03 ( 0.03)1.701.26 ( 0.20)13,17- ; 11,15-dimec390.90 ( 0.01)1.04 ( 0.14)1.361.44 ( 0.08)9,13,17-trimec390.41 ( 0.03)0.17 ( 0.09)0.110.20 ( 0.03)12- ; 10-mec400.38 ( 0.05)0.20 ( 0.01)0.350.26 ( 0.07)12,16-dimec400.27 ( 0.00)0.27 ( 0.02)0.340.32 ( 0.05)c41:13.31 ( 0.27)1.70 ( 0.04)2.791.58 ( 0.25)13- ; 11-mec412.78 ( 0.20)1.45 ( 0.06)2.591.74 ( 0.31)13,17- ; 11,15-dimec413.34 ( 0.13)2.90 ( 0.15)4.003.33 ( 0.19)9,13,17-trimec410.79 ( 0.02)0.30 ( 0.02)0.330.42 ( 0.19)12- ; 10-mec420.30 ( 0.03)0.15 ( 0.01)0.250.26 ( 0.27)12,16-dimec420.48 ( 0.04)0.37 ( 0.02)0.520.47 ( 0.12)c43:12.72 ( 0.28)1.25 ( 0.06)2.271.31 ( 0.26)15- ; 13-mec431.00 ( 0.14)0.46 ( 0.05)0.870.61 ( 0.16)13,17-dimec431.99 ( 0.16)1.18 ( 0.16)1.931.68 ( 0.28)14,18-dimec440.12 ( 0.02)0.07 ( 0.01)0.120.13 ( 0.06)c45:10.42 ( 0.09)0.16 ( 0.02)0.330.21 ( 0.07)13-mec450.000.000.000.04 ( 0.03)13,17-dimec450.37 ( 0.08)0.18 ( 0.02)0.340.43 ( 0.13)mean percent ( sd ) of total hydrocarbon composition
. compounds are listed in elution orderacronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y)these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .
( 2005)an isomeric mixture or two or more compounds co - eluted in this peak total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) mean percent ( sd ) of total hydrocarbon composition .
compounds are listed in elution order acronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .
( 2005 ) an isomeric mixture or two or more compounds co - eluted in this peak hydrocarbons from termites
n - alkanes and dimethylalkanes were the predominant classes of hydrocarbons in all castes and in fecal pellets ( table 2 ) , as previously reported by haverty et al .
n - alkanes comprised from 23.12% to 38.39% , unsaturated components from 15.21% to 19.03% , terminally branched monomethylalkanes from 3.90% to 5.32% , internally branched monomethylalkanes from 7.70% to 9.01% , and dimethylalkanes from 25.25% to 36.18% , of the total hydrocarbon content .
there was also a homologous series of trimethylalkanes , representing 8.26% to 9.48% of the total hydrocarbon content .
table 2relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbon classpseudergatesalatessoldiersfecal pelletsnormal alkanes38.3923.1229.7718.71olefins18.3821.1120.3120.66terminally branched methylalkanes3.904.495.325.19internally branched methylalkanes9.017.708.888.33dimethylalkanes25.2436.1829.7937.40trimethylalkanes5.097.405.929.71percent of total hydrocarbon compositionall peaks with co - eluting olefins and saturated compounds are summarized as olefinsthe hydrocarbons of i. minor characterized in this study included all of the compounds reported by haverty et al .
( 2005 ) , as well as 18 additional hydrocarbons ( table 1 ) .
these additional hydrocarbons were not abundant , never representing more than 0.85% of the total hydrocarbon ( as for the mixture of 12,16-dimec34 and c35:1 in soldiers ) content , and usually much less .
the detection of these additional hydrocarbons in this study are likely due to the greater concentration of the extracted hydrocarbons used or , possibly , colony to colony variation .
relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) percent of total hydrocarbon composition all peaks with co - eluting olefins and saturated compounds are summarized as olefins hydrocarbons from termite fecal pellets in general , the hydrocarbons from whole - body extracts of i. minor were represented in extracts of fecal pellets . as with whole - body extracts of i. minor , dimethylalkanes were the predominant class of hydrocarbon , followed by olefins and normal alkanes ( table 2 ) .
1 ) were found in termites and not in fecal pellets , but these were present in small quantities , less than 0.1% of the total hydrocarbon content .
the lack of these two hydrocarbons in fecal pellets may be a function simply of the concentration of the extracts .
changes in hydrocarbons of fecal pellets over time we identified 73 hydrocarbon peaks in fecal pellets of i. minor ( table 1 ) . of these peaks
( data from non - significant slopes not reported ) , 19 of 73 ( 26% ) had a significant change , either positive ( five ) or negative ( 14 ) , over time ( fig . 2 ) .
this is a much higher number of statistically significant slopes ( different from 0 ) , than would be expected by chance alone ( 5% of 73 peaks is < 4 peaks ) .
2changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant ( = 0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colonthe index , iage , for each of the three replications ( a , b , and c at 0 , 30 , 90 , and 365 day ) was plotted against the associated number of days ( fig .
the fitted line , with a 95% confidence interval for the means to show the variability of the replicates , had a high adjusted r (= 0.888 ) value , suggesting that it might be possible to predict fecal age by the index .
3fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .
values for 30 day are jittered a small amount so that all three data points can be seen changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant ( = 0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .
values for 30 day are jittered a small amount so that all three data points can be seen we do not know the basis by which peaks increase or decrease over the 1-year period .
in general , those that increased in relative abundance tended to have higher initial relative abundances ; i.e. , mono- , di- , or tri - methylalkanes with carbon chains of 31 , 32 , or 33 .
those that decreased were represented in five of the six classes of hydrocarbons , ranging from c26 to c45 .
the composition of the hydrocarbon mixture of insects is genetically controlled ( toolson and kuper - simbrn , 1989 ; kaib et al . , 1991 ; page et al . , 1991 ; coyne et al . , 1994 ) .
this composition can be slightly affected by diet and environmental conditions ( hadley , 1977 ; espelie et al . , 1994 ; chapman et al . , 1995
( 1996 ) demonstrated significant seasonal variation in quantities of some hydrocarbons of coptotermes formosanus shiraki from hawaii ; these differences were small and associated with the production of alates .
however , the qualitative mix of hydrocarbons has been shown to be stable over decades among species of cone beetles in the genus conophthorus ( page et al . , 1990 ) .
the motivation for our study was to devise a method for the evaluation of the success or failure of drywood termite treatments , including fumigation and local application methods .
with the recent classification of sulfuryl fluoride ( the only fumigant active ingredient registered in california ) as a greenhouse gas ( mhle et al . , 2009 ) and
the anticipated increase in local treatments , we felt that there would be considerable interest by the industry , regulatory agencies , and consumers for a simple and accurate means to determine whether or not a targeted colony / infestation is still in a structure .
our method , based on changes in hydrocarbon chemistry over time , shows promise to this end .
future research includes : ( 1 ) validating these findings for additional species of drywood termites and geographic populations of the same species , ( 2 ) validating these findings at different times of the year , and ( 3 ) exploring seasonality in pellet production and hydrocarbon quality in pellets . | hydrocarbon mixtures extracted from fecal pellets of drywood termites are species - specific and can be characterized to identify the termites responsible for damage , even when termites are no longer present or are unable to be recovered easily . in structures infested by drywood termites , it is common to find fecal pellets , but difficult to sample termites from the wood . when fecal pellets appear after remedial treatment of a structure , it is difficult to determine whether this indicates that termites in the structure are still alive and active or not .
we examined the hydrocarbon composition of workers , alates , and soldiers of incisitermes minor ( hagen ) ( family kalotermitidae ) and of fecal pellets of workers .
hydrocarbons were qualitatively similar among castes and pellets .
fecal pellets that were aged for periods of 0 , 30 , 90 , and 365 days after collection were qualitatively similar across all time periods , however , the relative quantities of certain individual hydrocarbons changed over time , with 19 of the 73 hydrocarbon peaks relatively increasing or decreasing .
when the sums of the positive and negative slopes of these 19 hydrocarbons were indexed , they produced a highly significant linear correlation ( r2 = 0.89 ) .
consequently , the quantitative differences of these hydrocarbons peaks can be used to determine the age of worker fecal pellets , and thus help determine whether the colony that produced them is alive or dead . |
there
is a need for computer methods that can calculate the aqueous solvation
free energies of solute molecules accurately and efficiently .
explicit - solvent models provide a physically
accurate and atomistically detailed model of solvent , but they can
be computationally expensive .
boltzmann
( pb ) , generalized born ( gb ) , and
weighted surface area ( wsa ) approaches , are computationally efficient because they treat
water as a continuum .
however , they are sometimes inaccurate , smearing
out the particulate nature of water molecules , and they may
have limited transferability to situations for which they have not
been optimized .
solvation modeling has
often been improved by combining the advantages of implicit- and explicit - solvent
models .
one such approach is the semi - explicit assembly ( sea ) water model . in the sea approach ,
water s solvation behavior is precomputed
in explicit - solvent simulations of water around model spheres that
are then combined together as building blocks to represent any given
solute structure at run time .
the solvation free energy gsolv for a target solute molecule is calculated
from a sum over solvating waters .
sea has been shown to predict efficiently
the air - to - water transfer free energies ( ghyd ) of small - molecule solutes in both prospective and
retrospective studies with reasonable accuracy .
even
so , the sea model does not give accurate solvation free energies of
ions or solutes having high charge density .
here , we describe
field - sea , a considerable improvement over sea , which gives accurate
free energies of transfer of ions and charged solutes , at no additional
computational cost and with no degradation of the predictions for
nonpolar and uncharged polar solutes . in overview , we studied full
md simulations of ionic and neutral solutes in tip3p water , described
below , and found that the results could be captured by using a solvation
free - energy field surface ( in field - sea ) , instead of using precomputed
waters ( as in sea ) .
moreover ,
we found that a weakly charged solute atom that is adjacent to a strongly
charged solute atom retains some of the restrictions of its solvating
water molecules that its neighboring charge has ; see figure 1 .
field - sea captures this effect using an adaptive
boundary method . a solvent water molecule around a solute molecule .
on the left , each solute atom ( weakly charged ) has a predefined radius ,
irrespective of its neighboring solute atoms , leading to the locus
of water centers shown by the black dashed curve . on the right ,
one
solute atom is strongly charged , leading to two consequences : its
own solvating water molecule is pulled in tightly , and neighboring
solute atoms have tighter water interactions too .
such effects may not be captured in simplified solvation
models that treat atoms as having fixed radii , independent of neighboring
atoms .
below , we describe the field - sea approach to computing solvation
free energies . the original sea model is described elsewhere , and our related explicit - solvent and linearized poisson
boltzmann
equation modeling ( lpbe ) are described
in the supporting information ( si ) . in both the original sea and the new
field - sea described here ,
first , in a precomputation step , various
model spheres are solvated in an explicit - solvent model , such as tip3p .
this provides a database of component free energies that are used
in the second step . in the second step , at the run time ,
any given
solute molecule of interest is assembled from an appropriate concatenation
of these spheres .
the solute s solvation free energy is computed
by summing the component sphere free energies . on the one hand , this
approach provides the speed advantages of simple additivity - based
models as we only need to perform the first step once for a given
solvent model . on the other hand , this procedure is more accurate
than additivity - based approaches because ( 1 ) sea sums are regional ,
not local , and ( 2 ) the database encodes the microscopic response observed
in explicit - solvent simulations .
the new field - sea instead captures
the solvation free energy using a continuous solvation field . to do this ,
the charging free energies for a series of lennard - jones
( lj ) spheres solvated in tip3p water were calculated with thermodynamics
integration ( ti ) in the set of precomputations ( see explicit solvent
free energy calculations in the si ) .
we
construct a free - energy contour ( see si figure s1 ) as a combination of the electric field at the first solvation
shell boundary ( e = q / rw , where e is the signed
magnitude of the electric field , q is the sphere s
charge , and rw is the distance from the
sphere center to the first peak of the water oxygen s radial
distribution function , rdf , around the given sphere ) , the curvature c = 1/rw at this boundary , and
the charging free energy of the spheres . within each charge step ( q ) , all of the data of free energy , electric field , and
curvature were fitted to a formula , which can be taken as an expansion
of the born model1 equation 1 will
be used to calculate the free energy associated with any surface spot
on an arbitrary solute solvent boundary ( esub ) .
this free energy could also be calculated from interpolation
between data points on the free - energy contour .
in addition
to a charging free - energy contour , we also need a contour for estimating
the explicit - solvent - accessible surface of a given solute molecule .
to generate this
, we calculated the rdf of water oxygen around each
charged sphere and picked the first peak in the rdf , denoted as the
boundary - sphere distance rw .
all of the rw values from these spheres and their charge
and lj parameters ( lj and lj )
were used to build an rw contour . to generate a solvent - accessible
surface for a given solute
, an rw for
each atom is first calculated from interpolation with its partial
charge , lj , and lj on the rw contour .
a lee richards surface is then constructed from the nonoverlapping
sections of these rw spheres centered
on their associated atom sites .
the fixed rw boundary is dependent upon only the individual parameters
of the surface atoms .
for solute molecules with multiple partial charge
sites ( especially molecular ions , where strong electric fields are
involved ) , a more physical representation of the explicit - solvent - accessible
surface would be one that responds to the whole solute s electric
field .
in other words , the surface atom distance , rw , is determined not only by the surface atom s
partial charge , but also by neighboring atoms charges .
taking
into account such collective electrostatic effects , we adjust the
fixed rw boundary in an adaptive manner ,
described by the following three - step procedure : ( 1)we cull
all surface segments within 1.4 of any solute atom . as we are
starting with a lee richards solvent - accessible surface , the
minimum rw possible in the surface construction
is half of a water molecule diameter ( corresponding to a solute atom
with a 0 radius ) .
this partial - culling process simply removes
potential numerical instabilities from surface sites overlapping solute
atom centers while providing some adjustable starting surface sites
that penetrate within the fixed rw boundary .
we calculate
the electric field at a given dot a about atom a2where n is the number
of solute atoms , ria represents a vector from atom i to surface
dot a , and ria is its length ; signa = 1 when raai=1n ( qi / ria3)ria 0 , and signa = 1 when raai=1n ( qi / ria3)ria < 0 , and raa is the vector from atom a to dot a. from the electric field , we
calculate the corresponding charge by3where raa is the length of vector raa . we interpolate with this new charge and
atom a s lj parameters to get a new rw , rw , new .
in addition ,
we assume that rw can not expand ( if rw , new > rw , original , we let rw , new = rw , original ) . this nonexpansion assumption is supported by solute
solvent
boundary plots of model diatomic solutes ( see si figure s2 ) . finally , the rw , new s of all atom s potential surface dots are averaged
to yield an rw for this atom , used in
the later culling process . ( 3 ) we cull the buried dots adaptively .
because each dot atom distance ( e.g. , daa ) is adjusted independently in the above
procedure , the shell of potential surface dots about an atom will
no longer be spherical .
thus , culling buried dots is no longer a simple
process of eliminating dots within a neighboring atom s rw , and an adaptive culling process is needed .
to adaptively cull
buried surface dots , when we check
whether a given dot a is buried by a neighboring
atom b , we first determine a corresponding charge
at dot a from the electric field at this dot .
this
is used along with atom b s lj parameters
to get a new rw , rw , badp , following the above rw adjustment procedure .
atom
distance , dab , with rw , badp , to determine if the
dot is buried in atom b. if dab is less than rw , badp , it is removed from the set of potential surface
dots .
we call the new dot surface resulting from the above culling
procedure the adaptive boundary .
our term field - sea refers both to
the field and to the adaptive boundary .
the adaptive nature of the
boundary only pertains to multiatom solutes ; the adaptive and fixed rw boundaries are identical for single - atom solutes ,
like monatomic ions . while our adaptation procedure could , in principle ,
be applied iteratively , we found no further improvement after a single
calculation .
the charging
free energy can be calculated for any given field - sea surface via4where ndot is the total number of surface - exposed dots , mi is the total number of dots
( exposed + occluded ) for corresponding atom i ( each
surface dot , j , of atom i , only
corresponds to 1/mi of
this atom sphere s total surface area or total solvation free
energy ) , and esub , j is
the subenergy associated with exposed surface dot j , calculated from the free - energy contour . here ,
esub , j is calculated from eq 1 using the signed magnitude of the electric field , ej , and the curvature at dot j , 1/rij .
ej is defined as negative when ejrij < 0 , where ej is the electric
field ( vector ) at dot j , rij is the vector from atom i to its surface dot j , and rij is this vector s length .
now ,
we take the total solvation free energy to be the sum of polar and
nonpolar components5 assuming the polar component of the free energy of transfer
( gpol ) can be uniquely described
by both the surface electric field and geometry of the solute , this
molecular gpol can be accurately
calculated from the simple summation of the surface dots energies
process described above .
we consider the gsolv of monatomic ions as an initial test . for molecular
solutes ,
to account for the role of solute conformation in the solvation
free energy , we average gsolv results
from calculations on 50 conformations ( though 10 conformations are
usually enough ; see the si ) . these solute
conformations were generated from 5 ns tip3p water md simulations
with a 100 ps snapshot interval .
we developed field - sea because of the errors that we
observed in simpler methods in solvating ions ; see figure 2 .
the explicit - solvent curve shows the hydration
asymmetry discussed by others , where positively charged solutes are less favored than negatively
charged solutes of similar size .
gsolv as the function of a model lj sphere (
= 0.22 nm , = 0.06538 kj / mol ) charge for tip3p , lpbe , and field - sea .
for comparison at infinite - dilution conditions ,
an ewald correction
is applied to the tip3p and field - sea results . here , we tested field - sea on the solvation free energies
of monatomic ions using ion parameters developed by aqvist or joung and cheatham ( table s2 , si ) .
these ions have different
sizes and charges ; therefore , they have different charge densities .
table 1 and figure s3 ( si ) show the results of field - sea calculations , compared with
lpbe and tip3p .
lpbe results using the lj surface do not capture quantitatively
the gsolv of ions with high charge
densities .
in contrast , field - sea reproduces the tip3p gsolv values regardless of the specific lj parameters
and charges .
these results indicate that field - sea is accurately reproducing
explicit - solvent free energies of solvation of monatomic ions , provided
that their charge and lj parameters are near or encompassed within
the range of the rw and free - energy contours .
a common approximation in simplified
solvation models is to suppose that atoms have fixed
atom
types , where any particular solute atom has a given value
of charge and radius , independent of its atomic neighbors . however ,
our tip3p explicit - solvent simulations described below show that this
approximation is a source of error . how the solvation surface is constructed
our explicit - solvent simulations show that the solvation shell is
not just a simple union of the spherical surfaces of all of the atoms
making up the solute molecule .
imagine a diatomic solute with partially
charged atom a covalently connected to partially
charged atom b. our tip3p simulations show that when
atom a attracts water molecules , it also shrinks
the solvation shell of waters around atom b. in this
way , the solvation surface around the diatomic molecule a b can be more complex than the simple union
of independent spherical solvation shells around atoms a and b. to address such situations , we developed
an adaptive method that gives a more explicit - like solvation boundary . to test our adaptive boundary method , we made up 22 fictitious
diatomic solutes ( see si table s4 ) , computed
their solvation free energies , and compared to their solvation in
tip3p .
we constructed these solutes by taking pairs of ordinary simulation
atom types , placing them at fixed covalent bond distance apart , and
giving each atom a charge and radius that we could vary systematically
over the series . because of the fictitious charges that we give them ,
these are not molecules observed in nature
however , they are physically
plausible molecules that provide us with a systematic series for learning
about how explicit - solvent models handle solvated charges .
figure 3 shows the computed free energies of these diatomic
solutes from field - sea when using the fixed rw and adaptive boundaries in comparison to explicit - solvent
tip3p simulations .
lpbe and original sea results are shown in the si instead of here for the sake of simplicity .
in summary
, we find that when the tip3p gsolv is weak , for example , when the charge density of solute
atoms is low , the fixed rw boundary works
fairly well .
when the gsolv grows
to 100 kcal / mol and larger ( more negative ) , it becomes increasingly
important to use an adaptive boundary .
these strongly solvated model
molecules often have large , unbalanced partial charges on the solute
atoms , and these situations lead to exaggerated distortions of the
explicit solvation shell .
field - sea gsolv for model diatomic solutes ( triangles : fixed rw ; circles : adaptive boundary ) compared to tip3p simulations .
figure 4 shows how a neighboring
solute atom can affect the solvation shell about another atom .
the
fixed rw and adaptive boundaries are identical
in figure 3a as both carbon atoms are weakly
charged and there is only minor collective electrostatic perturbation
on the boundary . however ,
when the solutes are more highly charged
and large charging energies are involved , as in figure 3b , the weakly charged carbon atom s solvation shell
will be distorted by its neighboring highly charged oxygen atom . in
this case ,
water molecules penetrate more deeply into the carbon s
solvent - accessible surface to better solvate the oxygen charge ( the
right part of the white curve in the dark blue region ) .
also , water
molecules pack more tightly around the carbon atom and reduce its
apparent rw ( the left part of the white
curve in the blue and light blue regions ) .
both of these effects are
captured well by adaptive field - sea boundaries , leading to field - sea
charging energies that are in excellent agreement with explicit simulation
results .
oxygen density around ( a ) weakly charged
and ( b ) strongly charged diatomic solutes .
the blue contours indicate
water density greater than the bulk value , with the darker blue regions
indicating the enhanced water probability density .
the black line
shows the nonadaptive fixed rw boundary .
for the weakly charged diatomic , the adaptive and nonadaptive boundaries
coincide .
the adaptive and nonadaptive boundaries differ . in order to establish that the accuracy of field - sea is not degraded
relative to sea on nonionic solutes , we applied field - sea with both
fixed rw and adaptive boundaries to a
standard set of 504 neutral small molecules .
this set contains an alchemically diverse
range of functional groups , for which solvation free energies are
available from experiments and tip3p simulations .
figure 5 shows 504 neutral solutes solvation free
energy from tip3p simulation , from the lpbe ( white diamonds ) and field - sea
( white circles ) ( see si figure s4 for nonadaptive
fixed rw field - sea results ) .
field - sea
shows a rmse ( root - mean - square error ) of 1kt with a negligible mse ( mean - signed error ) , comparable to the original
sea and considerably better than the
lpbe , which bears a systematically negative error .
while the fixed rw boundary field - sea results are comparable
to the lpbe ( table 1 ) , it overestimates the
free energy when weakly charged c atoms are neighbored by highly charged
o or n atoms , as in the cases of alcohols , amines , ethers , and esters
( si table s5 ) .
these errors arise because
the fixed rw boundary can not capture water
molecule penetration into the c atom s solvent - accessible surface
( see figure 4b ) , situations the adaptive boundary
handles directly . therefore , while these are simply neutral solutes ,
collective solute interactions still play a clear role in their overall
hydration .
figure s5 ( si ) compares
the total solvation free energy from field - sea with experimental results .
the mse / rmse of field - sea to experimental solvation free energy ( 0.67/1.45
kcal / mol , table s6 ( si ) ) is comparable
to that of the much more computationally expensive tip3p water model
( 0.66/1.22 kcal / mol ) .
these results indicate
that field - sea can accurately compute solvation free energies over
the full range from charge - dense ions to neutral polar and nonpolar
molecular solutes . here , we test field - sea on an expanded set of molecular
ions and biomolecules ( e.g. , acetate , butylammonium , etc . ; see si table s7 for the complete list and results ) .
these are ions that are larger and more complex than the simple
atomic and diatomic ions described above and should better test the
need for adaptive boundary considerations .
figure 5 compares solvation energies from lpbe ( orange diamond ) and
field - sea ( orange circle ; see si figure
s6 for field - sea results with a fixed rw boundary ) with tip3p results for 35 molecular ions .
the errors for
both lpbe and field - sea with a fixed rw boundary are around 10 kcal / mol ( table 1 ) .
while this might be regarded as acceptable in light of the nearly
100 kcal / mol span of free energies covered in the tip3p simulations ,
using an adaptive boundary with field - sea has half of this rmse .
field - sea
also performs well in multivalent molecular ion solvation ( si figure s7 ) and is as accurate as explicit - solvent
calculations compared to experimental values for ionic solute solvation
( table s6 and figure s8 , si ) .
md simulations
of gsolv for 504 neutral solutes
( white ) , 35 molecular ions ( orange ) , and 22 capped amino acid dipeptides
( cyan ) in tip3p water , compared to ( a ) lpbe and ( b ) field - sea .
we also tested our methods on 22 capped amino
acids ( n - acetyl - x - methylamide , x = glu , arg , leu ,
etc . ;
si table s8 ) , which are widely used
biological models in both theoretical studies and experiments .
these are useful precursor
structures for the foundation of hydrophobicity scales , used in estimating
the solvation of larger biomolecular structures . here
, we investigate the solvation
free energies of a full series of capped amino acids with field - sea .
as the size of solute increases , computing the total solvation
show , lpbe
( cyan diamond ) and field - sea ( cyan circle ) both yield solvation free
energies that agree well with tip3p calculations .
again , the adaptive
boundary helps field - sea considerably , cutting the rmse to half of
that seen from the lpbe or fixed rw cases .
these results indicate that the accuracy of field - sea does not degrade
as the solute size further increases .
the solvation boundaries
in field - sea are made from a set of surface dots .
above , we used 80 dots / atom ,
the same as was used in previous sea studies .
what is the minimum number of grid dots that we need to properly
represent the first - shell boundary ? to investigate this , we performed
an analysis of accuracy versus relative computational time as a function
of the granularity of the boundary .
this analysis indicates that the
rmse for field - sea results on the 504 neutral molecule set is essentially
uncompromised even down to a granularity of 5 dots / atom , without increasing
the rmse above 1 kcal / mol ( figure s9 and s10 ( si ) , the granularity does not affect the accuracy for charged
solutes either ) . at 5 dots / atom ,
field - sea is roughly 5-fold faster
than dipolar sea at 80 dots / atom , the minimum surface granularity
recommended for this method .
these results
indicate that while field - sea is sensitive to the physicality of the
solvation boundary , it is less sensitive to the granularity of its
depiction .
we have
described field - sea , a method for computing solvation free energies
of solutes in water .
it improves upon an earlier method called sea
( semi - explicit assembly ) .
sea captured the physics of solvation by
presimulating toy spheres in explicit water , collecting a database
of structural and energetic properties of those waters and then assembling
at run time the solvation physics as sums over appropriate toy spheres
to properly represent a given solute . in field - sea , this procedure
differs in using a solvation free - energy field , rather than explicit
waters .
furthermore , field - sea uses an adaptive boundary , allowing
solvating waters to approach a solute atom to different degrees depending
on neighboring atoms .
relative to sea , field - sea captures the solvation
free energies of ions and charged solutes accurately , is faster to
compute , and has no degradation of performance on nonpolar and polar
solutes .
both sea and field - sea offer advantages over implicit - solvent
modeling in that they entail no adjustable solute atom radii parameters .
sea and field - sea are built upon a corresponding force field and explicit - solvation
model . here
one of the
key observations that arises from our md simulations of charged solutes
in tip3p water , which is captured by field - sea , is that atoms that
are adjacent to charged atoms in solutes acquire partial characteristics
of those charged atoms .
for example , a weakly charged atom s
solvation shell is shrunk by its neighboring big charges .
an implication
of this for implicit - solvent modeling is that atomic radii should
not be treated as fixed for solvation in water ; an atom s radius
in implicit - solvent modeling can depend on the nature of its neighboring
atom . | previous
work describes a computational solvation model called semi - explicit
assembly ( sea ) .
the sea water model computes the free energies of
solvation of nonpolar and polar solutes in water with good efficiency
and accuracy .
however , sea gives systematic errors in the solvation
free energies of ions and charged solutes .
here , we describe field - sea ,
an improved treatment that gives accurate solvation free energies
of charged solutes , including monatomic and polyatomic ions and model
dipeptides , as well as nonpolar and polar molecules . field - sea is
computationally inexpensive for a given solute because explicit - solvent
model simulations are relegated to a precomputation step and because
it represents solvating waters in terms of a solute s free - energy
field .
in essence , field - sea approximates the physics of explicit - model
simulations within a computationally efficient framework .
a key finding
is that an atom s solvation shell inherits characteristics
of a neighboring atom , especially strongly charged neighbors .
field - sea
may be useful where there is a need for solvation free - energy computations
that are faster than explicit - solvent simulations and more accurate
than traditional implicit - solvent simulations for a wide range of
solutes . |
hydatidosisis term used to refer to infection with the parasite larva in humans and echinococcosis restricted to infection with the adult stage in carnivorous animals ( 1 ) .
recently , the genetics , structure and function of the human cytosolic gst enzyme with emphasis on their roles in the cellular metabolism has been defined ( 2 ) .
gst activity was detected in most mammalian tissues , especially in the liver which plays a key role in detoxification .
there are different classes of gst isozymes that diverge in their specificity to xenobiotic or endogenous substrates ( 3 ) .
gst enzymes involved in the cellular detoxification of a broad range of chemical substrates ( 4 ) . apart from reaction from their endogenous metabolism , gsts of helminth parasite may protect against exogenous xenobiotics as a result of immune effectors mechanisms from the host ( 5 ) .
significantly higher activity has been found in intestinal cestodes and digeneas , compared with parasitic nematodes ( 6 ) .
gsts activity assay has been demonstrated in the cytosol of protoscolices from sheep hydatid cysts ( 7 ) .
diagnosis of hydatid disease is done by a combination of clinical signs , imaging techniques , cyst fluid examination , serological tests and molecular techniques ( 9 ) .
the prevalence of liver hydatid cyst was reported 4.7% in people of peru country using recombinant antigen , epc1 glutathione s - transferase [ repc1-gst ] , in western blot technique ( 10 ) .
however , the hydatid cyst diagnosis technique is under developing due to specificity and sensitivity problems . in the present study , gst enzyme activity of hydatid cyst protoscolices ( parasite ) ,
healthy and cystic liver tissues were compared and mentioned gst enzyme importance for diagnostic biomarker in hydatid cyst disease .
ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .
parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) .
sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 .
then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) . the protein concentration in the extract solutions of protoscolices and sheep liver tissues
were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) .
gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .
protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .
cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette .
to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) .
sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .
molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands
was calculated , standard curve was prepared in excel software and finally proteins mw were determined .
independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues .
ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .
parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) .
sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 . then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) .
the protein concentration in the extract solutions of protoscolices and sheep liver tissues were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) .
gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .
protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .
cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette . to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) .
sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .
molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands
was calculated , standard curve was prepared in excel software and finally proteins mw were determined .
independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues .
the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .
protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .
significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .
statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) .
extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .
1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .
similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .
the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .
lanes 1 - 3 are healthy liver samples . lanes 4 - 6 are cystic liver samples .
lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .
64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )
the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .
protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .
significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .
statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) .
extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .
1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .
similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .
the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .
lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .
64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )
function of hepatocytesmay be disturbed in the presence of infections ( 15 ) . in our study
the reduction of protein synthesis as a result of hydatid cyst causes to decrease protein concentration .
gsts can make up to 10% of cytosolic protein in some mammalian organs ( 16 ) .
hepatic cells contain high levels of gst enzyme which has been found to be an indicator of hepatocyte injury in transplantation , toxicity and infections ( 17 ) .
the hydatid cyst infection stimulates oxidative stress and toxin production in hepatic cells . from the point of biochemical defense view gst enzyme
be able to neutralization of these toxins , therefore we expect to increase activity level of this enzyme .
increase of liver protein and gst in infected mice indicates the occurrence of oxidative stress in hepatocytes due to infection ( 18 ) .
serum gsta is a more sensitive marker than transaminases ( alanine aminotransferase , asparatate aminotransferase and alkaline transferase ) for monitoring and as an early analyst of hepatic damage ( 19 ) .
therefore gst activity difference between healthy and infected host liver tissue could be concerned for hydatid cyst diagnosis . however , other infections cause to hepatocyte damage , thus gst elevation must be evaluated with other clinical and paraclinical parameters of hydatid cyst disease .
mammalian cytosolic gsts are dimeric , with both subunits being from the same class of gsts , although not necessarily identical .
gst enzyme molecular weight of parasite is reported 24 - 27 kda ( 13 ) . in this
research protein bands with 24kda and 25kda were found in parasite and liver tissues respectively .
gst activity in cystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other parameters of hydatidosis . | abstractbackgroundthe aim of this study was to detect the glutathione s - transferase(gst ) enzyme activity of healthy / cystic liver as a diagnostic biomarker for hydatidosis . in order to compare with liver tissue ,
the level of the gsts enzyme activity of parasite was also determined.methodsparasites were collected from sheep liver tissue with hydatid cysts at a local abattoir and washed with pbs buffer .
collected parasites and liver tissues were sonicated or homogenized respectively .
extract solution samples were centrifuged and stored at - 20c .
gst enzyme activities were measured in the extract of parasite and liver tissue samples ( healthy and infected livers ) .
protein amounts and protein bands were detected using bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) methods respectively . to determine significant difference between two groups , two - sample t - test was performed.resultsgst specific activities of healthy / infected livers and parasites were estimated 304 , 1297 and 146 u / ml / mg respectively .
significant higher gst specific activities in cystic liver than healthy liver was observed ( p < 0.05 ) .
t - test analysis showed gst activity of parasite was lower than healthy liver tissue .
sds - page showed gst protein bands with 24 kda in parasite samples and25 kda in liver tissues.conclusiongst activity incystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other hydatid disease parameters . |
the risk of stroke and myocardial infarction is considerably increased in subjects with diabetes . already at the time of diagnosis of type 2 diabetes , many patients have manifest cardiovascular disease ( cvd ) .
this could be due to a long presymtomatic period with increased glucose level as evident by the increased cvd risk already present with impaired glucose tolerance ( igt ) .
thus , interest has focused on factors , not only glucose levels , in the prediabetic state that would increase the atherothrombotic process . a common , possibly genetic , antecedent of both type 2 diabetes and cvd has been proposed as the " common soil " hypotheses .
recently , markers of inflammation , such as highly sensitive c - reactive protein , have been found to predict type 2 diabetes in long - time follow up of population samples , although adjusting for measurements of obesity attenuates the relationship [ 8 - 12 ] .
such markers are weakly related to variables reflecting endothelial function such as adhesion molecules and von willebrant factor . in a large population - based study , also increased plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) were strongly related to the development of diabetes independent from insulin resistance and obesity .
the possibility of elevated pai-1 being a very early risk marker of the insulin resistance syndrome and diabetes was raised . furthermore , haemostatic variables related to endothelial function , such as von willebrant factor and factor viii , also predicted diabetes , especially in women . data are lacking regarding other aspects of the fibrinolytic system and the development of diabetes , such as the activities of pai-1 and tissue plasminogen activator ( tpa ) or the mass concentration of the endothelial - derived tpa ( " tpa antigen " ) , factors related to cvd [ 15 - 19 ] thus , there are two questions related to fibrinolytic variables and the development of diabetes .
firstly , do subjects with normal glucose tolerance , who later convert to diabetes , have disturbances in the fibrinolytic system and endothelial function that could explain their increased risk of cvd even before diabetes ensues ?
if so , do these changes predict future diabetes , independent of the influence of the insulin resistance syndrome , thereby pointing to other pathways to the diabetic state such as endothelial dysfunction ?
we studied fibrinolytic activity , as measured by tpa and pai-1 activity , and a marker of endothelial dysfunction , as measured by tpa antigen , in a population sample with normal glucose tolerance at baseline and analysed data according to conversion to diabetes or not over nine years of follow - up .
this study was performed within the framework of the northern sweden monica study . in 1990
a total of 2,000 randomly selected subjects aged 25 to 64 years were invited . in all , 1,583 persons participated ( 79.2 % ) .
a 75-gram oral glucose tolerance test ( ogtt ) was performed after an overnight fast in a randomly selected subset of subjects without known diabetes .
venous plasma glucose samples were analysed by the hexokinase method ( boehringer mannheim automated analysis for bm / hitachi system 717 ) .
glucose tolerance was classified according to who criteria from 1999 : normal glucose tolerance ( ngt ) fasting glucose < 7 mmol / l and post load glucose < 7.8 mmol / l , impaired glucose tolerance ( igt ) fasting glucose < 7 mmol / l and post load glucose 7.811.0 mmol / l , and diabetes fasting glucose 7.0
weight was measured with a balance scale to the nearest 0.2 kg , and height was measured to the nearest centimetre .
bmi was calculated as total body weight ( kg ) / height ( m ) .
waist circumference was measured midway between the lower rib margin and the iliac crest , which in most occasions was identical with the level of the umbilicus .
all measurements were done in a standing position while breathing normally , and participants were asked to wear light underwear and remove shoes . for the measurement of waist circumference , the recording was done after a gentle breath - out .
tpa and pai-1 activities were determined by chromogenic assays , spectrolyse / fibrin and spectrolyse / pl kit ( biopool ab , ume , sweden ) , respectively .
tpa antigen was measured by an enzyme - linked immunosorbent assay method ( tintelize tpa , biopool ab ) .
serum insulin was determined by radio immunoassay with a double - antibody solid - phase technique ( phadaseph insulin ria , pharmacia diagnostics ab , uppsala , sweden ) in 1999 , all participants , alive and still living in the area , were recalled for a follow - up examination . the same questionnaire and anthropometrical measurements as in the baseline survey were used .
altogether 1,148 ( 72.5 % ) subjects turned up for re - examination . subjects who previously had performed an ogtt with normal or impaired glucose tolerance , and answered no to the question "
participants , who denied having diabetes in the baseline survey and answered affirmative to the question " do you have diabetes mellitus ? " at follow up , were defined as incident cases of known diabetes mellitus according tho the who criteria from 1999 . in november 2002 , a questionnaire exploring details related to the diagnosis of diabetes , and to the type and duration of current hypoglycaemic treatment was sent to them .
case records were scrutinised to confirm diabetes diagnosis and time for start of insulin therapy .
subjects that started their insulin treatment within two years of diagnosis were classified as type 1 .
however , for subjects fulfilling these criteria but with a age of 40 or above at onset , current bmi had to be < 27 to be classified as type 1 .
the research ethics committee of ume university and the national computer data inspection board have approved the northern sweden monica study .
means or medians are given for those who remained non - diabetic and those who converted to diabetes . because the distributions for serum triglycerides , insulin and the fibrinolytic variables are highly skewed , we used logarithmically transformed values ( i.e. geometric means ) .
the risk of incident diabetes was compared across quartiles of the fibrinolytic variables , calculated from the total sample .
logistic regression was used for calculation of odds ratios and 95% confidence intervals ( ci ) for the development of diabetes comparing the quartile with highest risk with the other three pooled quartiles .
stepwise adjustment for age and sex , waist , serum insulin , triglycerides and diastolic blood pressure was used .
five hundred and fifty - one subjects who initially had normal glucose tolerance returned for re - examination . a new ogtt was done in 477 of those with normal glucose tolerance and without clinically diagnosed diabetes .
thus , there was no repeated ogtt in 13 % , mostly due to logistic reasons such as not being able to turn up in the morning after an over night fast . among persons with initially normal glucose tolerance , in all 15 subjects ( 2.7 % ) had type 2 diabetes diagnosed , either clinically ( n = 4 ) or by ogtt ( n = 11 ) after a 9-year follow - up or 4,959 person years .
fifty - one subjects ( 10,7 % ) worsened from normal glucose tolerance to impaired glucose tolerance .
converters from ngt to diabetes were 7 years older and had higher bmi and waist circumference .
diastolic blood pressure did not differ but both fasting triglycerides , insulin , fasting and post load plasma glucose were higher in subjects with subsequent diabetes .
plasma levels of tpa antigen and pai-1 activity were higher and tpa activity was lower .
baseline characteristics of subjects with normal glucose tolerance according to diabetes or not during 9 years of follow - up data are means ( sd ) , median ( interquartile range ) and [ geometric means ] .
the risk of developing diabetes increased with increasing quartiles of tpa antigen and pai-1 activity and decreased in a similar way for tpa activity ( figure 1 ) . in the highest quartile of tpa antigen ( above 8.7
g / l ) nearly 9 % had incident diabetes compared to none in the lowest quartile .
incidence of type 2 diabetes over nine years in subjects with normal glucose tolerance according to baseline quartiles of fibrinolytic variables .
the number of cases in each quartile for tpa activity was 7 , 4 , 3 and 1 .
corresponding numbers for tpa antigen was 0 , 1 , 2 and 12 and for pai-1 activity 2 , 1 , 3 and 9 , respectively .
p values for x(linear by linear association ) were 0.026 for tpa activity , < 0,001 for tpa antigen and 0,007 for pai-1 activity .
logistic regression was performed with an initial model adjusting for age and sex . in a second model
, waist circumference was added and in a third model , also diastolic blood pressure , serum insulin and triglycerides were added .
quartiles 13 of tpa antigen and pai-1 activity were contrasted with the fourth quartile and , for tpa activity , the first quartiles with quartile 24 ( table 2 ) . logistic regression analysis for fibrinolytic variables at baseline with incident type 2 diabetes as the dependent variable .
the lowest quartile of tpa activity was associated with a 3-fold increase in the risk of diabetes , which was attenuated after adjustment for waist circumference .
after further adjustment for diastolic blood pressure , fasting insulin and triglycerides , no prediction from low tpa activity on diabetes development was seen .
subjects in the highest quartile of tpa antigen experienced more than a ten - fold increase in risk , although the confidence intervals were wide due to few cases .
taking waist circumference into account , the odds ratio diminished to 6.7 and diminished further to 5.5 when adjusting for blood pressure , triglycerides and insulin .
this lead to somewhat higher age- and gender adjusted odds ratio ( 7.4 ; ci 1.830 , p value 0.005 ) . exchanging diastolic with systolic blood pressure did not change the findings .
furter adjustment for leisure time physical activity reduced the odds ratio from 5.5 to 5.1 ( p value 0.03 ) .
high pai-1 activity increased the risk of diabetes four - fold , although the result was attenuated to an insignificant or of 2.0 when all factors were adjusted for simultaneously .
the risk of developing diabetes or igt during follow up ( n = 66 ) increased stepwise across quartiles of tpa antigen , from 7.8% to 19.8% ( p = 0,003 , test for linear trend ) . when adjusted for age and sex , the odds ratio for the highest vs. the three lower quartiles of tpa antigen was 1.9 ( ci 1.011.6 , p = 0.04 ) but diminished further when also waist circumference was adjusted for , or = 1.3 ( ci 0.636.5 , p = 0.5 ) .
subjects with normal glucose tolerance , who subsequently develop type 2 diabetes over the ensuing nine years , are already at baseline characterised by impaired fibrinolysis and endothelial dysfunction .
low tpa activity and high pai-1 activity is mainly explained by the presence of abdominal obesity but also by other metabolic disturbances characteristic of the insulin resistance syndrome , such as high serum levels of triglycerides and insulin .
a novel finding , in this context , is that a high plasma level of the endothelial - derived tpa is only slightly explained by such factors .
thus , the development of type 2 diabetes is preceded by many years of perturbations in fibrinolytic and endothelial function , which increases the risk of atherothrombotic disease long before overt diabetes is present . pa1 - 1 is partly synthesised in fat cells and its activity is strongly related to abdominal obesity as mirrored by high waist circumference or whr .
high circulating levels of pai-1 inhibit tpa released from the vessel walls and lead to low levels of free tpa , i.e. low tpa activity and impaired capability of thrombolysis .
the only report of an association between fibrinolysis and the development of diabetes is a recent study where pai-1 antigen was an independent predictor of diabetes in american - mexicans that were followed up to 5 years .
there is no known pathway whereby impaired fibrinolysis would increase insulin resistance or decrease insulin release and our finding that high pai-1 and low tpa activity precedes diabetes probably mirrors the strong relationship with intraabdominal fat and circulating levels of free fatty acids .
a recent experimental study show an almost three fold increase in the level of pai-1 antigen with an infusion of triacylglycerol in healthy subjects without any impact on tpa .
this effect was noted with constant levels of insulin and glucose and also increased levels of soluble vascular cell adhesion molecules-1 was found .
this support our findings that adjustment for baseline triglyceride levels , which were increased in converters , did not significantly change the relationships noted for tpa antigen but did decrease those for pai-1 .
it is thus possible that also tpa concentration is an inflammatory marker although we found low correlations between tpa antigen and fibrinogen and highly sensitive crp ( own unpublished data ) .
recently , it was described in type 2 diabetes patients , that increased levels of tpa antigen , among other endothelial markers , predicted the development of urinary albumin excretion , a strong marker for cvd risk .
these findings should be related to recent reports where inflammatory markers predict the development of diabetes in longitudinal studies . in most cases ,
pro - inflammatory substances derived from abdominal fat tissue may be the common background for these findings .
we therefore find it plausible that the relation between high tpa and diabetes is a marker of some early disturbance in the vascular wall that operates independent of obesity and inflammation .
already borderline hypertension is associated with high concentrations of tpa antigen , even after adjustment for abdominal obesity , insulin and triglycerides .
thus , tpa mass may mirror an endothelial dysfunction that is not causally related to the insulin resistance syndrome but acts by a parallel pathogenetic mechanism leading to atherothrombosis [ 30 - 32 ] .
insulin resistance , on the other hand , is associated with endothelial dysfunction and impaired vasodilatation due to defects in endothelium - derived nitric oxide . taken together , these findings put focus on the role of the vessel walls in the pathogenesis of type 2 diabetes .
the relevance of impaired fibrinolysis , i.e. low activity of tissue plasminogen activator ( tpa ) or high activity of its inhibitor ( pai-1 ) , as independent risk factors for cvd is unclear , as these variables seem to mirror most components of the insulin resistance syndrome . in some studies , decreased fibrinolysis , as measured by high pai-1 activity , has been associated with cardiovascular events .
high levels of tpa antigen independently predict cardiovascular events both in a healthy population and in patients with prevalent coronary disease .
the major shortcoming of our study is the small number of incident cases of diabetes leading to wide confidence intervals . on the other hand , the odds ratios for tpa antigen are high and consistent and based on a truly representative population sample where basal normal glucose tolerance was well defined , as were the incident cases of diabetes .
thus , the risk of bias due to selection or case ascertainment should be low .
admittedly , the lack of a renewed ogtt in 13 % of the subjects could contribute to some misclassifiaction and dilute the results somewhat .
the early prediabetic state , with normal glucose tolerance , is characterised by both impaired fibrinolysis and endothelial dysfunction years before glucose levels increase .
changes in endothelial function are independent from the insulin resistance syndrome and point towards a distinctly different pathway both for cardiovascular disease and for the development of diabetes . to answer a classical question from 1990 yes
, the clock for coronary heart disease seems to start ticking before the onset of clinical diabetes !
bmi = body mass index ci = confidence interval crp = c - reactive protein cvd = cardiovascular disease igt = impaired glucose tolerance monica = monitoring of trends and determinants of cardiovascular disease ngt = normal glucose tolerance ogtt = oral glucose tolerance test pai-1 = plasminogen activator inhibitor-1 tpa = tissue plasminogen activator
me drafted the research on fibrinolysis within the monica project , performed the statistical analysis and drafted the manuscript .
bs and bl participated in the design of northern sweden monica study and participated in its design and coordination .
supported by grants from norrbotten and vsterbotten counties , by the joint committee of northern sweden health care region , the swedish public health institute and the swedish medical research council . | backgroundimpaired fibrinolysis is found in impaired glucose tolerance and type 2 diabetes , associated with components of the metabolic syndrome .
there are no data concerning fibrinolysis in subjects with normal glucose tolerance that convert to diabetes.methodswe studied the activities of tissue plasminogen activator ( tpa ) and plasminogen activator inhibitor-1 ( pai-1 ) and the levels of tpa antigen ( a marker of endothelial dysfunction ) in 551 subjects with normal glucose tolerance in 1990 in relation to incident diabetes during nine years of follow-up.resultssubjects with diabetes at follow - up ( n = 15 ) had significantly lower baseline tpa activity and higher pai-1 activity and tpa antigen than non - converters .
the risk of diabetes increased linearly across quartiles of pai - activity ( p = 0.007 ) and tpa antigen ( p < 0.001 ) and decreased across quartiles of tpa activity ( p = 0.026 ) .
the risk of diabetes with low tpa activity or high pai-1 activity persisted after adjustment for age and sex but diminished to a non - significant level after further adjustments .
the odds ratio of diabetes for high tpa antigen was 10.4 ( 95% confidence interval 2.740 ) adjusted for age and sex .
after further adjustment for diastolic blood pressure , waist circumference , insulin , triglycerides , fasting and post load glucose the odds ratio was 6.5 ( 1.333 , p = 0.024).conclusionsimpaired fibrinolysis and endothelial dysfunction are evident in subjects with normal glucose tolerance who later develop diabetes .
high tpa antigen is predictive of future diabetes independent from the metabolic syndrome . |
selective serotonin reuptake inhibitors ( ssris ) , serotonin and norepinephrine reuptake inhibitors , and noradrenergic and specific serotonergic antidepressants are categorized as first - line treatments in many countries , including japan.1,2 thus , health care providers have the opportunity to choose an antidepressant from a variety of first - line treatment options . to select the most appropriate treatment for each patient , it is important for clinicians to be aware of the differences in efficacy between comparable antidepressants at the doses approved in their countries .
one long - standing approach to comparing efficacy has been meta - analysis of several randomized controlled trials that compare widely used treatments.310 however , results have been inconsistent , perhaps partly due to the use of different methodologies .
recent meta - analyses have used mixed treatment comparisons in which both direct and indirect comparisons were used.11,12 duloxetine , a second - generation norepinephrine reuptake inhibitor that has demonstrated efficacy , safety , and tolerability in patients with mdd,1316 has been the subject of several recent mixed treatment comparisons . a recent analysis by cipriani et al12 compared the effects of 12 new - generation antidepressants in adults with mdd , and gartlehner et al11 conducted an analysis of 234 studies that included placebo as a comparator . however , these results are not applicable to clinical practice in japan , because they included data from patients taking duloxetine up to 120 mg / day , which is twice the maximum dosage ( 60 mg / day ) approved in japan .
another drawback was the inclusion of patients who took antidepressants not approved or not even available for use in japan .
therefore , it is uncertain whether these results can be applied to daily clinical practice in countries ( eg , japan ) where the approved dosage for duloxetine is 4060 mg / day and ssris are limited to four compounds , ie , paroxetine , sertraline , escitalopram , and fluvoxamine . to the best of our knowledge , there are no meta - analyses that compare the efficacy of duloxetine 60 mg / day with these ssris .
the primary purpose of the current study was to provide clinicians with efficacy results that would allow a comparison of duloxetine 60 mg / day with four approved ssris in japan .
the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .
we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .
for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,
a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,
patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .
common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 .
the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .
the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .
the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .
the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .
the study included 8 weeks of active treatment followed by a one - week dose - tapering period .
the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .
this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .
the primary endpoint was mean change from baseline in hamd17 total score at week 8 .
secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .
response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .
our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .
this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .
continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .
effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .
overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .
the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .
relative risk is presented with 95% confidence intervals ( ci ) and p - values .
the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .
absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .
safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .
reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .
safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .
the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .
we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .
for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,
a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,
patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .
common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 .
the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .
the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .
the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .
the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .
the study included 8 weeks of active treatment followed by a one - week dose - tapering period .
the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .
this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .
the primary endpoint was mean change from baseline in hamd17 total score at week 8 .
secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .
response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .
our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .
this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .
continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .
effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .
overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .
the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .
relative risk is presented with 95% confidence intervals ( ci ) and p - values .
the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .
absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .
safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .
reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .
safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .
at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .
the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .
completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .
adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .
discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .
a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population
, no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .
duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .
for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .
these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .
the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .
no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .
statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .
the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .
no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .
the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients ,
nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .
at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .
the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .
completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .
adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .
discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .
a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population
, no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .
duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .
for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .
these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .
the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .
no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .
statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .
the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .
no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .
the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .
our results show that duloxetine and ssri treatment within their respective approved dosage in japan demonstrated comparable efficacy with regard to overall depressive symptoms
. however , certain hamd17 subscale scores responded differently to duloxetine compared with ssris , and this may clarify the difference in clinical attributes among these antidepressants .
first , when assessing overall efficacy using the mean changes in hamd17 total scores from baseline , both duloxetine and ssri demonstrated greater efficacy than placebo , but did not indicate any significant difference between each other for either the total population ( hamd17 total score 15 ) or the more severe population ( hamd17 total score 19 ) . on the one hand , this is consistent with a prior report by thase et al,4 where all randomized patients across any severity were analyzed and no significant difference in efficacy between duloxetine and ssri was found .
however , on the other hand , this is inconsistent with prior reports that duloxetine 120 mg / day showed greater efficacy than ssris in patients with at least moderately severe depression.4,16 given this and the finding of a modest dose response trend for duloxetine efficacy by mallinckrodt et al16 this inconsistency may be explained by the upper limit on the dosage of duloxetine ( 60 mg / day ) in this study .
second , when assessing energy and interest using retardation subscale scores , duloxetine showed greater efficacy regardless of baseline depression severity ( hamd17 total score 15 or 19 ) .
this is consistent with previous reports by mallinckrodt et al16 and martinez et al.24 furthermore , katz et al25 reported that desipramine ( a norepinephrine reuptake inhibitor ) demonstrated better efficacy than paroxetine ( an ssri ) on psychomotor retardation symptoms .
these results may be explained by the effect of duloxetine on norepinephrine , which has been shown to be associated with arousal and activity.26 the current results show some inconsistency with a report by cipriani et al in which some ssris were significantly more efficacious than duloxetine .
this is partly due to the different methodologies used in that study and in our current study . in cipriani
et al,12 approximately one third of the comparisons were direct comparisons from randomized controlled trials and many of the pairwise comparisons were based on only one study .
degree of heterogeneity and statistical incoherence ( estimates based on the direct comparisons are not contained in the 95% ci for indirect comparisons ) in the study network were detected .
study conclusions based on direct comparisons were much more conservative than those based on indirect ones . on the other hand ,
the strengths of this database analysis are that it analyzed individual patient data , used the same analysis method and endpoint across studies , and included similar patients .
the adopted database analysis design allowed for a homogeneous patient population , with almost the same inclusion criteria and the exact same endpoint . although our analysis resulted in a comparatively smaller number of studies and patients , it achieved high internal validity and reproducibility .
in addition , direct comparisons of head - to - head data enabled us to avoid the uncertainty of how much the two different evidence levels are weighted when pooling direct and indirect comparisons .
the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated .
using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .
similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .
another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .
furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .
further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .
in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .
lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .
the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using
this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .
similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .
another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .
furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .
further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .
in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .
lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .
comparison between duloxetine and ssri demonstrated comparable efficacy within the japan - approved dose range in patients with mdd .
results of the hamd17 subscale analysis indicated that duloxetine might be superior to ssris in improving loss of energy and interest . | backgroundapproved doses of antidepressants in japan are usually lower than those in the usa and european union , but to date meta - analyses comparing antidepressants have all used the higher doses approved in the usa and european union and often have used indirect comparisons .
the purpose of this study was to conduct an integrated database analysis of patient level data to compare the effects of duloxetine with those of selective serotonin reuptake inhibitors ( ssris ) at the doses approved in japan.methodspooled data were analyzed from four randomized , double - blind , placebo - controlled studies that compared duloxetine at the dose range approved in japan ( 4060 mg / day ) with other ssris ( paroxetine 20 mg / day or escitalopram 10 mg / day ) and placebo in patients with major depressive disorder . in total , 1,694 patients were included in the analysis ( duloxetine , n=688 ; selective serotonin reuptake inhibitors , n=690 ; placebo , n=316 ) .
the primary outcome measure was the mean change from baseline at week 8 in 17-item hamilton rating scale for depression ( hamd17 ) total and subscale scores.resultsduloxetine and both selective serotonin reuptake inhibitors were superior to placebo in hamd17 total score at week 8 in both the all - randomized group and the more severe subgroup ( hamd17 total scores 19 ) .
duloxetine was superior to ssris in improving the hamd17 retardation subscale score ( least squares mean difference [ 95% confidence interval ] ) : all - randomized group , 0.33 [ 0.60 , 0.07 ] , p=0.015 ; severe subgroup , 0.45 [ 0.83 , 0.07 ] , p=0.020).conclusionwithin the dose range approved in japan for patients with major depressive disorder , duloxetine and selective serotonin reuptake inhibitors demonstrated comparable overall efficacy , with a possible advantage for duloxetine in improving loss of energy and interest . to the best of our knowledge , this analysis is unique not only in evaluating dosages specific to japan , but also in using individual patient data and the same endpoint across studies to allow for strictly direct head - to - head data comparisons as opposed to pooling direct and indirect comparisons . |
we all have a very different understanding of how we define a need for help .
this is most clearly felt when encountering individuals who , despite outside temperatures below zero , prefer sleeping rough in the street over the warm place in an overnight homeless shelter that a social worker or other care worker has offered them .
if they additionally show no response to medical diagnoses such as open ulcers , flu infections or severe breathing difficulties , we can safely say that this person s behaviour deviates fundamentally from the expectations and understanding that health professionals would normally associate with need for action .
being exposed to people who reject such support services , which seem well - motivated and necessary from a medical point of view , is only one of several fruitful challenges that are deliberately provoked as part of the new teaching concept in the institute for the history of medicine at giessen university .
the objectives are as follows : to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as bedarf / bedrfnis ( needs in objective vs. subjective terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students soft skills , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals . to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as bedarf / bedrfnis ( needs in objective vs. subjective terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students soft skills , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals .
these teaching objectives are implemented and pursued , among others , in the interdisciplinary field querschnittsbereich 2 / q 2 history , ethics and theory of medicine ,
that is part of the approbationsordnung fr rzte/(ao : regulations for licensing doctors ) ( ao , 2002 ) . as a matter of fact , some of the objectives
are also presented and discussed in some medical school - curricula in medical sociology and psychology , as well as in community medicine / public health , however , in these contexts , the primary emphasis here is on the methodological and conceptual repertoire from their related disciplines , sociology and psychology . -
similar teaching concepts as those presented in q 2 can also be found at an international level , where they tend to be associated with the concept of medical humanities ( " kulturwissenschaften in der medizin ) , .
medical faculties vary in their interpretation and weighting of these teaching concepts ; however , in the context of research and debate in medical education , the main focus is on the relationship between the three approaches ( history , ethics , theory : additive or integrative ; weighting of individual components ) , which are specified explicitly in the ao and which intend to introduce a reflective perspective on current medicine , .
this paper sets out to discuss the possibility of supplementing the above - described methods with methodological approaches and concepts from another discipline based in cultural studies , namely medical anthropology ( or medical ethnology ) , and to apply these in the context of a specifically designed form of experience to which students are exposed . outside of germany
, medical anthropology is an established discipline in university departments , degree courses and associations .
the seminar medical care on the fringes of society : participant observation and change in perspectives has been designed with these approaches and experiences in mind .
it has been offered as a seminar option in q 2 at the medical school of giessen university since the 2010 - 11 winter semester .
the new teaching concept was developed on the basis of the following findings and considerations : in the standard degree courses required by the regulations for licensing doctors medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result ,
the focus is on problems as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .
the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness ( patients from a doctor s point of view ) will often be new to this environment .
this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .
the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting.as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .
however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .
the priorities for action that result from the individual illness of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years
, the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances according to their own subjective criteria , .
however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training .
the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the risks of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .
the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair
bedarf/bedrfnis , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term bedarf presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term
bedrfnis is far more closely linked to the subjective perception of an individual or a particular predefined group.empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and problems as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary
, these problems tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .
if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .
moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .
such behaviour is frequently perceived as exotic and therefore attributed to the culture or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation
may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .
another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .
stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups . in the standard degree courses required by the regulations for licensing doctors
medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result , the focus is on problems as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .
the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness ( patients from a doctor s point of view ) will often be new to this environment .
this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .
the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting . as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .
however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .
the priorities for action that result from the individual illness of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years
, the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances according to their own subjective criteria , .
however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training . the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the risks of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .
the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair
bedarf/bedrfnis , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term bedarf presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term
bedrfnis is far more closely linked to the subjective perception of an individual or a particular predefined group .
empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and problems as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary ,
these problems tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .
if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .
moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .
such behaviour is frequently perceived as exotic and therefore attributed to the culture or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation
may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .
another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .
stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups .
the three findings outlined above provided the basis for a teaching concept that incorporates the educational potential provided by concepts and procedures used in medical anthropology .
moreover , it draws on the specific experience that students gathered working with people from socially marginalised groups , or more precisely : without a permanent address .
the aim was to systematically draw students attention to the following aspects and dimensions : the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught;the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed;getting to know and immersing themselves in the living environments , health problems and specific needs of people living on the fringe of society following the model of cultural immersion approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services .
the aim is to look closely at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory;exposing the problems of the concept need for help in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds , objective ) needs to be meaningfully and systematically complemented by including the patient s perspective ( subjective , in the context of their biography and life situation , partly ambivalence / rejection of support services ) .
the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught ; the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed ; getting to know and immersing themselves in the living environments , health problems and specific needs of people living on the fringe of society following the model of cultural immersion approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services . the aim is to
look closely at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory ; exposing the problems of the concept need for help in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds , objective ) needs to be meaningfully and systematically complemented by including the patient s perspective ( subjective , in the context of their biography and life situation , partly ambivalence / rejection of support services ) . in achieving these objectives , as briefly outlined above , in addition to the medical body of knowledge , concepts and methods from ( medical ) anthropology were introduced as central components of the seminar .
this essentially involves the method of field research including participant observation , : central to this method is that those at the focus of medical attention will be visited in their everyday living settings .
an additional effort is made to find out about their physical and social environment and their daily routines in order to comprehend the importance of these factors in relation to the patients perceptions of their body and state of mind , and in relation their help - seeking behaviour .
in addition to visiting the living environment of those affected , systematic monitoring and as far as possible participating in their everyday lives , interviewing informers from the group in focus , together with the systematic documentation of experiences and reflections , form essential elements of this method , .
this will enable students to get a better grasp , for example , of the behaviour displayed by the strangers they encounter ( which at first glance may often seem obscure and inadequate ) within the logic and conditionality of the particular contextual setting .
students will also be introduced to the central concept and methodological approach of counter - transference ( as modified by the ethnologist and psychoanalyst george devereux ) : according to this concept contrary to a widely shared assumption researchers and other professionals working with people too respond emotionally to these persons and the conditions or behaviours observed in connection with them .
such emotional responses range from sympathy to various feelings of ambivalence to fear or aggression , which can impact upon professional interaction as well as prolong or shorten it , or even lead to selective perception and specific interpretation of observed behaviours and even persons . following devereux , researchers or doctors
are asked to systematically observe and reflect upon their own subjectivity and employ their findings , as it were , as an examination tool in its own right that will in addition to other methods yield a more holistic and rounded image of the other .
encounters with homeless people can as a result of their often radically different way of living and occasionally extreme physical neglect etc . trigger particularly striking responses from those providing help ; a situation that serves well as an introduction to systematic self - reflection , among other things , on behavioural patterns that are likely to result from such emotional responses .
in addition to concepts and methods based in anthropology / ethnology and ethno - psychoanalysis , the seminar also teaches the basic principles about the general social , economic and legal conditions affecting the life of people without a permanent address .
cooperation with relevant institutions plays a major role in this : our main partner is frankfurter verein fr soziale heimsttten e.v . ( fv ) ,
in the summer semester we additionally have elisabeth strassenambulanz ( esa ) ( backed by : caritasverband frankfurt / m . ) on board .
the fv is an institution that has close ties with the city and offers an extensive range of services to different groups of homeless in frankfurt / main . during the winter months fv social workers drive the klte - bus to those spots where the homeless
are known to congregate and , if required , supply them with tea , blankets and sleeping pads .
the social workers are the first points of contact for the homeless and , if required , will arrange placements offering follow - on support services , including transport organisation .
the esa provides services aimed at improving the health of women and men in housing difficulties , bearing in mind the unusual living conditions of these people . that a profound knowledge of the particular needs of homeless people as well as of institutional and legal possibilities in dealing with homeless people plays a central role in designing and implementing meaningful teaching concepts
however , the focus there was on teaching concepts that concentrate mainly on providing medical treatment for socially marginalised groups , and less on the meta - level of systematic self - reflection and the ability to take on a different perspective , which is the focal point of the project presented here .
the seminar is structured into a preparatory theoretical - methodological module , a practical module , namely the field work ( in the winter semester with the klte - bus , in the summer semester visiting social work in the context of the esa ) , and a follow - up module .
the seminar groups consist of around 10 students ; this figure is based upon the limited number of individuals when accompanying the klte - bus and in the context of the esa ( one student per appointment ) .
the first part of the preparatory sessions ( 2 hours ) takes place in the classrooms at giessen university . here
students are informed about the legal , social and medical situation of people on the fringe of society ; previous experience and expectations from students are likewise discussed .
in addition , students are familiarised with the experience of patients rejecting their offer of support , which can often be disconcerting ; this includes introducing them to the different perceptions people have of illness , and to the concept of counter - transference .
the structure of the responsible social institutions , particularly those based in frankfurt ( fv , esa ; among others ) is presented and practical information on the planned field trip given .
people on the fringe of society : the field work starts now . the second part of the preparatory session ( 2 hours ) takes place on site in the rooms of the fv in frankfurt a preliminary stage of the field trip : the students leave their familiar environment in order to gain new and unfamiliar impressions . in the rooms of the overnight shelter , amidst the homeless persons staying there , fv social workers provide in - depth information interspersed with specialist and personal contributions .
they also shed light on the medical and social situation of the individuals in the shelter and discuss expectations and particularities as regards the organisation of this part of the field work with the students .
the field work proper does not take place in the group setting , but students go out invidudually : one student at a time accompanies the fv or esa social workers on their on - site visits to homeless people .
a 6-hour period is scheduled for this hands - on experience ; in the winter months it includes a trip on the klte - bus between 8 p.m. and 2 a.m. ; in summer the field trip lasts just as long but takes place during the day or in the evening .
students accompany the social workers on their visits on foot or by bike or in the car ; some students get to attend call - outs on the esa s medical bus . during the feed - back sessions ( 2x2 hours )
students relate the different impressions they have had , contextualise them and explain how they felt , thus creating a wide spectrum of different impressions , evaluations and responses , which provide ample opportunities to reflect upon ways of action and potential changes in perception when dealing with patients .
possible essay topics ( the seminar is assessed by essay ) begin to take shape ; these are discussed in - depth and in relation to how individual students approach their work , as well as formal specifications . in the essay students
are asked to present their own personal observations and experiences during the field trip ( e.g. , feeling insecure in the unfamiliar environment , personal responses to coming face to face with the living environment , the appearance and behaviour of homeless people ) ; in a second step the aim is to reflect upon these observations and their significance for medical thinking and acting .
given the limited number of participants ( ten students per semester ) and the relative short period for which the seminar has been available ( since the 2010 - 11 winter semester ; only three seminar groups have fully completed the class ) we are thus far only able to qualitatively comment on the course ; the total number of participants is still too low to allow for any meaningful quantitative - statistical analysis . to date
, 31 students overall have participated in the seminar : 16 women and 15 men .
the anonymous written evaluation contained three open questions ( positive aspects include ; negative aspects include ; what do you take out of this experience for your further studies ? ) and nine questions to be answered by way of a graded scale ( school grades a to f ) . among these ,
three questions were of a general nature ( please assess how much you have learned ; relevance for training as a medical doctor ; overall school grade ) , six related to specific aspects of the seminar , such as design and structure of the teaching concept , preparation of the lecturer , availability and comprehensibility of relevant information / literature or learning environment . the overall feedback on the structure of the course , on the preparation of the lecturer ( b.m . ) and on the learning environment was between positive and very positive ( grade b or a in analogy to the school grading system ) .
the questions was the seminar interesting ? and the seminar encouraged reflection received the top grade ( a ) .
somewhat in contrast likewise an unanimous perception the relevance for hands - on practical application ( relevant for my training as a doctor ) was recognised but felt to be less positive ( grades b and c ) . in answering the open questions , the on - site experience with social workers and ( in a different response ) the
individual tuition as a result of this was perceived to be particularly worthwhile .
another positive comment was that attention was drawn to how a person s concrete living environment affected their health .
finally , the opportunity to look outside the box was felt to be a profound experience that would shape the students further course of studies .
the essays too contained statements that reflected the intensity of the seminar and the potential inherent in the anthropological approach complemented by field work and participant observation .
the following statement has been taken from the discussion section of one of the essays :
never before did i look at frankfurt s bahnhofsviertel in this way [ ] .
another participant in the seminar felt that the field research method opened up an exotic world [ for him ] .
the human being that crosses our daily horizon as a penniless and neglected figure on the periphery suddenly gets to be at the heart of our attention .
both in the evaluation and occasionally also in the essays the wish was expressed to actually extend the practical part of the seminar . arrangements for receiving written feedback from seminar participants at predefined intervals ( one , two and five years ) have been made , and interviews about the long - term effects of learning and reflection processes are planned with focus groups .
the preliminary experiences we gained with the teaching concept providing medical care on the fringe of society : participant observation and change in perspective illustrate that the spectrum of methodological approaches in q 2 ( ao ) can indeed be complemented by concepts from medical anthropology , in combination with exposing students to people in extreme social situations .
the objective of getting students to reflect systematically upon historical , ethical - normative and theoretical foundations in medicine can therefore be extended and intensified in particular as regards the following three aspects : making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent . making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent . at a more general level students
will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine .
the teaching project therefore ties in with international models that likewise seek to integrate these teaching objectives in their medical training .
the method of field research ( or inspired by this in the context of medical training that of cultural immersion ) has proven to be very helpful for understanding the perceptions and needs of socially or ethically marginalised groups , and in principle all other groups in health care , , , , [ . in relation to this in the context of the new teaching concept the attention on social determinants of health and how they impact on the pathogenesis and course of health conditions
yield synergies with other teaching concepts , for example in medical sociology , community medicine / public health or general medicine , , which focus on similar matters .
collaborations with community medicine practitioners working for example at technical colleges / fachhochschulen or other institutions outside universities could likewise bear relevance .
, it will be necessary to know and familiarise oneself with the central ( medical ) anthropological concepts and methods .
further , opportunities will have to be created that will enable cooperation with such institutions that are willing to set up the organisational framework and make available their expertise to enable the direct contact of students with people on the fringe of society in their everyday living environments .
the new curricular option described here could only be realized through the co - operation and engagement of many members of staff at the frankfurter verein fr soziale heimsttten e.v . as well as the elisabeth - straen - ambulanz frankfurt / m .
( esa ) . we want to express special thanks to heiko ewald and christine heinrichs , together with the staff of the kltebus-project , the ostpark - project , the beratungszentrum fr aufsuchende sozialarbeit , as well as dr .
| this paper examines the new teaching concept providing medical care on the fringe of society : participant observation and change in perspectives in the context of the interdisciplinary field of querschnittsbereich 2/q 2 ( the transdisciplinary section under ao , the german regulations for licensed physicians ) that explores the history , theory and ethics of medicine .
the disciplinary approach usually adopted in q 2 is supplemented with concepts from medical anthropology ; in addition students will be exposed to people in extreme social situations . the aim is to make students aware of and invite them to reflect upon : the importance of participant observation in the specific on - site setting of medical thinking and acting ; the importance of the subjectivity of all those involved in doctor / patient interaction ; and the fact that key medical terms ( such as the need as seen by the physician vs. the need as seen by the patient ) are essentially context - dependent in their interpretation . at a more
general level students will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine . |
foodborne liver and intestinal flukes are important groups of parasites from the public health point of view [ 1 - 4 ] .
lao peoples democratic republic ( lao pdr ) is located in the middle of the indochina peninsula , and the mekong river runs through the whole length of the country from north to south . until the 1990s , the liver fluke opisthorchis viverrini and soil - transmitted nematodes , including ascaris , trichuris , and hookworms , were the major helminths prevalent among the laotian people [ 5 - 8 ] . in 1991 ,
haplorchis taichui adult flukes were recovered for the first time from 5 laotian students studying in czechoslovakia . since then , other intestinal flukes , including haplorchis pumilio , haplorchis yokogawai , prosthodendrium molenkampi , phaneropsolus bonnei , centrocestus formosanus , echinostoma revolutum , artyfechinostomum malayanum , echinochasmus japonicus , and euparyphium sp .
have been recovered from infected residents of vientiane municipality , phongsaly , saravane , khammouane , and savannakhet province [ 10 - 16 ] .
the predominating species and the the intensity of infection with each fluke species varied by locality .
for example , o. viverrini infection was predominant over h. taichui in vientiane municipality , whereas h. taichui infection was dominant over o. viverrini in saravane province . on the other hand , in phongsaly province , located in the northernmost area of lao pdr , high prevalences of h. taichui and h. yokogawai were detected with no detection of o. viverrini .
after completion of the international collaboration project between the korea association of health promotion and ministry of public health ( moph ) , lao pdr on the control of intestinal helminthiases in lao pdr from 2000 - 2004 , a new 5-year project plan was started in 2007 to control foodborne trematode infections by the korea foundation for international healthcare and moph , lao pdr . in this new project ,
the primary goal was to control opisthorchiasis in endemic areas and to reduce the incidence of cholangiocarcinoma in the mekong river basin .
for the successful implementation of this project , a desirable pre - requisite should be the elucidation of the actual status of infection with o. viverrini in each target village .
the recovery of eggs only in the feces is not sufficient for this purpose , because the eggs of o. viverrini and those of minute intestinal flukes are practically indistinguishable in routine fecal examinations .
the present study was performed to determine the actual status of infection with foodborne trematodes by recovery of adult flukes from riparian people of saravane and champasak province , lao pdr .
saravane province ( capital city , saravane ) is located about 500 km south from vientiane , and champasak province ( capital city , pakse ) is located nearby and just south of saravane province ( fig .
1 ) . small riverside villages , each having 200 - 300 residents , located near the mainstream or tributaries of the mekong river were selected for this study , which included 12 villages ( in 6 districts ) in both saravane and champasak province .
most villagers were agricultural workers , and some had caught freshwater fish from small streams and ponds and consumed them raw or improperly cooked . a total of 1,460 fecal samples ( 1 sample per person ) were collected from the villagers ( 717 men and 743 women , 12 - 82 years - of - age ) from december 2009 to june 2010 .
samples were transported to the malaria stations in saravane and champasak province within 2 - 3 days of collection and were stored at 4 until examined .
trematode eggs 23 - 32 m in size were collectively recorded as small trematode eggs ( ste ) .
fecal examinations and anthelmintic treatments were officially approved by the ministry of public health , laos , under the terms of the korea - laos agreement on foodborne trematodiases control in laos ( 2007 - 2011 ) .
thirty people who were positive for ste were selected for adult worm recovery at the malaria station . after obtaining informed consent ,
they were treated with a single oral dose of 40 mg / kg praziquantel ( distocide@ ; shinpoong pharm . ,
worms were counted and some were fixed with 10% formalin under cover slip pressure , acetocarmine - stained , and morphologically identified using a light microscope .
fecal examination results were analyzed using the student 's t - test and the chi - square test .
the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .
there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .
the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .
the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .
paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .
other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .
the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) .
the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .
a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .
intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .
the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .
the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .
small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .
two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .
the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province .
the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .
there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .
the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .
the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .
paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .
other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .
the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) .
the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .
a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .
intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .
the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .
the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .
small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .
two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .
the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province .
an extremely high predominance of intestinal flukes , in particular , h. taichui , was evident in saravane and champasak province . the liver fluke , o. viverrini , constituted only a very small portion of the helminths infected among the riparian people in the surveyed localities .
therefore , we could confirm that the real o. viverrini heavy endemic areas in lao pdr include vientiane municipality and savannakhet province , based on the previous results from our research group .
dominate in khammouane , saravane , and champasak provinces with only a low - grade endemicity of opisthorchiasis , based on our previous observations and those of the present study .
phongsaly province and luang prabang and xiengkhouang provinces are also haplorchis - dominant areas without o. viverrini endemicity ( to be published ) .
the individual worm load of h. taichui was extremely high in this study , particularly in saravane province .
it was 67 times higher than in khammouane province ( 324 vs. 21,565 ) , 209 times that of savannakhet province ( 103 vs. 21,565 ) , and 799 times that of vientiane municipality ( 27 vs. 21,565 ) .
indeed , it was even 3-times higher than that of our previous study in saravane province .
this may have been due to the fact that different districts were surveyed in our earlier and present studies .
the present report of intestinal flukes , including h. taichui and h. pumilio , from people in champasak province is novel .
however , recovery of o. viverrini was previously reported in 3 people ( 5 , 10 , and 395 specimens ) residing near pakse , the capital of champasak province .
therefore , in the present study , it was surprising to see that the worm load of h. taichui and h. pumilio was markedly higher than that of o. viverrini in champasak province .
one patient , a 35-year - old man , expelled as many as 129,159 specimens of h. taichui , and thus he represents the most heavily infected case with h. taichui ever recorded in the literature .
the hyperendemicity of h. taichui infection among riparian people in saravane province was closely correlated with the report of metacercarial prevalence in fish in saravane province .
h. taichui metacercariae were detected in various freshwater fish species from saravane that included hampala dispar , cyclocheilichthys enoplos , mystacoleucus greenwayi , and puntioplites proctozysron with the average metacercarial density per fish of 1,532 ( range 16 - 6,050 ) , 346 ( 14 - 986 ) , 337 ( 8 - 1,625 ) , and 175 ( 2 - 863 ) , respectively .
however , the metacercariae of o. viverrini were also detected heavily in h. dispar with the average number per fish of 453 .
no or very few o. viverrini metacercariae were detected in other species of fish examined in saravane province .
it is strongly suggested that h. dispar is an important source of infection both for h. taichui and o. viverrini in this area .
the present study indicated that the sole use of fecal examinations to detect helminth eggs does not fully allow the determination of the actual prevalence and intensity of liver and intestinal flukes in lao pdr .
for example , 2 recent studies performed in champasak province reported the results of fecal examinations on the residents [ 18 - 20 ] ; the ste were interpreted only as o. viverrini eggs , and the possibility for them to be eggs of intestinal flukes , in particular , haplorchis spp .
, was overlooked . the adult worm recovery after chemotherapy and purgation , like our studies
[ 10,11,14 - 16 ] and others , is helpful to determine the actual and relative predominance of liver and intestinal flukes in each area , although it is a tedious work and needs experts who have much experience and skill . it would be preferable to have a proper technique to distinguish the species of ste in fecal specimens .
several diagnostic techniques have been reported to be useful for morphologic discrimination of ste in fecal samples [ 21 - 23 ] .
iodine staining was helpful for discriminating lecithodendriid eggs ( p. molenkampi and p. bonnei ) from those of o. viverrini .
potassium permanganate temporary staining , using 1% ( w / v ) solution for 1 min , was also applied to discriminate the eggs of o. viverrini , h. taichui , and p. bonnei .
molecular techniques have been applied to detect o. viverrini dna in fecal samples [ 24 - 26 ] . in khon
kaen , thailand , a 98% specificity and a 100% sensitivity of the pcr technique were obtained in cases of moderate to severe infections ( > 1,000 epg ) but only a 68% sensitivity was shown in light infections ( < 200 epg ) . in lao pdr ,
the pcr sensitivity was lower ; only around 50% in cases even with high epg counts > 1,000 .
cetyltrimethyl - ammoniumbromide was used to remove such inhibitors , which resulted in an enhanced sensitivity of pcr technique to detect fecal o. viverrini dna , from 44.8% to 79.3% .
it was stated that the primers used for pcr did not cross - react with haplorchis spp . .
in this respect , the possibility of mixed infections with o. viverrini with minute intestinal flukes , including haplorchis spp .
, was raised as a reason for low sensitivity of pcr technique to detect o. viverrini dna .
subsequently , pcr assays to discriminate o. viverrini from those of h. taichui and h. pumilio were developed using the internal transcribed spacer ( its ) regions , its1 and its2 .
the pcr amplicons of o. viverrini , h. taichui , and h. pumilio were 800 , 930 , and 1,250 bp , respectively , for its1 , which successfully differentiated the 3 species , whereas those for its2 were 380 , 530 , and 380 , respectively , which could not discriminate o. viverrini and h. pumilio .
the sensitivity of its1 pcr using fecal samples , however , was relatively low ( 76.2% ) .
it may be argued that not all of the o. viverrini worms were expelled from the subjected cases by a 40 mg / kg single dose treatment with praziquantel .
however , in our previous studies in vientiane municipality and savannakhet province , the same protocol as in the present study was applied , and 3 - 315 o. viverrini specimens in vientiane and 1 - 1,350 o. viverrini specimens in savannakhet were recovered individually in the diarrheic stools of the subjects within 5 - 6 hr ( 4 - 5 successive stools ) after praziquantel administration .
these observations indicate that if a person is infected with o. viverrini , a majority , if not all , of the worms are expelled by this procedure . a previous study in thailand supports this hypothesis ; in 9 patients treated with 40 mg / kg praziquantel in a single dose followed by purging and whose diarrheal feces were collected at least 24 hr after purgation , 95% of all worms were expelled within the first 4 hr . a study performed in khon
kaen , thailand , a hyperendemic area of opisthorchiasis , reported that the worm load of o. viverrini among 181 human autopsy cases averaged 182.4 and ranged from 1 - 2,946 per individual . in our previous studies in savannakhet and vientiane , the average worm load measured by worm recovery after praziquantel treatment and purging was 115.4 ( n=29 ; 0 - 1,350 per individual ) and 57.8 ( n=18 ; 3 - 315 per individual ) , respectively
therefore , it is suggested that the procedure we used is a reliable method for recovery of both liver and intestinal flukes from humans . | in this study , we found that haplorchis taichui , a heterophyid intestinal fluke , is highly prevalent , with heavy worm loads , among riparian people in saravane and champasak province , lao pdr .
fecal specimens were collected from 1,460 people ( 717 men and 743 women ) in 12 riparian ( mekong river ) districts and were examined by the kato - katz fecal smear technique .
the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively .
the positive rate for small trematode eggs ( ste ) , which included h. taichui and other heterophyids , opisthorchis viverrini , and lecithodendriids , was 69.9% and 46.3% in saravane and champasak province , respectively . to obtain adult flukes ,
30 ste - positive people were treated with 40 mg / kg praziquantel and then purged .
whole diarrheic stools were collected 4 - 5 times for each person and searched for fluke specimens using a stereomicroscope .
mixed infections with various species of trematodes ( h. taichui , haplorchis pumilio , o. viverrini , prosthodendrium molenkampi , centrocestus formosanus , and echinochasmus japonicus ) and a species of cestode ( taenia saginata ) were found .
however , the worm load was exceptionally high for h. taichui compared with other trematode species , with an average of 21,565 and 12,079 specimens per infected person in saravane and champasak province , respectively , followed by h. pumilio ( 41.9 and 22.5 , respectively ) and o. viverrini ( 9.4 and 1.5 , respectively ) .
these results show that diverse species of intestinal and liver flukes are prevalent among riparian people in saravane and champasak province , lao pdr , with h. taichui being the exceptionally dominant species . |
for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information a misdiagnosis , an incomplete medication list , the wrong demographic data
thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality . building on a framework for data quality ,
as the source of patient - centered data , consumers have important roles to play in reviewing and confirming their own health information and should be engaged partners in efforts to understand and improve the quality of electronic health data .
for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information a misdiagnosis , an incomplete medication list , the wrong demographic data
thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality .
consumers are not only the foundation of the data quality framework kahn et al . , but have important roles to play in reviewing and confirming their own health information along the chain of data stewardship . recognizing patients and families as sources of valuable information and powerful forces for change in identifying and preventing errors will also help to foster a culture of safety and build a sense of partnership between providers and patients in the mutual pursuit of better health and care . building on kahn et al.s figure following the chain of data stewardship ,
i have overlaid opportunities for patient engagement at each step of the process , underscoring the critical role of individuals as the original data stewards ( see figure 1 ) .
the number of people with online access to their electronic health information is growing by leaps and bounds . before the health information technology for economic and clinical health ( hitech ) act , patients had a right to receive a copy of their health records within 3090 days under the hipaa privacy rule .
now , thanks to meaningful use , patients have the ability to view online , download , and transmit their own records within 36 hours after a hospital discharge , or within four business days after a doctor receives the health data.1 health information is increasingly available at the click of a button with blue button .
blue button is an easy , secure method for individuals to download their medical records in digital form from a secure website offered by doctors , insurers , pharmacies or other health - related services.2 implementation of these policy changes is underway , and there are undoubtedly individuals who still struggle to access their own health information ( electronically or otherwise ) , and work is necessary to improve the experience and utility of online access for patients .
nonetheless , online access to clinical information is an opportunity to partner with patients and families to assess and enhance the quality of health care data .
as patients and families get easier access to their medical records and health data , possibly for the first time , it is inevitable that they will have feedback corrections , additions , and observations to share .
in fact , the absence of health information produced , shared , and reviewed by patients themselves contributes to poor data quality .
patients and their caregivers can provide information critical to high - quality research and care delivery , including family health history , treatment side effects , changes in health status or functional status , and even treatment outcomes.3 too often , records are missing key data about health outcomes did the patient get better after the recommended course of treatment ? corrections and amendments are another important type of pghd .
patients are often the first to identify errors in their own records . increased access by individuals to their own health information
these could include updated medication lists , nontolerated medications , other allergies , and more .
these and other patient generated health data complement clinical information generated by care teams to provide a comprehensive view of an individual s health , thus enhancing data quality , as well as enabling more meaningful conclusions to be drawn from secondary uses of this data . with greater online access to their health information and new tools to electronically communicate and share information with providers , patients and families can help improve the accuracy and reliability of data stored in medical records .
i recently heard a speaker at the office of the national coordinator ( onc ) s consumer summit affectionately refer to the benefits of this new capability as an army of free fact checkers .
a 2014 nationwide survey found that patients with online access to their health information overwhelmingly use this capability : 86 percent log on at least once a year , and more than half ( 55 percent ) log on three or more times per year .
survey data show that online access has a positive impact on patients knowledge of their health , ability to communicate with doctors , and ability to correct information ( for 63 percent of patients ) .
more frequent online access has an even greater impact on ability to correct information ( 71 percent for frequent users , compared with 48 percent for less frequent users).4
as the original generators and stewards of their own health data , individual patients and consumers are the foundation in the data stewardship chain outlined in kahn et al.s data quality framework .
consumers also want to continue to be involved , considered , and consulted in all aspects of data capture , assessment , and use .
figure 1 takes kahn s figure and illustrates the potential data contributions of individual patients and consumers , as well as opportunities to support critical data management activities at multiple points along the chain of data stewardship . in particular , there are opportunities for consumer and patient involvement in improving the quality and integrity of data stored in electronic health records ( ehrs ) for clinical use ( data originator box ) . for example , providers could share their computer screen with patients during clinical encounters , allowing patients to see their medication lists or medical records .
this gesture not only engages patients in the data entry , but also offers an opportunity for patients to contribute corrections or new information to their record in person . the growing ability for patients to view , download , and
patients and their families can view their health records and supply missing data or correct inaccurate data via patient portals or secure messaging with their providers .
individuals can review and verify data further down the stewardship chain via a portal offered by a health information exchange ( hie ) .
hies collect data from multiple institutions , and some portals allow the patient to see the collective longitudinal record and to offer corrections or amendments to the whole .
finally , at the end of the data stewardship chain , consumers could review and offer feedback on the relevance or usefulness of the analysis and visualizations being created by clinical researchers and other investigators .
of course , questions remain about how best to collect and ingest information from patients into the electronic medical record .
questions , for example , such as how to authenticate data coming from patients prior to use ( how to confirm when the data were created , whether they were modified in some way , what instrument was used to collect them , etc . ) .
policies and procedures are still necessary to ensure that providers can receive , review , record , and respond to pghd shared by patients and families.5
a comprehensive data quality reporting framework for assessing and describing data quality may help to validate pghd and incorporate them into the medical record , and use them to inform clinical care or health care policy . we must also more effectively engage patients and families in an online health environment and educate them about the impact of data quality on their health and health care .
underlying changes to clinical culture are also necessary : moving toward viewing patients as active partners in their care , rather than as passive recipients.6 great strides are being made in patient - centered outcomes research , as exemplified by the patient centered outcomes research institute ( pcori ) , which funds research and supports patients and other stakeholders to meaningfully partner with researchers throughout the research continuum . looking forward , we must also broaden our focus beyond current data types and quality issues to consider relevant health data outside traditional clinical settings , such as social determinants of health ( building on the institute of medicine s recommendations),7 and their place and impact in the data quality framework .
consumers have much to gain from , and contribute to , activities to assess and improve data quality.8 efforts to adopt and implement a comprehensive data quality framework should preserve and reinforce the role of individuals as the original data stewards , generating and sharing critical information to inform better research , safer clinical care , and ultimately improved health outcomes . | introduction : for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information a misdiagnosis , an incomplete medication list , the wrong demographic data care safety , quality , and outcomes suffer and patients and families suffer.opportunity for change : thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality .
building on a framework for data quality , there are numerous opportunities for patients to be part of
feedback loops to understand and improve data quality are presented.foundation for the future : as the source of patient - centered data , consumers have important roles to play in reviewing and confirming their own health information and should be engaged partners in efforts to understand and improve the quality of electronic health data . |
adverse drug reactions ( adrs ) , including interactions , in older people are a common cause of admission to hospital1 and are also a common cause of disease in hospitalized patients .
frail elderly patients appear to be particularly at risk of adrs . in some cases , this is because insufficient account is taken of the effect of age and frailty on the pharmacokinetic and pharmacodynamic of the drug , especially in relation to hepatic2 and renal elimination.3 although many adrs are recognized during the drug development process and before licensing , this is not always the case , especially when they are uncommon or rare.3 moreover , patients with severe cognitive impairment may not be able to refer symptoms related to an adr , making the recognition of this clinical condition a medical problem.4 rhabdomyolysis is a clinical and laboratory syndrome that is caused by various etiologies , involving the skeletal muscle.5 rhabdomyolysis is characterized by the leaking of myoglobin and other intracellular proteins and electrolytes into the circulation , and may be associated with a wide variety of diseases , injuries , medications , and toxins.5 it is important to recognize drug - induced rhabdomyolysis because clinical effects are usually reversible.4 rhabdomyolysis related to macrolide - statin interaction has previously been described.68 clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins like simvastatin and atorvastain.69 to date , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.10 we report the case of a 90-year - old caucasian male , admitted to the university hospital of pisa for dyspnea , who experienced rhabdomyolysis related to clarithromycin treatment .
a 90-year - old caucasian male was admitted to the university hospital of pisa for dyspnea .
he had experienced dyspnea for 2 weeks , and showed signs of pneumonia after a chest x - ray was performed .
the patient was affected by alzheimer s disease , in advanced phase , and did not take any home - medication as referred by his general physician . at admission in the emergency room ,
clinical workup revealed mental confusion ( the relatives referred progressive cognitive impairment for several months ) , sinus tachycardia ( 115 beats per minute ) , basilar wheezes , and rhonchi .
body temperature was 36.6c with normal systemic blood pressure ( 140/80 mmhg ) , diuresis , and blood oxygen saturation ( so2% 96.2% ) .
routine laboratory exams , including complete blood cell count , thyroid hormones , liver ( aspartate aminotransferase and alanine aminotransferase ) and muscle ( creatine kinase [ ck ] ) enzymes , as well as the coagulation pattern , resulted within normal limits .
serum brain natriuretic peptide was measured and a cardiac markers curve ( including myoglobin ) was also started , ruling out cardiogenic dyspnea .
blood potassium and creatinine concentrations were 4.96 meq / l ( reference range 3.55 meq / l ) and 122.8 mol / l ( reference range 4590
a diagnosis of acquired pneumonia was made and treatment with ceftriaxone ( 1 g four times daily , intravenous ) , hydration ( crystalloid 1.5 l / day ) , aerosol with salbutamol and oxitropium , o2 inhalation ( 3 l / minute ) , and dalteparin ( 2500 ui , subcutaneous ) was started .
after the geriatric ward admission , clarithromycin ( 500 mg two times daily , intravenous ) was also started .
on the second day of hospitalization , for continuing sinus tachycardia , in the absence of any evident causes ( fever , hypotension , arterial - venous fistula , thyrotoxicosis , suspected drug treatments , etc ) beta - blocker treatment ( bisoprolol 1.25 mg , daily ) was added and a cardiac markers curve was restarted ( figure 1 ) .
the second point of the curve , almost 6 hours after the third clarithromycin dose , revealed a huge myoglobin increase ( 1144 ng / ml ; 8.5 times the upper normal range [ 28138 ng / ml ] ) without electrocardiogram modifications .
u / l ; 6.5 times the upper normal range [ 38180 u / l ] ) along with a mild creatinine increase ( 152.9 mol / l ) , in the absence of liver enzyme alterations ( aspartate aminotransferase , alanine aminotransferase ) .
urine color concomitantly changed toward dark brown and hemogasanalysis revealed a slight respiratory insufficiency type 1 with normal ph .
a diagnosis of rhabdomyolysis was made and clarithromycin was stopped , while the concomitant therapy with ceftriaxone , bisoprolol , and aerosol with salbutamol and oxitropium was continued .
the patient was immediately treated with intravenous crystalloid hypotonic solution ( 80 ml / hour ) and corticosteroid ( 40 mg methylprednisolone , intravenous ) .
serum ck and myoglobin levels decreased within a few days and a chest x - ray showed significant improvement .
the patient was discharged home on the sixth hospital day ( figure 1 ) with the diagnosis of iatrogenic rhabdomyolysis and acquired pneumonia of unknown etiology .
indeed , serum and urinary antigen and antibody exams excluded the presence of legionella pneumophila , streptococcus pneumoniae , and mycoplasma pneumoniae while virus infection assessments were not performed .
clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins as simvastatin and atorvastatin.3,69 rhabdomyolysis related to macrolide and statins interaction has previously been described ; however , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.8,10 in our case , there was an evident temporal sequence between clarithromycin exposure - withdrawal and the onset - recovery from rhabdomyolysis biochemical signs , while other potential causes of muscle damage were reasonably excluded .
accordingly , the naranjo probability scale confirmed a probable relationship ( score 6/9).13 a general physician and a nurse followed the patient at home , thus we are confident the home care was carried out in a correct manner , ruling out mistakes and accidental drug intake .
moreover , an interaction with other concomitant medications was improbable considering the rapid resolution after clarithromycin withdrawal and the elimination pathways of the concomitant drugs ( both bisoprolol and ceftriaxone have hepatic and renal elimination routes).11,12 in addition , bisoprolol was administered at the minimal dose ( 1.25 mg ) and both bisoprolol and ceftriaxone , as single therapy , were never related to an adverse drug reaction like rhabdomyolysis .
we ruled out infection as the possible cause of muscle damage considering the sequence of the events .
in fact , the patient was admitted to the hospital for dyspnea developed at home since 2 weeks , while serum ck suddenly increased 2 days after hospital admission .
if rhabdomyolysis had been caused by the infection we would have expected that serum ck increased earlier , thus being already evident at hospital admission . moreover ,
the diagnosis of rhabdomyolysis , unless there is a high index of suspicion , can be missed since muscular pain , swelling , and tenderness may not be prominent or even absent .
the definitive diagnosis should be made by laboratory tests including serum ck and urine myoglobin.5 in our case , the patient had advanced cognitive impairment making him unable to easily refer any symptom , and the diagnosis was made by the huge elevation of biochemical markers ( 6.5 times the upper normal value for ck , 8.5 times for myoglobin ) .
overall , these findings allowed us to make the diagnosis of rhabdomyolysis with sufficient warranty .
although clarithromycin associated rhabdomyolysis is mainly due to pharmacokinetic interactions , a direct muscle toxicity might be postuled.5,10 | rhabdomyolysis is a clinical and laboratory syndrome that is caused by various etiologies , involving the skeletal muscle .
clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins .
rhabdomyolysis related to macrolide statin interaction has previously been described . to date , rhabdomyolysis induced by clarithromycin has been described in only one previous report .
we describe the case of a 90-year - old caucasian male , admitted to the university hospital of pisa for dyspnea , who developed rhabdomyolysis associated with clarithromycin administration . |
supravalvar mitral ring is a circumferential ridge or membrane arising from the left atrial wall overlying the mitral valve and frequently attached to the mitral valve.1 variable in thickness and extent , the ring ranges from a thin membrane to a thick discrete fibrous ridge.2 the membranous variety may be difficult to detect because the membrane often adheres to the anterior mitral valve leaflet while remaining just proximal to the posterior mitral leaflet .
adhesion to the valve may impair the opening of the leaflets , and this impairment may be the main mechanism of mitral valve inflow obstruction in some patients .
in other patients , the ring may be large enough to protrude into the mitral valve inflow and cause obstruction .
the supramitral ring may initially be incomplete and eccentric , allowing for an unobstructed flow through the mitral valve .
however , turbulence can cause a progressive increase in the supravalvar membrane or ridge thickness , worsening mitral inflow obstruction.3 the same mechanism is responsible for the acquired variety of supravalvar mitral stenosis , which occurs after mitral annuloplasty for the repair of mitral regurgitation .
we herein present a case of a supraventricular ring in a 17-year - old male , who presented with syncope .
about 15 years ago , he had undergone a cardiac operation for ventricular septal defect ( vsd ) closure and mitral valve repair . in chest x - ray
both lungs were clear , and the electrocardiogram result showed normal sinus rhythm , had undergone right bundle branch block , and right ventricular hypertrophy .
transthoracic echocardiography , transesophageal echocardiography , and finally cardiac catheterization revealed ejection fraction of 5055% , pulmonary hypertension with pulmonary artery pressure ( pap ) of 80/40 mm hg , pulmonary capillary wedge pressure of 23 mm hg , gradient in the mitral valve plane of 40 mm hg , no residual vsd , no patent ductus arteriosus ( pda ) , bicuspid aortic valve , no aortic insufficiency , no aortic stenosis , no sub - aortic web , normal coronary arteries , mild mitral regurgitation , moderate to severe mitral stenosis , supra mitral valve ring , normal left ventricle size , moderate right ventricle enlargement , and moderate systolic dysfunction .
there was a free floating high redundant obstructive membrane in the left atrium that was attached to the mid portion of the anterior mitral valve leaflet and also in the medial and base of the posterior mitral valve leaflet and large papillary muscles in the left ventricle ( figure 1 ) .
it also extended just adjacent to the lower limb of the left atrium appendage mouth with a small orifice ( 6 mm ) in the lateral side and also multiple small fenestrations ( by 2 dimensional echocardiography ) , resulting in a severely restricted diastolic flow ( mean peak gradient = 9.3 mm hg ) .
this web was not found in mri cine turbo - flash images acquired on a 1.5 t scanner ( magnetom vision - siemens ) in the four - chamber view ( 3a ) and the transverse plane , short - axis view ( figure 2 ) . the patient s ascending aorta , descending aorta , and aortic arch were tortuous with moderate narrowing of the descending aorta distal to the subclavian artery with a systolic gradient of about 30 mm hg , which was suggestive of insignificant coarctation of the aorta .
after heparinization , extracorporeal circulation was established between the venae cava and the ascending aorta . following a median sternotomy ,
there was a long supravalvar muscular band , which was resected ( figure 3 ) .
supravalvar mitral ring rarely occurs as an isolated defect ; other congenital heart defects coexist in 90% of patients .
the mitral valve itself is often abnormal and stenotic at the valvar or subvalvar level ; fusion of leaflets , a small valve orifice , and abnormal papillary muscles are common abnormalities .
shone s complex is a combination of four congenital heart defects : supravalvar mitral ring , parachute mitral valve , subvalvar aortic stenosis , and aortic coarctation .
other common associated lesions in patients with supravalvar mitral ring are ventricular septal defect ( vsd ) , pda , atrioventricular ( av ) canal defect , and tetralogy of fallout.4 uncommonly associated defects include atrial septal defect , cor triatriatum , left superior vena cava , unroofed coronary sinus , partial anomalous pulmonary venous drainage , pulmonary venous obstruction , double - orifice mitral valve , and wolff - parkinson - white syndrome.5 lesions such as transposition of the great arteries , atrioventricular discordance , and double - outlet right ventricle are occasionally complicated by a supravalvar left atrioventricular valvular ring .
obstruction to the mitral inflow results from the reduced area of the mitral valve orifice .
when clinically significant , a diastolic pressure difference occurs between the left atrium and the left ventricle . left atrial and pulmonary venous pressures increase , leading to exudation of fluid into the pulmonary interstitial , which increases lung stiffness .
breathlessness and tachypnea are secondary to the interstitial edema and diminished pulmonary compliance.6 in severe cases , frank pulmonary edema can occur.7 an associated atrial septal defect may decompress the left atrium , reducing or masking the severity of the mitral valve obstruction .
associated lesions such as vsd or pda , which increase the left ventricle output , exacerbate the manifestations of mitral inflow obstruction.8 conversely , a supravalvar mitral ring may be difficult to detect in conditions with a diminished pulmonary blood flow such as tetralogy of fallot.9 persistently elevated pulmonary venous hypertension leads to pulmonary arterial hypertension , rise in pulmonary vascular resistance , and eventual failure of the right ventricle and tricuspid regurgitation .
it also deserves of note that predilection for race , age , and sex has not been found yet .
treatment is the surgical resection of the web , but balloon dilatation is reported to have been successfully performed under fluoroscopic and transesophageal echocardiographic guidance by some authors.10
preoperative identification of a supravalvular mitral ring is the target for obtaining good surgical results . for the identification of this congenital heart disease , | supravalvar mitral ring is a rare congenital heart defect of surgical importance .
the condition is characterized by an abnormal ridge of the connective tissue on the atrial side of the mitral valve .
it often substantially obstructs the mitral valve inflow .
we herein introduce a case of a supravalvar mitral ring in a 17-year - old male , who was admitted to our hospital with cardiac syncope .
he had undergone a cardiac operation for ventricular septal defect ( vsd ) closure and mitral valve repair 15 years before .
transthoracic echocardiography , transesophageal echocardiography , and finally cardiac catheterization revealed a neglected supravalvular mitral ring .
the ring was resected in a second operation , and the patient was discharged from the hospital symptom free . |
in the recent millennium , the constant trend of change in the demands of the community as well as transforming the trend of knowledge production has highlighted the necessity for researchers to adopt a more comprehensive approach .
increasingly , many academic disciplines are utilizing qualitative research ( qr ) as the qualitative method investigating the why and how of the process of a developed concept ( 1 , 2 ) .
qualitative research is sometimes defined as interpretive research , and as interpretations can be incorrect or biased , the findings may be controversial ( 3 ) . however , qualitative research is not only useful as the first stage of quantitative research , but can also play a key role in validating it or in providing a different viewpoint on the same social phenomena ( 4 ) .
qualitative studies tend to use methods that result in text production rather than numerical outputs .
given that the researcher is considered to be the research instrument , and the plan of inquiry needs to be developed and altered as the study progresses , a qualitative researcher can not depend upon traditional approaches to address certain concerns such as bias and credibility .
therefore , learning from a series of mistakes is often considered an integral part of qualitative research ( 5 , 6 ) . in this study ,
a literature review was carried out in international electronic databases including pubmed , web of sciences , cumulative index to nursing and allied health literature ( cinahl ) , scopus , ebsco , embase and science direct without any time limitation , using the search terms qualitative research ,
these keywords were also searched on national electronic databases including scientific information database ( sid ) , iran medex and medical articles library ( medlib ) using the same strategy .
authors of the present article endeavor to shine a light on the ethical issues affecting researchers and propose strategies to face the ethical challenges of qualitative studies , so as to provide applicable and trustworthy outcomes
. this could be the basis for the formulation of specific ethical guidelines in this regard .
up to the 1970s , qualitative research was solely employed by anthropologists and sociologists . during the 1970s and 1980s ,
however , it was favored by various disciplines and experts of different branches of science and humanity such as health care , psychology , nursing , management , political science , education , and communication studies ( 2 , 7 ) .
qualitative research has been conducted in the field of nursing in order to identify , describe and explain related concepts , experiences and phenomena and to develop the nursing knowledge .
, qualitative research has been performed to achieve the concepts of patient care and other main perceptions in the nursing profession .
qualitative studies provide nurses with sensitivity to the lived experiences of individuals from different nursing care aspects ( 4 , 8) .
in the case of nurses who perform qualitative research , ethical issues are raised when the nurse - patient relationship in the research area leads to some degree of therapeutic communication for the participants ( 9 ) .
thus , nurse researchers must be aware of the impact of the questioning on the participants , and in order to decrease such harmful effects on human subjects , the reflexive approach is recommended ( 10 ) . in qualitative studies researchers
are often required to clarify their role in the research process ( 11 ) . in the qr procedure
the researcher is involved in all stages of the study from defining a concept to design , interview , transcription , analysis , verification and reporting the concepts and themes .
therefore , whenever instruments are involved in qualitative research , a human being will be an integral part of the process ( 12 ) .
it is argued that humans have increasingly become the instrument of choice for naturalistic research due to certain characteristics : they are highly responsive to environmental stimuli , have the ability to interact with the situation , pull together different pieces of information at multiple levels simultaneously , and perceive situations holistically ; moreover , they are able to process findings the instant they become available , can present immediate feedback , and feel unusual responses .
nevertheless , researchers need to improve the abilities that make them appropriate human instruments and consequently , their interpersonal skills are of major importance in natural settings and study processes ( table 1 ) ( 13 , 14 ) .
the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .
ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .
in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term
confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .
the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .
when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .
it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .
informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .
the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .
clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .
informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .
many people consider it necessary to participate in research that their peers , community and/or society may benefit from .
therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .
the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .
therefore , researchers can interpret people s experiences because they are involved in human activities .
the principle of no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects .
obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .
there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .
researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .
it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .
researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .
furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .
therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .
overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 ) . in qualitative research ,
data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .
the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .
the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .
data collection needs to be as overt as possible , and findings should be recorded .
although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research , the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods , ethnography has singular characteristics .
when a researcher aims to study the culture of certain people , living amongst them is inevitable .
this method of collecting data is a subject of debate from an ethical point of view .
participants should always be aware of the information that has been obtained and is being recorded , and consent to it .
sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .
the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .
for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .
ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .
researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35 37 ) . in order to gain explicit data ,
these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .
researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .
the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .
interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .
it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40 42 ) .
the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .
ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .
in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term
confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .
the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .
when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .
it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .
informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .
the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .
clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .
informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .
many people consider it necessary to participate in research that their peers , community and/or society may benefit from .
therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .
the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .
therefore , researchers can interpret people s experiences because they are involved in human activities .
the principle of no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects . obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .
there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .
researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .
it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .
researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .
furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .
therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .
overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 )
in qualitative research , data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .
the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .
the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .
data collection needs to be as overt as possible , and findings should be recorded . although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research
, the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods ,
ethnography has singular characteristics . when a researcher aims to study the culture of certain people , living amongst them is inevitable .
this method of collecting data is a subject of debate from an ethical point of view .
participants should always be aware of the information that has been obtained and is being recorded , and consent to it .
sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .
the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .
for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .
ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .
researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35 37 ) . in order to gain explicit data , ethnographers need to know the role of instrument details .
these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .
researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .
the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .
interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .
it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40 42 ) .
it is argued that qualitative research that deals with sensitive topics in depth can pose emotional and other risks to both participants and researchers .
clear protocols for dealing with distress should be in place so that both parties involved in research can use them if necessary .
it is not usually easy to predict what topics are likely to lead to distress , and researchers should therefore receive sufficient training in predicting traumatic situations .
preventive measures for researchers who carry out sensitive qualitative studies should include official arrangements for a peer support program consisting of a list of researchers who are involved , or a constellation of researcher support activities aiming at improving psychological fitness in the form of a professional confidence building module .
other such measures include offering adequate supervision to provide opportunities for self - development and self - care , and facilitating the process of self - reflection and self - monitoring .
strategies for emotional distancing need to be considered and adopted if the research topic or participants have the potential to be emotionally challenging .
an appropriate planning should be in place before the commencement of the fieldwork , and it must be perfectly clear how the study should be conducted and what level of relationship development is necessary .
measures must also be taken so that levels of self - disclosure , objective displays of emotion during the interviews , and strategies to end the relationships are well defined and communicated .
one of the most prominent tasks of qualitative researchers is to minimize the flaws in observation and endeavor to gain truthful knowledge .
therefore , it is necessary for researchers to continuously update their investigation skills in terms of methodology and find novel techniques to better carry out studies in the field of health and sociology . as explained before ,
qualitative research is carried out in natural settings , which requires researchers to work in close collaboration with other members of the team and under direct supervision to discuss and resolve issues as they arise .
therefore , development of practical strategies and communicating them to researchers can be of great benefit and assist them in conducting more perceptive qualitative studies .
it is noteworthy that such research should be directed towards making a difference in people s lives , improving care delivery in different settings and at all levels , and providing a framework for health sciences without any ethical disturbances . as a result of the extensive body of research in the field of medical sciences ,
research ethics committees are formed to provide independent advice to participants , researchers , funders / sponsors and healthcare organizations on the extent to which research proposals comply with universally endorsed ethical standards . in the history of social and medical science , there have been a few research studies that seriously injured people , and many more in which their welfare was not sufficiently protected .
to return to the matter of privacy , the researcher should not rely solely on the informant to identify possible intrusion , but needs to work at anticipating it in advance .
confidentiality does not necessarily preclude intrusion , as anonymity by itself is not enough to protect a person s privacy or prevent disclosure of personal issues .
investigators should refrain from soliciting private information that is not closely related to the research question .
considering the aforementioned challenges , it is recommended to conduct further research in order to provide meticulous and explicit ethical protocols , guidelines and codes with respect to qualitative studies . | considering the nature of qualitative studies , the interaction between researchers and participants can be ethically challenging for the former , as they are personally involved in different stages of the study .
therefore , formulation of specific ethical guidelines in this respect seems to be essential .
the present paper aimed to discuss the necessity to develop explicit guidelines for conducting qualitative studies with regard to the researchers role . for this purpose ,
a literature review was carried out in domestic and international databases by related keywords.health care providers who carry out qualitative research have an immense responsibility .
as there is no statistical analysis in qualitative studies , the researcher has to both evaluate what he or she observes and to interpret it .
providing researchers with the necessary skills and applying stringent supervision can lead to better extraction of reliable information from qualitative studies .
this article presents a debate in order to illustrate how researchers could cover the ethical challenges of qualitative studies and provide applicable and trustworthy outcomes.researchers face ethical challenges in all stages of the study , from designing to reporting .
these include anonymity , confidentiality , informed consent , researchers potential impact on the participants and vice versa .
it seems of paramount importance that health care providers , educators and clinicians be well informed of all the different aspects of their roles when acting as qualitative researchers .
hence , these adroit roles need to be well defined , and the use of practical guidelines and protocols in all stages of qualitative studies should be encouraged . |
since the discovery of penicillin in 1928 by a scottish scientist alexander fleming followed by the release of many other earlier drugs onto the market to treat infection , the development of drug resistance in various sectors including aquaculture has been reported [ 13 ] .
the misuse and abuse of the antimicrobial drugs are among the important factors that have contributed to the rise of resistant microbes around the world .
antibiotics , which have saved millions of lives and were also known as miracle drug in the past , are no longer the ultimate way for the treatment of infections because bacteria have continued to develop multiple resistance towards many different types or classes of the drugs .
antimicrobial agents have been widely used in fish farming for either therapeutic , prophylactic , or other purposes .
the antibiotics are normally used to increase growth as well as feed efficiency in the animals . however , some of the antibiotics have been frequently used in both veterinary and human medicine such as sulfonamides , chloramphenicol , tetracycline , nitrofurans , oxytetracycline , neomycin , erythromycin , streptomycin , prefuran , and enrofloxacin .
the evolution of bacteria towards antibiotic resistance has been accelerated distinctly by selective pressure due to inappropriate and overuse of the antibiotics [ 3 , 9 ] . in the efforts to cope with this problem
, scientists have accelerated the search for alternative antimicrobial agents by screening many potential sources including medicinal plants [ 10 , 11 ] and microbes [ 12 , 13 ] .
aquaculture is an important sector in the agriculture industry and is rapidly growing to meet the world 's demands for protein source .
this sector is challenged with the diverse type of diseases and bacterial infections ; and antibiotics are an excellent tool to circumvent the problem [ 14 , 15 ] . the presence of bacteria with multiple antibiotic resistance found in food products has become a threat to public health as there is potential that the carried or acquired genes are transferred to other bacteria of clinical significance [ 1618 ] . some antibiotics which are commonly used in food - producing animals are also used in human medicine , reducing the antibiotic 's efficiency when treating infections and increasing the morbidity and mortality associated with diseases .
the resistance limits the choice of antibiotics for the disease treatment [ 4 , 16 , 19 , 20 ] .
the use of antibiotics needs to be monitored from time to time to evaluate the emergence and spread of bacterial resistance towards antimicrobial agents [ 5 , 21 ] .
there is limited data on the antibiotic resistance of bacteria in fish and other cultured organisms sampled directly from fish farms as well as the aquaculture environment .
sampling was carried out at aquaculture farms located at selected districts in sarawak , malaysian borneo , including kuching , bintulu , limbang , miri , and sampadi ( lundu ) .
three types of samples were collected which were the sediment , water , and cultured species .
cultured species refers to fish or shrimp . in kuching , bintulu , and miri ,
the cultured organisms collected were the fish while in limbang and sampadi ( lundu ) , the cultured organisms collected were shrimps .
the isolation , designation , and identification of the isolates were carried out as reported by kathleen and coworkers earlier .
ninety - four bacterial isolates from 17 different genera were assessed for their susceptibility to different antibiotics utilizing the disk diffusion method according to method described by clinical and laboratory standards institutes ( clsi ) on mueller - hinton agar ( mha ) .
the bacterial groups and the antibiotics tested are listed in table 1 . briefly , fresh bacterial culture with 0.5 mcfarland turbidity was swabbed onto the mha surface using sterile cotton buds .
commercial antimicrobial discs ( oxoid , uk ) were evenly embedded onto the inoculated agar incubated at 37c for 18 to 24 hours .
escherichia coli strain from american type culture collection ( atcc ) 25922 was used as control .
the diameter of complete inhibition zone formed around the antibiotic discs was measured to the nearest whole millimeter using standardized ruler .
the results obtained were analyzed as resistant or susceptible according to standard interpretative table by clsi and bonnet .
multiple antibiotic resistance ( mar ) index was then determined for each isolate by dividing the number of antibiotics to which an isolate is resistant with the total number of antibiotics tested .
the mar index is an indicator to identify the risk contamination that is potentially hazardous to human .
calculated value of more than 0.2 indicates that the isolates were isolated from high risk sources .
in this study , commonly used antibiotics in both veterinary and human medicine were selected for the antibiotic susceptibility testing .
the antibiotic selection also depends on the bacterial genera because different bacterial genera require different classes of antibiotics for optimal antibacterial activity .
some of the antibiotics were not tested in this study because some bacterial genera are naturally resistant to certain classes of antibiotics ; hence the antibiotics were excluded from the analysis .
the antibiotic profile of the bacterial isolates from the aquaculture fish and shrimps and their environment is shown in table 1 .
the bacterial isolates showing the top five highest percentages of resistant were towards streptomycin ( 85% , n = 20 ) , followed by ampicillin ( 56.8% , n = 74 ) , penicillin ( 47.1% , n = 51 ) , erythromycin ( 43.1% , n = 51 ) , and cephalotin ( 42.3% , n = 71 ) . the bacterial isolates showing the top five highest percentages of susceptible were towards gentamicin ( 1.1% , n = 90 ) , followed by tobramycin ( 2.2% , n = 90 ) , chloramphenicol ( 4.0% , n = 75 ) , norfloxacin ( 5% , n = 80 ) , and amikacin ( 5.6% , n = 90 ) . the other bacterial isolates and their percentage of resistant are shown in table 1 . in this study , the antibiotic resistant patterns for all isolates were also determined to monitor the spread of antibiotic resistance .
sixty - one different antibiotic resistance patterns were observed among the isolates through this study ( data not shown ) .
the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .
. isolated from water in limbang aquaculture farm was resistant towards 12 out of 19 antibiotics tested , which was the highest amount of antibiotic resistance observed in this study .
most isolates ( 53.2% , n = 94 ) possess distinctive pattern . however , there are also patterns ( 11 patterns ) shared by 2 or more bacteria isolates .
the pattern shared by most isolates ( 19 isolates , n = 94 ) is 0% resistance ( mar index equal to 0 ) .
there is at least an isolate with 0% resistance towards all antibiotics tested in all sampling locations except for bintulu .
there is difference in the resistance pattern for bacteria isolated from water , sediment , and the cultured organisms .
bacteria that were isolated from the same pond and same source of origin ( sediment , water , or cultured organisms ) possess different antibiotic patterns .
the antibiotic resistant patterns of the bacterial genera are more influenced by the location from where the isolates were isolated rather than their genus .
this analysis has been used to group the different sources from where the bacteria were recovered using the frequency of antibiotics resistance .
isolates with mar < 0.2 were determined as isolates recovered from low risk sources of contamination while isolates with mar > 0.2 were from high risk sources . in this study ,
analysis on overall isolates regardless of sampling location revealed that 63.1% ( n = 94 ) isolates belong to group mar < 0.2 while 36.9% ( n = 94 ) isolates belong to group mar > 0.2 .
bintulu ( btl ) has the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources while sampadi ( spd ) has the lowest percentage ( 11.7% , n = 17 ) .
assessment of the antibiotic resistance among aquaculture bacteria against antimicrobial agents is important for update on the bacterial antibiotic resistance patterns .
it is part of a surveillance system aiming at monitoring emerging antibiotic resistant bacteria and their widespread .
isolation of antibiotic resistant bacteria from aquaculture products and aquaculture environment indicates the health risk associated with the aquaculture .
there had been reports on detection of antibiotic resistance genes in bacteria isolated from aquaculture products that can be transferred to human microbiota .
this matter is becoming critical if the resistance genes are transferrable to human pathogens . providing effective treatment towards this infection becomes a problem to the medical practitioners as the choice of antibiotics is limited .
thus , any source of antibiotic resistant bacteria must be carefully monitored . in antibiotic resistance analysis
, the history of antibiotic application in particular area is reflected by the percentage of bacterial resistance to antibiotics .
the frequency of antibiotics usage is related to the level of resistance among bacteria [ 2 , 29 ] . in this present study ,
similarly , lim and kasing and hatha et al . in their respective studies observed that almost none of the bacteria tested were resistant to gentamicin and chloramphenicol .
low frequency of antibiotic resistant bacteria may indicate the less activity associated with the contamination of antibiotics in the area .
the use of chloramphenicol in aquaculture has been banned in certain countries including malaysia , korea , and japan since 1983 .
this is because of the adverse effect of chloramphenicol in humans , even at very low dosage , which can cause other side effects like severe or fatal blood problems .
the problems associated with blood are like anemia and grey syndrome , a syndrome of cyanosis and cardiovascular collapse , which occurs particularly in newborn babies .
this poses risks to the workers handling the products containing this antibiotic [ 5 , 31 ] .
banning of antibiotics has aided in reducing the number of antibiotic resistant bacteria in an environment .
reported the significant reduction in the frequency of vancomycin - resistant enterococci from broiler after the banning of vancomycin in denmark in 1995 .
high percentage of streptomycin , ampicillin , and penicillin g resistance was observed in this study .
similarly , high ampicillin and streptomycin resistance were also observed by zhang et al . in their study on antibiotic resistance detection in e. coli strains isolated from two different aquaculture systems in south china .
also recorded high resistance of ampicillin . in another study , identified isolates from mangrove soil in malaysia were 100% resistant towards ampicillin and penicillin while 77.8% of the isolates were resistant towards streptomycin .
a study carried out by akinbowale et al . recorded 54.8% ampicillin resistance of aquaculture bacteria in australia .
in contrast with the results in this study , low resistance of streptomycin ( 21.2% ) and ampicillin ( 6.1% ) was observed in a study on 33 marine bacteria isolates by you et al . .
in this study , farmers in the aquaculture farms where the sampling was carried out stated that there was no history of utilization of antibiotics in their farms . despite the absence of antibiotics as medicines or in feeds ,
high resistance was observed to commonly used antibiotics such as streptomycin , ampicillin , and penicillin .
high resistance of ampicillin and streptomycin in this study and other researches was not surprising as these antibiotics were among the first antibiotics introduced since the discovery of penicillin .
although antibiotic usage in the studied farms has been stopped decades ago , antibiotic contamination is still possible as there may still be residues of antibiotics left in the environment .
bacteria isolated from the sediment of the aquaculture pond may have acquired antibiotic resistance characteristics through unconsumed foods and the cultured organism 's faeces that contain the remaining antibiotics [ 3941 ] .
the unconsumed foods and faeces will be deposited in the sediment and the composition of sediment microbiota will be altered due to selective pressure .
fish feeds were a possible reservoir for antibiotic resistant genes in the farm sediments . in this present study ,
bintulu ( btl ) recorded the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources ( mar > 0.2 ) while sampadi ( spd ) aquaculture farm recorded the least number ( 11.7% , n = 17 ) of mar index > 0.2 isolates .
the aquaculture farm is located at an area that has many agriculture activities ( e.g. , pig farming , duck farming , and dragon fruit cultivation ) surrounding it .
there is the possibility that antibiotics from the animal feeds or medications were absorbed into the sediment causing bacterial selection in the nearby environment .
multiple antibiotic resistant bacteria might have travelled through water from these agriculture activities to the aquaculture ponds .
buschmann et al . in their study suggested that antibiotic resistant bacteria in mariculture farm may be transported by water current which flows from surrounding farms that utilize antibiotics excessively .
antibiotic resistance pattern may vary depending on the geographical locations and selective pressure [ 43 , 44 ] and these patterns change rapidly from time to time .
the different patterns exhibited by different strains or species suggest how complex is the understanding of the antibiotics resistance in the study area . in this study ,
the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .
awareness on antibiotic resistance threat should be instilled in the community regardless of age as precaution and prevention step against dissemination of antibiotic resistant bacteria .
many surveillance programs had also been introduced to monitor the emergence and spread of antibiotic resistant bacteria .
it has also been suggested by son et al . that plasmid screening should be considered as an additional procedure in the monitoring programs to trace antibiotic resistance dissemination .
alternatives to treatment using antibiotics like probiotics , vaccines , and antimicrobials from plants should be also considered .
however , most of the alternatives could not really effectively replace antibiotics , so they act as additional preventive measures rather than alternatives .
the mar indexing has revealed that 63.1% of the isolates came from low antibiotic usage area .
although antibiotic resistance in aquaculture in the malaysian borneo is still in its infancy , the need for continuous monitoring of the antibiotic resistance patterns should not be overlooked and the community should be educated on the awareness of antibiotic resistance and its implication on human health and environment . | the administration of antimicrobials in aquaculture provides a selective pressure creating a reservoir of multiple resistant bacteria in the cultured fish and shrimps as well as the aquaculture environment .
the objective of this study was to determine the extent of antibiotic resistance in aquaculture products and aquaculture 's surrounding environment in sarawak , malaysian borneo .
ninety - four identified bacterial isolates constituted of 17 genera were isolated from sediment , water , and cultured organisms ( fish and shrimp ) in selected aquaculture farms .
these isolates were tested for their antibiotic resistance against 22 antibiotics from several groups using the disk diffusion method .
the results show that the highest resistance was observed towards streptomycin ( 85% , n = 20 ) , while the lowest resistance was towards gentamicin ( 1.1% , n = 90 ) .
the multiple antibiotic resistant ( mar ) index of the isolates tested ranged between 0 and 0.63 .
it was suggested that isolates with mar index > 0.2 were recovered from sources with high risk of antibiotic resistant contamination .
this study revealed low level of antibiotic resistance in the aquaculture bacterial isolates except for streptomycin and ampicillin ( > 50% resistance , n = 94 ) which have been used in the aquaculture industry for several decades .
antibiotic resistant patterns should be continuously monitored to predict the emergence and widespread of mar .
effective action is needed to keep the new resistance from further developing and spreading . |
with an incidence rate of 0.92% , maternal puerperal lower extremity nerve injuries are rare .
lateral femoral cutaneous neuropathy ( meralgia paraesthetica ) is the most common , followed by femoral neuropathy .
nerve injuries are more likely to occur in nulliparae , in cases of prolonged stages of labour and assisted vaginal deliveries .
the presented case involved a 32-year - old woman with bilateral heel numbness due to bilateral neuropathy of the medial calcaneal nerve due to external compression during vaginal delivery .
a 32-year - old woman presented to our neurology outpatient clinic with tingling and numbness in both heels .
she continuously complained about these sensations ever since she had given vaginal birth to her first child 3 months earlier in a hospital .
spinal analgesia ( lumbar level , bupivacaine / sufentanyl bolus , followed by a continuous administration of 8 ml / h ) was used during labour .
the first stage of labour was prolonged , and because of fetal compromise during the expulsion phase , a vacuum delivery system ( palmpump kiwi ) , together with episiotomy , was used for fetal extraction .
after delivery , the epidural analgesia was continued during the closure of the episiotomy with sutures .
the patient did not use any other medication and had no history of neurological disorders or risk factors for neuropathy ( diabetes , hypothyroidism or hereditary liability to nerve pressure palsy ) . on examination , she had hypoaesthesia in the area of both heels .
the strength of both the leg and foot muscles was normal , and so were the deep tendon reflexes .
, a near - nerve needle conduction study of the medial calcaneal nerve was not performed .
a diagnosis of bilateral neuropathy of the medial calcaneal nerve , most likely due to longstanding pressure on both heels with epidural analgesia as a predisposing factor , was made .
three months after presentation ( 6 months after delivery ) , her numbness had diminished , but had not completely disappeared .
bilateral neuropathy of the medial calcaneal nerve due to external compression during delivery using epidural analgesia is very rare .
only 1 patient with numb heels has been reported in a large study , describing 2,615 women who received epidural anaesthesia during delivery .
however , no details about delivery or analgesia in that patient were mentioned . in epidural analgesia during delivery , a motor block is unwanted , and thus voluntary muscle movements are possible , which makes longstanding external compression of nerves uncommon . in our patient ,
prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy probably masked signs of external compression on both heels .
the medial calcaneal nerve arises from the tibial nerve at the medial side of the ankle , perforates the laciniate ligament , travels downwards , passing the bony projection below on the inner side of the ankle , and supplies the skin over the medial aspect of the heel .
patients with risk factors , such as diabetes , are at a greater risk of developing neuropathy during epidural anaesthesia .
however , also without any risk factors , the clinician should be aware of the absence of the normal reaction to longstanding pressure on a nerve during epidural analgesia .
preventative measures like soft gel pads or intermittent posture changes could possibly prevent neuropathy of the medial calcaneal nerve in long - lasting epidural analgesia .
bilateral medial calcaneal nerve neuropathy as a result of external compression is a rare complication of epidural obstetric analgesia .
| we describe the case of a 32-year - old woman who developed bilateral heel numbness after obstetric epidural analgesia .
we diagnosed her with bilateral neuropathy of the medial calcaneal nerve , most likely due to longstanding pressure on both heels .
risk factors for the development of this neuropathy were prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy .
padded footrests decrease pressure and can possibly prevent this neuropathy . |
haemotropic mycoplasmas ( haemoplasmas ) are bacterial organisms without cell walls that attach to and grow on the surface of red blood cells .
three feline haemoplasma species are described : mycoplasma haemofelis ( mhf ) , candidatus mycoplasma haemominutum ( cmhm ) , and candidatus mycoplasma turicensis ( cmt ) .
these feline haemoplasmas are the causative agents of infectious anemia in cats and in several mammalian species .
there is also potential interspecies transmission of some of these agents as recorded from cats to immunocompromised dogs .
their zoonotic potential has recently been substantiated by the molecular identification of a feline haemoplasma isolate ( mhf ) in an hiv - positive immunocompromised human patient .
the three feline haemoplasma species have different pathogenicities , mhf often resulting in haemolysis and severe anaemia in contrast to cmhm and cmt which are less pathogenic .
although several studies worldwide have reported on the epidemiology of feline hemoplasmosis in sick or healthy client - owned pet cats with prevalences ranging from 7.2% to 45.8% [ 519 ] and few studies have focused on stray cat ( with prevalences from 11.5% to 60% [ 2024 ] ) , there have been no studies investigating stray cats in northern italy .
information on regional prevalence of haemoplasmas could be used to limit the spread of diseases in feline populations and for predicting the likelihood of infection in cats presented with anemia .
the vector potential of ctenocephalides felis has been demonstrated in experimental mhf infections , and stray cats may play a role in multiplying the organism in fleas that then infest pet cats and dogs and human beings .
direct transmission , by aggressive interaction of cats , or interspecies transmission might play a role in the epidemiology of these organisms .
in addition , haemoplasmas can be directly transmitted through intravenous infusion of infected blood [ 4 , 26 ] and have been shown to survive for up to 1 week in stored blood products .
as administration of fresh or stored whole blood becoming more common in feline medicine , the knowledge of the regional prevalence of blood transmitted pathogens in owned and stray cats that share the same environment and parasites is increasingly important .
this would provide useful information for evaluating the risks of transmission of blood - borne infections from potential blood donors and in the development of optimal screening protocols in blood donors .
the aim of this study was to evaluate , using a conventional pcr assay , the prevalence of mhf and cmhm infections in stray cats from colonies in milan and to identify possible risk factors for these infections .
during a 2-year collection period ( january 2008 to january 2010 ) , blood samples were taken from 260 stray cats from urban colonies in milan ( northern italy ) , under a trap - neuter - release ( tnr ) program approved by the local authority of the city council .
age ( estimated based on dentition , animals < 6 months of age were considered juvenile , whereas all others were considered adult ) , gender ( male or female ) , origin ( provenance area of colonies ) , and body condition score ( bcs 46 , indicating normal weight , 13 underweight ) were recorded together with data obtained from physical examination of the cats ( healthy or unhealthy ) .
unhealthy cats were defined as cats with one or more of the following clinical abnormalities : lymph node enlargement , pale mucous membranes , stomatitis , or signs of ocular and respiratory infections . the seasonal analysis based on the date of sample collection
was grouped as follows : winter ( january , february , and march ) , spring ( april , may , and june ) , summer ( july , august , and september ) , autumn ( october , november , and december ) .
the cats were not systematically examined for the presence of ticks or fleas and so rates of ectoparasitism were not recorded .
blood samples were collected aseptically from the jugular vein while cats were anaesthetized for neutering and placed in edta - treated tubes and in serum separator tubes . within 24 h of sample collection , a complete blood count ( cbc )
was performed on whole blood using an advia 120 system ( siemens healthcare diagnostics , milan , italy ) .
cats were categorized in terms of presence or absence of anaemia ( hct < 24% ) , leukopenia ( wbcs count < 10,570/l ) , leukocytosis ( wbc > 14,390/l ) , and thrombocytopenia ( plt < 200,670/l ) .
following separation , serum samples were tested for antibodies to fiv ( relative to gp40 and p24 fiv antigens ) and for felv p27 antigen with a commercial enzyme - linked immunosorbent assay ( elisa ) kit ( snap felv / fiv combo plus test ; idexx laboratories , hoofddorp , the netherlands ) .
toxoplasma gondii sera igg antibodies were detected using indirect fluorescent antibody tests ( ifat ) performed with a commercial kit ( fuller - laboratories , fullerton , ca , usa ) .
titres 1 : 64 were considered seroreactive and , therefore , indicative of t. gondii exposure .
the results of these serological tests have been already published and were reanalyzed with the present results .
conventional pcr was performed on blood samples to amplify dna associated with haemoplasmas ( mhf and cmhm ) .
the reaction mixture included 2 l of template dna , 0.25 mm dntps , 0.4 mm of each primer , 1x reaction buffer , and 2.5 u tap dna polymerase ( gotaq dna polymerase , promega , madison , wi , usa ) .
primers were used that target part of the 16s rrna gene , producing a 170 base pair ( bp ) product from mhf and 193 bp amplicon from mchm ( forward primer , 5-acg aaa gtc tga tgg agc aat a-3 and reverse primer 5-acg ccc aat aaa tcc gra taa t-3 ) [ 6 , 31 ] .
pcr products were resolved using 2% agarose gels and fragment size was estimated using a dna molecular weight marker ( 50 bp dna ladder ; promega madison , wi , usa ) .
control reactions were done in the absence of template dna to rule out contaminations during pcr .
association between the three groups of haemoplasma status ( haemoplasma positive , mhf positive only , and cmhm positive only ) and categorical variables ( age , gender , colony of origin , bcs , season of sampling , health status , presence or absence of selected clinical and cbc abnormalities , felv / fiv status , and t. gondii test results ) were analysed by univariate analysis using the chi - squared test ( cell frequencies of > 5 ) or fisher 's exact test ( cell frequencies of 5 ) .
any parameters statistically linked to positive pcr results were used in a logistic regression model to test for independent risk factors associated with infection .
descriptive statistics ( including minimum , maximum , mean , median , and standard deviation ( sd ) ) were obtained for the continuous variables rbcs count , hct , hb , wbc count , and plt count values .
distribution of the data for normality was assessed with kolmogorov smirnov test , and a t - test or a mann - whitney u test , respectively , was used to test the differences between the feline haemoplasma positive and negative cats depending on whether data was normally distributed or not .
associations were considered statistically significant when p < 0.05 . both the p value and odds ratio ( or ) with 95% confidence interval ( ci ) are reported .
haemoplasmic dna was detected in 33.1% ( 86/260 , 95% ci 26.540.9% ) of blood samples . of the positive samples , 28 cats ( 10.8% , 95% ci 7.215.6% )
were infected with mhf and 58 cats ( 22.3% , 95% ci 16.928.8% ) were infected with cmhm .
characteristics of the population and association between risk factors and haemoplasma pcr status ( negative or positive ) are reported in table 1 and between mhf alone and cmhm alone positive results in table 2 .
none of the risk factors were associated with the pcr results for haemoplasmas ( tables 1 and 2 ) , with the exception of negative associations at univariate and multivariate analysis between winter season of sampling and haemoplasma positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.140.61 , p = 0.001 ) and cmhm positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.120.70 , p = 0.01 ) .
no significant associations were found with cbc abnormalities and haemoplasma pcr - positive results ( figure 1 ) or in the number of anaemic and nonanaemic cats using a t - test .
we present the first study investigating the prevalence of haemoplasmas in urban stray colony cats from the city of milan in northern italy .
the prevalence of haemoplasma infection in our sample showed a similar pattern to that reported worldwide in client - owned [ 57 , 915 , 1719 ] and stray cats [ 1923 ] .
cmhm infection is reported to be the most common infection , ranging from 8% in arizona to 41.6% in portugal ; mhf infection was less common ranging from 0.5% in switzerland to 12.8% in portugal and dual infection was absent or rare ( no more than 5.4% as found in korea ) .
the prevalence of haemoplasmas in stray cats in our study ( 33.1% ) was higher than that reported in other studies on stray cats performed worldwide ( table 4 ) .
differences in prevalence among countries can be explained by geographical variation , such as climate , vector distribution , and the cat population surveyed .
moreover , direct comparisons of prevalence results are of limited value because of the characteristics of sample of cat populations investigated ( number of cats included in the study , healthy versus sick cats ) , inclusion criteria , diagnostic techniques ( molecular tools versus microscopical detection ) , different statistical methods , stage of infection ( acute versus chronic ) , or a combination of all them , resulting in differences between studies .
stray cats in this study had higher rates of infections than those reported for pet cats in northern italy , in which 18.9% of pcr tested cats were reported to be haemoplasma positive .
a high incidence of haemoplasmas in stray cats is not surprising as outdoor access is a recognized risk factor for infection .
for example , in a study on haemoplasma in client - owned cats from barcelona ( spain ) , outdoor access was found to be a risk factor for infection ( or = 3.8 ) .
additionally , in a study involving feline blood donors from the usa , the prevalence of haemoplasmas was 19.7% in domestic cats with outdoor access and only 3.6% in domestic cats not allowed outdoors .
free - ranging cats may have more exposure to bloodsucking arthropods and exhibit a higher fighting activity than owned indoor cats ; thus they might experience a higher infection risk for haemoplasmas . in our study
there was a negative statistical association between sampling in the winter season and haemoplasma pcr - positive status and cmhm positive status .
this may be due to reduced outdoor activity of fleas in winter seasons , although this hypothesis is purely speculative .
there were no statistically significant differences or any relationship between the presence of haemoplasma infection and anaemia status or hct levels between positive and negative haemoplasma group .
this finding was somewhat surprising and was in agreement with studies undertaken in client - owned cats in switzerland and in italy , which also found no association between haemoplasma infection and anaemia or hct variations .
in addition , other studies have failed to demonstrate a significant difference in prevalence rates of haemoplasma between healthy and anaemic cats [ 10 , 32 ] .
these results could be explained by the higher prevalence of cmhm ( a less pathogenic species than mhf ) in this and previous studies or by the fact that the stage of infection is not known in our cats with a positive pcr result , since chronically infected cats that recover from acute illness may be asymptomatic .
another explanation for the lack of association between infection status and the presence of anaemia in the present study could be that the hct was known only for 150 of the 260 cats tested .
epidemiological data on haemoplasma are particularly important in areas where feline blood donor programmes are active , as in milan where a donor programme has been running since 2010 at university veterinary transfusion unit ( rev , reparto di medicina emotrasfusionale veterinaria ) .
feline haemoplasmas can be directly transmitted by intravenous infusion of fresh edta - anticoagulated blood , heparinized blood , and by infusion of blood stored in cpda-1 solution for 1 h ( mhf ) and 1 week ( cmhm ) .
cats do not reliably eliminate the organism following infection and most infections with cmhm are chronic and asymptomatic .
a significant number of asymptomatic cats are positive for haemoplasma infection and may play a role , along with infected cats , in the maintenance of haemoplasma infection within a population .
all these characteristics of feline haemotropic mycoplasma infection need to be considered when choosing potential blood donors . the limitations of this study include the lack of information on candidatus mycoplasma turicensis in our study population .
lastly , statistical analysis was limited in some groups because of the sample size and so some conclusions or associations may be affected by type i errors ; that is , no difference between haemoplasma positive and negative groups was observed due to insufficient sample size ; for example , no association was recorded between anaemia and a positive haemoplasma result because of the low number of mhf positive cats ( 28/260 ) .
regardless of these limitations , we believe that this study provides new and useful information on feline haemoplasma infections in stray cats in italy .
from this study it can be concluded that feline haemotropic mycoplasma mhf and cmhm are common in the stray cat population of milan .
indeed , pet cats with outdoor access in this region should be regularly monitored and treated for ectoparasites to minimize health risks .
importantly , feline blood donors in this area should undergo pcr screening for these infections before donations and preferably donors should be drawn from exclusively indoor cats that receive regular flea control . | this study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in milan , northern italy .
blood samples from 260 stray cats were evaluated , with conventional pcr , for the presence of dna associated with mycoplasma haemofelis ( mhf ) and candidatus mycoplasma haemominutum ( cmhm ) .
odd ratios ( or ) were calculated to identify risk factors for haemoplasma infections .
pcr was positive in 86 out of 260 subjects ( 33.1% ) , with a prevalence of 10.8% ( 28/260 cats ) for mhf and 22.3% ( 58/260 cats ) for cmhm .
no coinfections were registered .
there were significant associations between infections and season of sampling , that is , a negative association between winter sampling and a haemoplasma positive status ( or = 0.29 , p = 0.001 ) , or cmhm positive status ( or = 0.29 , p = 0.01 ) .
haemoplasma infections are common in stray cats in milan .
thus , domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition . moreover , as these infections are transmitted via blood , feline blood donors from this area should be screened by pcr and preferably be drawn from a population of indoor cats regularly treated for fleas . |
in a case - control study , 140 patients including two equal groups of traumatic due to traffic injuries and non - traumatic patients were evaluated . using the persian version of the conners adult adhd rating scale ( caars ) self - report ( screening version )
the study was performed in two university hospitals in tabriz , iran during a 13- month period - august 2008 to september 2009 .
the sample size was calculated using sas software version 9.1 based on proportion comparison tests to ensure 80% statistical power and type 1 error less than 0.05 .
the required data for sample size calculation were obtained from a previous study with conservative rounding of figures to come up with possible dissimilarities ( 11 ) .
inclusion criteria were as follows : physical trauma , motor vehicle traumas ( motorcycle and car ) , being the driver / rider when injured , age range of 18 to 65 years , and ability to complete the conner 's questionnaire .
the exclusion criteria were as follows : brain trauma resulting in decreased consciousness , non - driver / rider victims of motor vehicle accidents , psychiatric comorbidity , factors affecting the attention and concentration such as drug and alcohol use while driving , and illiteracy .
patients with physical trauma ( case group ) and those patients admitted due to non - trauma reasons without previous history of trauma hospitalization were recruited during the study period .
adhd diagnosis was performed using caars , and the psychiatric comorbidities were assessed according to dsm - iv - tr using scid , and those with a psychiatric comorbidity were excluded .
all of the patients were informed about the design and purpose of the study , and a written consent form was obtained from each volunteer patient .
the assessed factors in both groups included age , sex , educational level , job , accident history , daily driving amount , weekly driving amount , adhd index , a subscale ( attention deficit symptoms ) , b subscale ( hyperactivity - impulsivity symptoms ) , and c subscale ( total adhd symptoms ) .
in addition , type of vehicle , light intensity at accident time , and the location of the accident were recorded in the case group .
the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics
is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .
this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,
the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches of 0.83 . in case
the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .
the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) .
comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .
in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant .
the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics
is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .
this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,
the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches of 0.83 . in case
the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .
the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) .
comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .
in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant .
no significant statistical difference was found between case and control groups on age , gender , educational level and occupation . with respect to type of vehicle , 25 cases had a car accident ( 35.7% ) and 45 had a motorcycle accident ( 64.3% ) .
the light condition was bright in fifty four ( 77.1% ) , semi - bright in eight ( 11.4% ) and dark in eight cases ( 11.4% ) .
accident location in the case group was within the town in 30 cases ( 42.9% ) , side road in 20 ( 28.6% ) , main road in 13 ( 18.6% ) , and highway in 7 cases ( 10% ) .
accordingly , the percent of subjects with accident history was significantly higher in case group ( p= 0.24 ) . furthermore , the mean driving hours in case group was significantly more compared to controls .
no significant difference was observed between the two groups with respect to other factors .
comparison of measured variables in two groups *
the data are shown as mean standard deviation and frequency ( percent ) . in self - report questionnaire of conner 's adult adhd rating scale ,
subscales ' scores higher than 70 were considered positive.adhd index was positive in 3 ( 4.3% ) subjects in both case and control groups .
adhd total symptoms were positive in 7 ( 10% ) and 10 ( 14.3% ) cases in both case and control groups respectively p= 0.438 , or= 1.5 ( 0.5- 4.2 ) , ci : 95% . in a subscale
, inattentive symptoms were observed in 5 ( 7.1% ) and 6 ( 8.6% ) participants in case and control groups respectively p= 0.753 , or=1.2 ( 0.4- 4.2 ) , ci : 95% . in b subscale ( hyperactive - impulsive symptoms were observed in 7 ( 10% ) and 6 ( 8.6% ) participants in case and control groups respectively pv= 0.771 , or= 0.8 ( 0.3- 2.7 ) , ci : 95% .
in this study , the frequency of adult adhd was compared between traumatic adult patients ( due to traffic injuries ) and a control group . accordingly , adhd index was positive 4.3% in both case and control groups , adhd symptoms total were positive 10% , 14.3% in case and control groups respectively .
a subscale ( inattentive symptoms ) was 7.1% , 8.6% in case and control groups respectively ; and b subscale ( hyperactive / impulsive symptoms ) was 10% , 8.6% in case and control groups respectively .
hechtman et al ( 1984 ) first described the higher rate of traffic injuries in children with adhd ( 15 ) .
the next studies performed by barkley et al . , and jerome et al . , accredited the initial report ( 1619 ) .
the association of adhd and traffic injuries is so important that driving center of canada was listed the uncontrolled adhd as an item for giving certificate to the drivers .
the higher frequency of each type of accident in adhd patients was compared to healthy subjects ( odds ratio 1.7 ) and the car accidents are a subgroup of these events ( 20 ) . despite of numerous studies about the association of adhd and car accidents ,
ludolph et al . , reported twofold higher rate of car accidents in these patients compared with the normal population ( 21 ) . in barkley
studies and cox et al ( 2000 , 2006 ) , this ratio has been different from two to six ( 16 , 2224 ) .
sobanski et al . , evaluated 27 patients with adult adhd and 27 age and sex matched subjects as a control group .
the frequency of car accidents in first group was 2.6 fold higher than the control group patients , the other related factors in this field were also ( 25 ) . besides the higher rate of car accidents in adhd higher . among these
, we can point to the higher frequency of losing driving certificate , driving after alcohol and substance use , exceeding the speed limit , and other abnormal traffic behaviors , car accidents trauma , high rate of traffic punishments and rate of suspension of driving certificate and higher rate of being responsible for the accidents ( 2630 ) .
besides the epidemiological studies , there are numerous clinical trials showing the strong association between adhd and car accidents .
cox et al . , in two different studies showed that the frequency of car accidents in adults with adhd receiving methylphenidate was lower than the group without treatment ( 23 , 31 ) .
barkley et al . , in a similar study demonstrated that treatment of adult adhd may result in decreased rate of car accidents and related injuries ( 32 ) .
in a study on selegiline in comparison with methylphenidate in adhd children and adolescents showed that selegiline is also effective and well tolerated for adhd ( 33 ) .
, in a study on modafinil as a treatment for adhd in children and adolescents found that this drug significantly improves symptoms of adhd and is well tolerated ( 34 ) . on the other hand , it is shown that high risk behaviors of adhd patients during driving would persist despite controlling other factors .
in other words , high risk behaviors during driving are an independent factor associated with adhd ( 25 , 30 ) .
different mechanisms are suggested including attention deficit , inability to control impulsivity , early fatigue , anger and aggressiveness ( 25 , 3537 ) . according to the mentioned studies
, it may be concluded that there is a significant association between adult adhd and car accidents .
it should be mentioned that in all studies , the patients with adhd and controls were either under investigation for driving - models or their driving situation while we compared the frequency of adhd in subjects with and without car accidents .
the current study is the first report from tabriz- iran on the association of adhd to traffic injuries .
the lack of difference between two groups regarding frequency of adhd may be due to the fact that the two groups were matched for job and educational level in this study .
it was previously reported that adult adhd is one of the main causes of educational and job problems ( 30 ) .
hence , matching of two groups for these variables may be a kind of selection bias . also driving skill level
unfortunately , we could not evaluate this condition in our sample . according to available reports ,
the low driving skill level and lack of sufficient information about driving rules ( especially in young person ) is one of the main causes for car accidents in our country ( 38 , 39 ) . in other words ,
high frequency of car accidents in our society is mainly due to lack of sufficient training of young drivers and lack of sufficient respect to the driving rules .
hence , it is very difficult to determine the role of adhd in this field .
high frequency of motorcycle accidents in this study ( 64.3% ) is a congruent factor because of higher rule destruction in this group .
difference in driving duration is another major factor and the mean daily driving hours were significantly higher in patients with car accidents .
on the other hand , it has previously been shown that the mortality or major injuries and severity of injury in adhd patients experiencing car accidents is more than healthy subjects ( 26 , 27 ) . despite the mentioned restrictions ,
accordingly , it seems that most car accidents involving driver injury are not related to adhd .
this study showed that the odds of adult adhd was not significantly higher in drivers with traffic injuries compared with the matched controls . therefore ,
larger scale studies stratified for different types of traffic accidents are recommended to further investigate such an association
most of the traffic injuries cause death but these case were not included in our study . | objectivenowadays , it is well known that the attention - deficit / hyperactivity disorder ( adhd ) is not confined to children and adolescents .
recent data showed that a considerable portion of the general adult population may be affected by adhd . on the other hand ,
the impact of adhd on driving performance , a major area of adult life , has gained enthusiasm .
more recent studies revealed an association between adult adhd and undesirable driving problems .
this study was performed to determine the association between presence of adult adhd and traffic injuries.methodsin this case - control study , in a 13 month period , 140 subjects ( 70 drivers / riders injured in traffic accidents , and 70 age- and sex - matched non traumatic controls ) were selected to participate in the study and were placed in two groups .
subjects with psychiatric comorbidities were excluded .
the conners adult adhd rating scale ( caars ) self - report ( screening version ) was used for screening adult adhd in both groups .
finally , the occurrence of this condition was compared between the case and control groups.resultssixty- nine males and one female were recruited in each group with a mean age of 29.319.32 ( 18 - 61 ) years in the case and 29.039.07 ( range : 18 - 60 ) years in the control groups ( p > 0.05 ) .
previous history of traffic accident was significantly higher in the case group ( 15.7% vs. 4.3% , p=0.024 ) .
the mean driving time in a day was also significantly higher in the case group . in the case group ,
the scores of caars ( the adhd index , adhd symptoms total , inattentive subscale and hyperactive / impulsive subscale ) were positive ( higher than 70 ) in 4.3 , 10 , 7.1 and 10 percent of patients respectively . among the controls ,
the corresponding proportions were 4.3 , 14.3 , 8.6 and 8.6 percent respectively .
the two groups were comparable for the mentioned rates.conclusionthe results of this study revealed no significant association between adhd and traffic injuries in tabriz- iran . |
crh is a 41-amino - acid peptide hormone synthesized in the paraventricular nucleus of the hypothalamus .
its function in the central nervous system is to stimulate the hypothalamic - pituitary - adrenal ( hpa ) axis as part of the stress response .
crh is also produced in peripheral tissues including the placenta of humans and hominine primates [ 13 ] .
placental crh secretion results in an exponential rise of crh concentration in maternal plasma during the third trimester of gestation .
the rate of the increase is related to gestational length , since the rise of crh level is accelerated in pregnancies ending with preterm birth , while the increase is retarded in pregnancies that continue after term .
because of this relationship , it is believed that the regulation of placental crh production is linked to the mechanism that determines the length of pregnancy and triggers labour and delivery .
the mechanism regulating the exponential increase in placental crh expression remains unclear although positive feedback by glucocorticoids and increasing numbers of syncytial cell nuclei are suggested explanations [ 5 , 6 ] .
spontaneous and agonist - induced syncytium formation by cytotrophoblasts is associated with crh expression with camp being a strong stimulant of both syncytial differentiation and crh gene activity .
molecular studies using crh promoter - reporter constructs indicated that transcription factor complexes bound to a consensus cyclic - amp response element ( cre ) at 224 bp upstream of the major transcription initiation site mediated the camp - stimulation , although a nonconsensus second promoter site was also implicated in the cyclic nucleotide response [ 811 ] .
it has been suggested that these molecular interactions are involved in the gestational age dependent control of crh gene activity .
epigenetic chromatin modifications define cell - specific gene expression potential and alter gene expression patterns during cell differentiation and development .
methylation of cytosines at cpg dinucleotides in dna is a well - characterized epigenetic chromatin modification generally associated with closed chromatin structure and gene repression .
furthermore , methylation of cpg sites within particular transcription factor binding sequences may modify transcription factor binding affinity and alter regulatory changes in gene expression [ 1417 ] .
the human crh proximal promoter contains 9 cpg dinucleotides with one located within the methylation sensitive cre sequence .
in addition , the promoter is within 1000 bp distance from an intragenic cpg island , which corresponds to a cpg island shore
region , the methylation of which is related to tissue specific gene expression . therefore , in the present investigation we have explored the possibility that methylation of the promoter contributes to the control of crh expression in trophoblast cells .
we used the bewo choriocarcinoma cell line in the experiments , which is a well - characterized trophoblast model exhibiting dynamic dna methylation as well as ability to syncytialise and increase crh expression when stimulated with camp [ 1921 ] .
the bewo human choriocarcinoma - derived cell line was obtained from the american type culture collection ( manassas , va , usa ) ( atcc # ccl-98 ) .
the culture medium was dmem / f12 ( with hepes and l - glutamine , without phenol red ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 1x antibiotic - antimycotic ( gibco / life technologies , mulgrave , vic , australia ) .
cells were cultured at 37c in a humidified atmosphere of 5% co2 in air . at approximately 80% confluence cells
were detached with trypsin / edta solution ( gibco ) , washed , counted , and transferred into six - well plates at a density of 0.8 10 cells / well .
cultures reaching 50% confluence were incubated with fresh medium with or without 8-br - camp ( 8-bromoadenosine-3,5-cyclic monophosphate , 2.5 10 mol l ) and/or 5-aza - dc ( 5-aza-2-deoxycytidine , 5
10mol l ) ( sigma - aldrich , sydney , nsw , australia ) followed by harvesting for rna and dna isolation .
drug concentrations were optimised in previous studies and showed no toxic effects in bewo cells at the concentrations and exposure times employed [ 19 , 22 , 23 ] .
rna was extracted from cells using the rneasy mini kit ( qiagen , chadstone centre , vic , australia ) according to the manufacturer 's protocol .
rna was eluted from the rneasy spin columns with 30 l of rnase - free water and quantified using a nanodrop 1000 spectrophotometer ( thermo fisher scientific australia , scoresby , vic , australia ) .
contaminating dna was removed by dnase treatment using the turbo dna - free kit ( ambion / life technologies ) following the routine protocol .
the total reaction volume was 20 l including 2 l of 10x dnase buffer , 1 l of dnase , and up to 2 g of rna .
rna integrity in all samples was assessed by agarose gel electrophoresis . prior to reverse transcription , the rna samples were spiked with 5 10 copies of alien rna transcript ( supplied with the alien qrt - pcr inhibitor alert kit , stratagene / integrated sciences , chatswood , nsw , australia ) per microgram rna .
the alien rna transcript served as a reference rna of equal abundance in all samples and pcr runs .
rna was reverse transcribed using the superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) with random hexamer primers .
real - time pcr was performed using an applied biosystems 7500 real - time pcr system with reagents supplied by the manufacturer ( applied biosystems / life technologies ) .
the amplification reaction contained template cdna from 20 ng of reverse - transcribed rna , 6 10 moles l forward and 3 10 moles l reverse primer , sybr green master mix , and milliq water to a total volume of 25 l .
the crh cdna primers were designed and optimised by sehringer et al . and are listed in table 1 .
primer sequences for amplifying alien cdna are proprietary and were used according to the manufacturer 's instructions ( stratagen / integrated sciences ) .
the temperature sequence was 50c for 2 min , 95c for 10 min , 40 cycles of 95c for 15 s , and 60c for 1 min , followed by melting curve analysis .
no - template control and no - reverse transcriptase controls for all samples were included to detect residual genomic dna .
expression levels of the crh mrna were determined relative to alien rna according to the ct method .
cells grown in six - well plates were collected in 750 l pbs ( phosphate - buffered saline ; 137 10mol l nacl , 2.7 10 mol l kcl , 8 10mol l na2hpo4 , and 2 10 mol l kh2po4 , ph 7.4 ) using cell scrapers and centrifuged for 5 min at 300 g .
cell pellets were resuspended in 200 l of pbs and processed for dna isolation as per the manufacturer 's instructions .
genomic dna was eluted from the mini spin columns with 200 l milliq water , quantified using the nanodrop 1000 spectrophotometer , and stored at 4c .
up to 300 ng of genomic dna was bisulfite - converted and purified using the methylseqr bisulfite conversion kit ( applied biosystems ) .
the crh proximal promoter regions were pcr - amplified using the toptaq master mix kit ( qiagen ) and two sets of nested primers designed with the methyl primer express software v.1.0 ( applied biosystems ) .
pcr reactions contained purified bisulfite - converted dna template , 25 l of 2x toptaq master mix , 4 10 mol l of each primer , and milliq water up to 50 l final volume .
the conditions for the first pcr amplification included an initial step at 94c for 3 min , followed by 30 cycles of 94c for 30 s , 54c for 30 s , and 72c for 1 min and a final extension step at 72c for 10 min .
one microliter of a 20-fold diluted aliquot of the first pcr reaction was used as template for the second pcr amplification using the nested primer set .
pcr conditions were 94c 30 min , 30 cycles of 94c for 30 s , 50c for 30 s , and 65c for 1 min and an extension step at 65c for 10 min . following amplification , 20 l of the pcr reaction mixture
was separated by agarose gel electrophoresis and the amplification product was visualised with ethidium bromide .
the gel slice containing the amplified dna fragment was excised , extracted , and purified using the wizard sv gel and pcr clean - up system ( promega , auburn , vic , australia ) .
the dna was collected in 50 l milliq water by the centrifugation of the sv minicolumn .
the bisulfite - converted and pcr - amplified dna was ligated into the pgem - t easy vector using reagents provided by the manufacturer ( promega ) . the 10 l reaction mixtures contained ligation buffer , 3 weiss units of t4 dna ligase , 50 ng of pgem - t easy vector , and pcr product at 3 : 1 insert : vector molar ratio .
a positive control using the control dna provided and a negative control ( no pcr product ) were also included .
the ligation mixture was used subsequently to transform jm109 competent cells ( promega ) according to the manufacturer 's protocol .
fifty l of transformed cell suspension was spread onto duplicate lb / ampicillin / iptg / x - gal plates and incubated at 37c overnight .
the white streak colonies were picked the next day and cultured in 5 ml luria broth at 37c overnight .
plasmids were isolated from the minicultures using the genelute plasmid miniprep kit ( sigma - aldrich , castle hill , nsw , australia ) .
the presence of inserts was verified by digesting an aliquot from each plasmid preparation with ecor i ( promega ) followed by agarose gel electrophoresis .
plasmids containing the expected size inserts ( 300 bp ) were sequenced from both directions by the australian genome research facility ( agrf , brisbane , qld , australia ) .
the sequencing primers were designed by promega and produced by invitrogen / life technologies ( forward : 5-tatttaggtgacactatag-3 , reverse : 5-tatttaggtgacactatag-3 ) .
quality control was automatically performed and any sequence with an unacceptably low conversion rate or high number of sequencing errors was excluded .
ten randomly selected clones , representing individual gene copies , were processed for methylation frequency analyses from each dna sample using the chi - square test and fisher 's exact test as appropriate .
significant one - sided tests are reported in cases when the two - sided tests showed borderline significance . the stata ( college station , tx , usa ) software package was used for the statistical calculations .
as shown in figure 1 , abundance was not significantly different between 0 h and 24 h and between 24 h and 28 h of incubation
. between 48 h and 72 h , a 2.9-fold increase ( p = 0.029 ) was observed , which was coincident with the reported spontaneous syncytialisation of bewo cells . in the presence of 8-br - camp
( 2.5 10 mol l ) , a powerful stimulant of syncytial differentiation , significant increases of crh mrna level were detected between 0 h and 24 h ( p < 0.0001 ) , 24 h and 48 h ( p = 0.03 ) , and 48 h and 72 h ( p = 0.049 , one - sided t - test ) with a maximum at 72 h , which was 23.2-fold higher than the 0 h level ( figure 1 ) . in cultures treated with the dna methyltransferase inhibitor 5-aza - dc ( 5 10mol l ) a slight , but significant , increase of crh mrna abundance
was observed between 0 h and 24 h ( 1.43-fold , p = 0.028 , one - sided t - test ) , but there was no further change between 24 h and 48 h and between 48 h and 72 h. combined treatment with 8-br - camp and 5-aza - dc resulted in robust increases of crh mrna levels between 0 h and 24 h ( p < 0.0001 ) , 24 h and 48 h ( p = 0.0026 ) , and 48 h and 72 h ( p = 0.039 ) reaching a 86.4-fold rise at 72 h compared to 0 h ( figure 1 ) .
8-br - camp increased crh mrna abundance relative to vehicle at all time points ( 24 h , p = 0.0042 ; 48 h , p = 0.0066 ; 72 h , p = 0.0004 ; figure 1 ) , which confirmed previous findings of the stimulatory effects of 8-br - camp on crh gene expression in bewo cells .
5-aza - dc treatment had no effect at 24 h and 48 h and reduced crh mrna abundance relative to vehicle at 72 h , effectively blocking the increase seen between 48 h and 72 h in vehicle - treated cells ( p = 0.0021 , figure 1 ) . combined treatment with the cyclic nucleotide and the dna methyltransferase inhibitor upregulated crh mrna expression at all time points beyond the level reached in response to 8-br - camp alone ( 24 h , p < 0.0001 ; 48 h , p = 0.0002 ; 72 h , p = 0.0029 ; figure 1 ) .
the significant effects of 5-aza - dc on crh mrna expression suggested that dna methylation was involved in the control of crh gene activity . to explore this further
, we have determined the effects of 8-br - camp and 5-aza - dc on the methylation of the 9 cpg sites present in the crh proximal promoter .
bisulfite sequencing revealed partial methylation ( 38% of the 9 cpg sites combined ) at 0 h , before treatments commenced ( figure 2 ) , which increased spontaneously to 57% ( p = 0.001 ) by 72 h of culture .
treatment with 8-br - camp ( 250 m ) resulted in a similar increase of methylation ( to 61% ) , not significantly different from the vehicle - treated control .
treatment with 5-aza - dc for 72 h reduced promoter methylation to 23% , which was significantly less than the control ( p = 0.001 ) . combined treatment with 5-aza - dc and 8-br - camp increased the level of methylation compared to 5-aza - dc alone ( to 33% , p = 0.011 ) but did not reach the methylation level observed in cells treated with 8-br - camp only ( p = 0.001 , figure 2 ) .
clonal bisulfite sequencing determines cytosine methylation with single base resolution in individual alleles ( gene copies ) .
the technique enabled us to determine the particular cpg sites that undergo methylation changes under the treatment conditions that influence methylation levels overall , as presented in figure 2 .
the scheme in figure 3 shows the positions of the 9 methylatable cpg dinucleotides in the human crh proximal promoter .
the two major transcription initiation sites and the two sequence regions implicated in the camp - response are also indicated with cpg2 residing within the cre [ 811 , 29 ] .
the heatmap in figure 4 illustrates the methylation levels of the 9 cpgs under the treatment conditions employed .
methylation levels were significantly different among the cpg sites ranging from 10% to 70% at 0 h and from 13% to 80% at 72 h of culture ( p < 0.001 , figure 4 ) indicating site - specific differential methylation .
the methylation level of cpgs 1 , 2 , 3 , and 7 , but not of cpgs 4 , 5 , 6 , 8 , and 9 increased significantly during the 72 h culture period demonstrating that methylation was dynamic at these sites .
figure 5 shows the methylation of each cpg in the cloned copies of the crh proximal promoter .
the scattered distribution of methylated and unmethylated cpg sites suggests that the partial methylation observed was allele independent both at 0 h and at 72 h of culture .
cells treated with 8-br - camp and 5-aza - dc had similar scattered distribution of methylated cpgs in individual alleles ( not shown ) .
in cells treated with 8-br - camp for 72 h , the cpg sites remained differentially methylated ( from 23% to 73% , p = 0.004 ) , and the methylation levels of individual cpgs were not significantly different from the corresponding sites in the vehicle - treated control ( figure 4 ) .
treatment with the dna methyltransferase inhibitor 5-aza - dc decreased methylation at all cpg sites compared to vehicle , except for cpg 4 , where methylation was relatively low . furthermore , 5-aza - dc abolished the differences between the methylation levels of the individual cpgs .
the methyltransferase inhibitor eliminated the methylation differences among individual cpgs in the presence of 8-br - camp as well ( figure 4 ) .
cotreatment with 8-br - camp prevented , however , the demethylating action of 5-aza - dc at cpgs 1 , and 8 , but not at cpgs 2 , 3 , 5 , 7 , and 9 ( camp versus camp + aza in figure 4 ) .
finally , no individual cpg site exhibited a statistically significant methylation difference in 8-br - camp + 5-aza - dc - treated cells compared to treatment with the methyltransferase inhibitor alone ( aza versus camp + aza in figure 4 ) despite the small , but significant , overall increase in methylation ( aza 72 h versus camp + aza 72 h , in figure 2 ) .
the aim of this study was to explore the involvement of promoter methylation in crh gene regulation in human trophoblast cells .
placental crh expression is predictive of gestational length and is influenced by pregnancy disorders [ 4 , 30 ] .
dna methylation is a developmentally regulated epigenetic modification influenced by environmental inputs [ 31 , 32 ] , which can potentially control crh gene expression during pregnancy and in response to pathogenic factors . in our experiments we used the choriocarcinoma - derived bewo cell line , which is a well - characterized trophoblast model exhibiting increased crh gene expression during spontaneous and camp - induced syncytial differentiation similar to normal trophoblasts .
our dna sequencing data show that the crh proximal promoter sequence is identical in bewo cells and in normal trophoblasts including all reported transcription factor response elements and the methylation sensitive cpg sites ( figure 3 ) .
we have shown by clonal bisulfite sequencing that the cpgs within the crh proximal promoter are partially methylated with significant differences among the individual sites .
the size of the analysed sequence ( 261 bp ) and the methylation level correspond to an intermediate methylation region , which is a genomic feature implicated in tissue specific epigenetic gene regulation .
this is similar to normal trophoblasts , which also exhibit allele independent partial and differential methylation in the crh promoter region .
dna methylation increases in bewo cells during culture and the dna methyltransferase inhibitor 5-aza - dc changes cell phenotype and gene expression levels [ 21 , 22 , 35 ] .
our results show that the crh promoter follows this general trend , which is different from primary trophoblasts , where crh promoter methylation does not change in culture and remains unaltered by 5-aza - dc and 8-br - camp under the conditions where bewo cells show methylation changes . for this reason , bewo cells
are uniquely suited to explore the relationship between crh promoter methylation level and gene activity .
methylation of cpg island promoters is associated generally with the repression of gene activity [ 12 , 14 , 21 ] .
the crh promoter is located in a cpg island shore region relative to a crh intragenic cpg island ( chr8:67,089,250 - 67,089,962 in the grch37/hg19 assembly , ucsc genome browser ) .
our results showed , however , that neither the spontaneous nor the 8-br - camp - evoked increase of crh gene activity was associated with the demethylation of the crh promoter ( figures 1 and 2 ) .
treatment with 5-aza - dc decreased crh promoter methylation and abolished the cpg site - specific methylation differences as expected ( figures 2 and 4 ) , but it also blocked the spontaneous increase of crh gene expression in culture ( figure 1 ) .
8-br - camp - stimulated crh expression strongly in the presence of 5-aza - dc ( figure 1 ) , but the cyclic nucleotide actually increased methylation in 5-aza - dc - treated cells ( aza 72 h versus camp + aza 72 h in figure 2 ) .
thus , crh expression was directly , and not reciprocally , related to promoter methylation under these conditions
. this relationship may be unexpected in view of the well - documented global association between gene repression and promoter methylation , but genome wide trends do not necessarily predict the behavior of individual genes .
in fact , the methylation level of the cpg - poor class of promoters was found to be uncorrelated with gene activity .
the crh promoter falls into the cpg - poor class according to established criteria , with partial methylation in both the hypothalamus and the trophoblast [ 34 , 37 , 38 ] ( figure 2 ) .
methylation reduces crh gene expression in the hypothalamus as expected ; in trophoblastic bewo cells , however , promoter methylation appears to have the opposite effect as detailed before .
there is evidence to suggest that this cell - specific regulation may result from the methylation - dependent change of the functional properties of the camp - response element ( cre ) in the crh promoter ( figure 3 ) .
the cre is critical in regulating the activity of transfected crh promoter - reporter constructs in trophoblast cells [ 911 ] .
it contains a cpg dinucleotide that , when methylated , reduces the affinity of cre to its cognate transcription factor , creb , and renders it unresponsive to camp - stimulation [ 15 , 39 ] . at the same time , the methylated cre has increased affinity to bind the transcription factor c / ebp - alpha , which often activates tissue specific genes during differentiation .
the cpg within the cre was 30% methylated under basal ( 0 h incubation ) conditions ( cpg2 in figure 4 ) .
culturing for 72 h increased cpg2 methylation to 60% concomitantly with enhanced gene expression , while treatment with 5-aza - dc reduced cpg2 methylation to 23% and diminished crh gene activity ( figures 1 and 4 ) .
considering that c / ebp - alpha is expressed in bewo cells , it is reasonable to conjecture that methylation - evoked changes in the transcription factor binding specificity of the cre may have contributed to the enhanced crh expression observed in association with increased promoter methylation .
the cpg2 in the cre was partially methylated under all experimental conditions , which suggests that the unmethylated portion could have mediated stimulation by 8-br - camp using the canonical , creb - dependent pathway .
moreover , the crh proximal promoter contains a second , noncanonical camp - response element ( figure 3 ) , which contributes to the regulation of the gene specifically in the trophoblast [ 9 , 11 ] .
methylation of the cre may thus function to influence the relative contribution of the two camp - response elements , their associated transcription factors , and the coupled signaling pathways to the overall activity of the crh gene under basal and camp - stimulated conditions .
cotreatment with 5-aza - dc strongly augmented the stimulation of crh mrna expression by 8-br - camp , although the dna methyltransferase inhibitor alone had no stimulatory effect under the same culture conditions ( figure 1 ) .
cotreatment with 5-aza - dc , however , decreased cpg methylation in the cre ( cpg2 , figure 4 ) to 43.3% from 70% measured after 8-br - camp treatment ( p = 0.034 , fisher 's exact test , one - sided ) .
the increased proportion of unmethylated cres in the cell population could explain , at least partially , the augmented response to 8-br - camp - stimulation .
moreover , 5-aza - dc decreases dna methylation globally increasing or repressing the activity of numerous genes [ 4143 ] .
this suggests the possibility that 5-aza - dc may potentially influence crh expression indirectly through intervening gene products generating synergy between 8-br - camp and 5-aza - dc .
although gene activation occurs in 5-aza - dc - treated bewo cells [ 22 , 23 , 35 ] , it remains to be established whether transcription factors or other gene products controlled by dna methylation regulate crh expression in trophoblasts .
we have utilized the dynamic changes of dna methylation in the bewo cell line to explore the relationship between this epigenetic chromatin modification and crh gene expression in a trophoblastic cell type .
clonal bisulfite sequencing revealed the cpg site - specific and allele independent partial methylation of the crh proximal promoter and classified it as an intermediately methylated region of the genome .
the data suggest that promoter methylation determines the contribution of the cre , its various associated transcription factors , and a trophoblast specific alternative camp - response element to crh gene regulation .
furthermore , our results are consistent with the possibility that dna methylation controls crh expression indirectly , but any intervening factor that may regulate crh expression by a dna methylation - dependent mechanism remains to be determined . | corticotropin releasing hormone ( crh ) production by the human placenta increases exponentially as pregnancy advances , and the rate of increase predicts gestational length .
crh gene expression is regulated by camp in trophoblasts through a cyclic amp - response element ( cre ) , which changes its transcription factor binding properties upon methylation .
here we determined whether methylation of the crh proximal promoter controls basal and camp - stimulated crh expression in bewo cells , a well - characterized trophoblastic cell line .
we treated the cells with 8-br - camp and the dna methyltransferase inhibitor 5-aza-2 deoxycytidine ( 5-aza - dc ) and determined the effects on crh mrna level and promoter methylation .
clonal bisulfite sequencing showed partial and allele independent methylation of cpgs in the crh promoter .
crh mrna expression and the methylation of a subset of cpgs ( including cpg2 in the cre ) increased spontaneously during culture .
8-br - camp stimulated crh expression without affecting the increase in methylation .
5-aza - dc decreased methylation and augmented 8-br - camp - stimulated crh expression , but it blocked the spontaneous increase of crh mrna level .
we conclude that the crh promoter is a dynamically and intermediately methylated genomic region in bewo cells .
promoter methylation did not inhibit crh gene expression under the conditions employed ; rather it determined the contribution of alternative camp - independent pathways and camp - independent mechanisms to crh expression control . |
the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig .
1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .
1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig .
1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .
these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .
1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .
the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .
the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .
lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .
the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .
however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .
lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case
gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .
lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .
also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .
unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .
one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .
2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation
. bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .
the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .
ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .
they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .
rajeswaran et al . solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,
there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .
their crystal structures as well as the structure of the native ribosome are available through the protein data bank ( pdb ) .
these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .
two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .
theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .
although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,
no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .
the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .
these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .
the methods applied in the calculations are described in the first section of the paper .
we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .
four structures of clindamycin are available in the protein data bank ( as of may 2011 ) .
three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .
the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome clindamycin complexes of deinococcus radiodurans ( pdb code 1jzx ) and haloarcula marismortui ( pdb code 1yjn ) .
therefore , based on the known x - ray structures of clindamycin ( fig .
2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .
, we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig .
2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .
the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .
the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .
no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called exp - opt , were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .
to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .
therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,
the solvent is described by a dielectric constant , which was set to 80 in our work .
the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .
the positions of these point charges were obtained from the x - ray structures of the ribosome
the coordinates of residues within 10 of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .
next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .
the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .
nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .
shielding constants were calculated using the b3lyp / aug - pcs-1 method and the giao routine [ 2630 ] .
the chemical shifts of the i - th nuclei were calculated as \documentclass[12pt]{minimal }
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\begin{document}$$ \delta ( i ) = \sigma_i(x ) - \sigma_i(c ) , $ $ \end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .
spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .
the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .
the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .
data were analyzed using gabedit , and the visualizations were carried out with vmd and xdrawchem .
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 . the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol )
are presented in fig . 5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,
the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .
to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .
in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig . 6 ) . the numerical values for the electron density ( bcp ) and
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions
. however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure
, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .
as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and
its chemical shift is lower for the b conformer than for the a conformer of clindamycin .
the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer
. however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method
in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 .
the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol ) are presented in fig .
5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize
, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig .
the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table 4 .
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9 h39c152.3700.01460.0131cp7c17cl28
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .
bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .
however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as
chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group ,
the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer .
however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer . this value is large enough to be measured experimentally.fig .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .
internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,
we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin spin coupling constants to the conformation of clindamycin .
the two most stable conformers of clindamycin exhibit c = oh o intramolecular hydrogen bonds . according to nbo and aim analyses ,
the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water . | lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens .
this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis .
currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna .
we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set .
the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties .
for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters .
based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o h o is the most stable conformer of clindamycin .
the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin . |
the rising prevalence of alzheimer s disease and related dementias ( henceforth referred to as dementia ) is emerging as a leading global health system challenge .
effective early diagnosis and management models are required to mitigate its impact on patients , caregivers , and health - care systems . enhancing primary care capacity
however , dementia care predominantly resides with geriatric specialists , who are in short supply in canada and elsewhere , delaying access to care . to enhance the care of persons with dementia ,
many of these have been established as primary care - based memory clinics ( pcmcs ) .
initial evaluations suggest that pcmcs can provide timelier assessment , lead to a high degree of satisfaction among referring physicians , patients , and caregivers , and streamline access to specialists . in order to retain the fidelity of such programs and consistency with initial training and practice guidelines , and
practice drift and ensure ongoing high quality of care , quality assurance frameworks are needed .
quality indicators ( qis ) , based on best practices , define achievable benchmarks and a quality assurance framework facilitates practice improvement through targeted , educational quality improvement mechanisms . to our knowledge
, there is no quality assurance framework specific to dementia care in primary care - based settings .
this paper describes the results of a consensus approach to identify qis and quality improvement mechanisms in an ontario - wide network of interprofessional pcmcs , and identify potential barriers and facilitators to the implementation of a quality assurance framework .
a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .
the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .
we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .
respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .
links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .
after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .
qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .
data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .
student s t - test was used to identify significant differences between pcmc clinicians and specialists .
spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .
two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .
a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .
the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .
we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .
respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .
links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .
after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .
qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .
data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .
student s t - test was used to identify significant differences between pcmc clinicians and specialists .
spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .
two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .
two survey rounds were conducted between april and june 2014 . in the first round , 842 surveys were distributed with a response rate of 21.3% .
the majority of respondents were physicians , and nurses and other health professionals equally represented the remainder of respondents ( table 2 ) .
respondents had an average of 14.6 10.1 years of clinical practice with older adults . among pcmc respondents ,
31% were from urban centres , 42% rural settings , and 25% from mixed urban / rural populations .
in contrast , 73% of specialists worked in urban settings , and 24% served mixed populations .
only 11 neurologists responded to the first survey , and their specialty was not included in round 2 . in round 2 ,
respondents had an average of 17.42 10.08 years of clinical practice with older adults and practice settings were similar to round 1 .
a third delphi round was not conducted due to the substantial drop in response rate between rounds 1 and 2 .
table 3 presents the results of the consensus process and table 4 presents the final list of quality indicators .
quality improvement mechanisms characterized by specialist integration , including case discussions , shared care , observerships , and mentorships , ranked highly ( table 5 ) .
other preferred quality improvement mechanisms included standardized electronic charting forms , self - directed learning activities , and interactive programs .
survey respondents recommended that between 1030% of patients seen in a pcmc also be reviewed by a specialist .
descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process . almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] . integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it
a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist [ specialist , round 1 ] .
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform . having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease .
palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced
. family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process .
almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] .
integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it [ pcmc clinician , round 2 ] . a second issue identified in relation to the implementation of the quality assurance framework
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform . having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning .
while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease .
palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .
family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .
referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] .
integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it
a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist [ specialist , round 1 ] .
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .
having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance
, many expressed that such planning discussions were not appropriate for patients with less advanced disease . palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .
family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
this study used a consultative process among primary and specialty providers to identify a core group of qis and preferred quality improvement mechanisms for dementia care .
quality assurance can be an effective method to ensure fidelity to best practices and maintain a high standard of care quality .
selected qis address care processes , assessment and reassessment , medication review and management , investigations , non - pharmacological management , pharmacological management , and managing concomitant conditions .
selected quality improvement mechanisms emphasize a desire for multimodal education and closer collaboration with specialists . assessing care quality and publically reporting the findings are increasingly commonplace .
however , quality assurance still mainly focuses on specific care sectors or episodes , rather than on overall care for conditions that require coordination and collaboration across multiple sectors . in that context
critical considerations include educating clinicians about the role of quality assurance , improving role clarity among providers involved in dementia care , expanding access to allied health professionals , and creating standardized electronic medical record templates to simplify qi documentation .
quality assurance must not create additional burden on clinicians often working with limited resources and time , a burden that could also impact the quality of the clinician - patient interaction .
respondents proposed that 1030% of patients seen in pcmcs be reviewed with a specialist , preferably in a shared care approach .
closer integration of urban specialists with rural pcmcs represents a tremendous opportunity to extend quality dementia care , particularly to rural patients .
, it will be necessary to identify barriers ( e.g. , allocation of funding ) and facilitators ( e.g. , resources for coordination , electronic medical records , telemedicine ) .
another important finding was the relatively low rankings of qis related to end - of - life planning with dementia patients .
in addition to potential discomfort among clinicians to address such issues , this finding may also stem from the understandable desire to maintain hope and engagement early in the course of the illness , though undue delays may leave patients and caregivers less able to meaningfully participate in such discussions . the most important identified barrier to the implementation of the quality assurance framework
is the need for greater clarity on responsibilities of referring family physicians , pcmc clinicians , and specialists , particularly with respect to follow - up , physical examination , and care of complex comorbidities .
quality assurance often targets relatively simple conditions , such as hypertension , or restricts its scope to specific aspects , processes or locations of care .
in contrast , dementia , like all major chronic conditions , follows a course of progressive decline punctuated by increasingly frequent health complications ( related to dementia itself , as well as to exacerbations of concurrent comorbidities ) , multiple care transitions , progressive caregiver stress and health service utilization , and ultimately death .
optimal dementia care thus requires a systems approach of integration and coordination . addressing greater role clarity among all dementia stakeholders , a task that with proper resources could be coordinated from within pcmcs , requires immediate attention , in order to ensure a stable clinical infrastructure that is able to safely and effectively address the needs of these patients , wherever they may be and whenever they arise . until such integration is achieved
first , only two delphi rounds were conducted because the response rate fell markedly after the first .
however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .
a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .
as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .
the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .
fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .
fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .
finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .
first , only two delphi rounds were conducted because the response rate fell markedly after the first . however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .
a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .
as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .
the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .
fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .
fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .
finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .
while this study has identified qis and quality improvement mechanisms to assess care quality for dementia , findings underscore the importance of system integration for the provision of quality dementia care , with specifically defined and mutually understood roles among stakeholders and , where necessary , the reallocation of existing resources to support this approach to care .
an approach whereby clear roles are negotiated among dementia stakeholders can provide sufficient flexibility to meet regional needs ( especially in rural areas where access to specialists is more limited ) , foster more effective collaboration and accountability , and thus facilitate the delivery and measurement of care quality for dementia . within that context , proper field - testing , validation , and evaluation of selected qis and quality improvement mechanisms
this work has significant implications on the organization of care for aging patients with complex conditions .
primary and specialist providers share the responsibility of providing and supporting integrated dementia quality care . as such
, the care of persons with cognitive impairment would be enhanced by the development of a practical and realistically feasible quality assurance framework , under whose umbrella both pcmc and specialist services are integrated , and which ensures high fidelity to intended design and best practices , and thus maintains a high level of care quality . | backgroundprimary care - based memory clinics ( pcmcs ) have been established in several jurisdictions to improve the care for persons with alzheimer s disease and related dementias .
we sought to identify key quality indicators ( qis ) , quality improvement mechanisms , and potential barriers and facilitators to the establishment of a quality assurance framework for pcmcs.methodswe employed a delphi approach to obtain consensus from pcmc clinicians and specialist physicians on qis and quality improvement mechanisms .
thirty - eight candidate qis and 19 potential quality improvement mechanisms were presented to participants in two rounds of electronic delphi surveys .
written comments were collected and descriptively analyzed.resultsthe response rate for the first and second rounds were 21.3% ( n = 179 ) and 12.8% ( n = 88 ) , respectively .
the majority of respondents were physicians .
fourteen qis remained after the consensus process .
ten quality improvement mechanisms were selected with those characterized by specialist integration , such as case discussions and mentorships , being ranked highly .
written comments revealed three major themes related to potential barriers and facilitators to quality assurance : 1 ) perceived importance , 2 ) collaboration and role clarity , and 3 ) implementation process.conclusionwe successfully utilized a consultative process among primary and specialty providers to identify core qis and quality improvement mechanisms for pcmcs . identified quality improvement mechanisms highlight desire for multi - modal education . system integration and closer integration between pcmcs and specialists were emphasized as essential for the provision of high - quality dementia care in community settings . |
symptomatic supravesical obstructive uropathy in a patient with dual functioning kidneys is classically characterized by bilateral hydroureters / hydronephrosis and an empty urinary bladder .
this obstruction may be secondary to metastatic abdomino - pelvic and retroperitoneal malignancies , ureteric calculi and retroperitoneal fibrosis [ 25 ]
. the evidence for obstruction may be partially or totally absent on ultrasound or computerized tomography , in one or both kidneys [ 68 ] .
our recent experience with a uraemic 56-year - old caucasian showing only unilateral moderate right - sided hydronephrosis is presented .
a 56-year - old caucasian male patient with a past medical history that included hypertension and coronary artery disease and a recent serum creatinine of 88.4 mol / l , was diagnosed 10 days earlier with urinary tract infection ( uti ) .
two days before presentation to us , he developed worsening anorexia , nausea with vomiting and oliguria .
his father had prostate cancer and died at age 75 years from colon cancer ; a sister had breast cancer and a maternal uncle had throat cancer . physical examination revealed a blood pressure of 165/89 mmhg , pulse rate of 56/min and respiratory rate of 16/min .
he was not dehydrated nor orthostatic . except for trace ankle oedema and bilateral costo - vertebral angle punch tenderness ,
mol / l , co2 14 mmol / l and potassium 5.5 mmol / l .
urinalysis showed 510 wbc per high - power field ( phf ) and 25 rbc phf .
alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and total creatine kinase ( ck ) levels were normal .
a post - void urinary bladder scan had demonstrated only 60 ml of residual urine .
a renal ultrasound showed unilateral moderate right - sided hydronephrosis with suspected mass effect on the inferior urinary bladder .
the left kidney appeared normal , measuring 16.2 cm 8.1 cm , with preserved cortical thickness .
the next day , with increasing vomiting , serum creatinine 894.6 mol / l , phosphorus 2.6 mmol / l , potassium 6.2 mmol / l and oliguria ( table 1 ) , he started daily haemodialysis .
a non - contrast computerized tomography examination , on day 2 , again showed unilateral right - sided hydronephrosis , bilateral nephric stranding and urinary bladder wall thickening suspicious for transitional cell cancer ( figure 1 ) .
cystoscopy , on day 3 , revealed a sessile urinary bladder tumour , which was resected .
the right ureter was successfully cannulated and a right ureteric stent was placed with prompt urine drainage .
the next day , with a strong push from nephrology , the patient consented to a percutaneous left nephrostomy procedure despite the normal appearing left kidney .
a percutaneous left nephrostogram revealed a previously unrecognized mild hydronephrosis / hydroureter with obstruction at the ureterovesical junction .
the pathology report revealed high - grade urothelial carcinoma , grade 3 of 3 , with invasion of the muscularis propria .
a patient with dual functioning kidneys presenting with uraemic symptoms and suspected to have obstructive uropathy must be presumed to necessarily have bilateral renal obstruction [ 610 ] .
this is without prejudice to the findings on conventional renal imaging with ultrasound or computerized tomography [ 3,610 ] .
there are false negative tests with these imaging modalities , the so - called syndrome of non - dilated obstructive uropathy or non - dilated obstructive nephropathy [ 610 ] .
clinical conditions associated with the absence of hydronephrosis on ultrasound and computerized tomography despite obstructed kidney(s ) include acute early obstruction , the presence of retroperitoneal fibrosis or infiltrative metastatic abdomino - pelvic cancers , dehydration or septic shock and severe oliguria [ 210 ] .
our patient was not dehydrated and was not hypotensive but was severely oliguric ( table 1 ) .
we note that we did not rule out the presence of retroperitoneal fibrosis in our patient .
the classic picture of bilateral hydronephrosis with hydroureters and an empty urinary bladder , in symptomatic uraemia following supravesical obstruction , in patients with dual functioning kidneys , is well acknowledged .
however , the presentation of new - onset symptomatic uraemia concurrent with only unilateral hydronephrosis / hydroureter on conventional imaging ( ultrasound , computerized tomography ) should raise the plausibility of non - apparent obstruction of the contra lateral kidney .
in such instances , the more sensitive albeit invasive percutaneous nephrostogram of the apparently normal appearing kidney is therapeutic and will lead to greater renal salvage [ 68 ] .
we would like to remind practicing providers that symptomatic uraemia presenting in the setting of suspected obstructive uropathy must be assumed to imply bilateral renal obstruction , regardless of the results / interpretations of any form of conventional renal imaging . therefore , necessarily , every attempt to decompress both kidneys must be the rule .
this approach would result in early and improved renal salvage . left undiagnosed and therefore untreated
, this potentially reversible cause of renal failure can lead to irreversible renal failure if bilateral , , or to significant residual loss of renal function if missed on one side only .
we note that we were not able to carry out any split renal functional testing after recovery as the patient 's primary attention at this point was to find out treatment options for his cancer . | bilateral hydronephrosis is classic for supravesical obstructive uropathy causing uraemia with dual functioning kidneys . recently , a patient presented with uraemia and metastatic urinary bladder carcinoma but only unilateral right - sided hydronephrosis .
a right ureteral stent was placed retrograde and no further intervention was planned since the left kidney appeared normal , and since the left ureteric orifice was not visualized .
we insisted on a left percutaneous nephrostomy which was successful with prompt urine return .
a left nephrostogram revealed unrecognized hydroureter / hydronephrosis .
following haemodialysis , kidney function normalized at 3 weeks . for symptomatic
uraemia from obstruction , an antegrade and/or a retrograde decompression must be attempted bilaterally to improve renal salvage . |
in 1985 , smith pioneered phage display technology , an in vitro methodology and system for presenting , selecting and evolving proteins and peptides displayed on the surface of phage virion . since then , phage display has developed rapidly and become an increasingly popular tool for both basic research such as the exploration of protein - protein interaction networks and sites [ 24 ] , and applied research such as the development of new diagnostics , therapeutics , and vaccines [ 510 ] .
usually , the protein used to screen the phage display library is termed as target and the genuine partner binding to the target is called template .
peptide mimicking the binding site on the template and binding to the target is defined as mimotope , which was first introduced by geysen et al . .
one type of the most frequently used targets is monoclonal antibody . in this situation ,
the template is the corresponding antigen inducing the antibody , and the mimotope is a mimic of the genuine epitope .
. goes a mimotope is defined as a molecule able to bind to the antigen combining site of an antibody molecule , not necessarily identical with the epitope inducing the antibody , but an acceptable mimic of the essential features of the epitope .
mimotopes and the corresponding epitope are considered to have similar physicochemical properties and spatial organization .
the mimicry between mimotopes and genuine epitope makes mimotopes reasonable solutions to epitope mapping , network inferring , and new diagnostics , therapeutics , and vaccines developing . powered by phage display technology , mimotopes can be acquired in a relatively cheap , efficient and convenient way , that is , screening phage - displayed random peptides libraries with a given target .
however , not all phages selected out are target - specific , because the target itself is only one component of the screening system . from time to time , phages reacting with contaminants in the target sample or other components of the screening system such as the solid phase ( e.g. , plastic plates ) and the capturing molecule ( e.g. , streptavidin , secondary antibody ) rather than binding to the actual target are recovered with those target - specific binders ( displaying mimotopes ) during the rounds of panning .
peptides displayed on these phages are called target - unrelated peptides ( tup ) , a term coined recently by menendez and scott in a review .
the results from phage display technology might be a mixture of target - unrelated peptides and mimotopes , and it can be difficult to discriminate tup from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the initial panning experiment .
therefore , target - unrelated peptides might be taken into study as mimotopes if the researchers are not careful enough .
obviously , target - unrelated peptides are not appropriate candidates for the development of new diagnostics , therapeutics , and vaccines . for mimotope - based epitope mapping ,
if tup is included in the mapping , the input data is improper and the result might be misleading .
there are now quite a few programs for mimotope based epitope mapping , none of them , however , has a procedure to scan , report and exclude target - unrelated peptides [ 1523 ] . in this study , we describe a web server named sarotup , which is an acronym for scanner and reporter of target - unrelated peptides .
sarotup was coded with perl as a cgi program and can be freely accessed and used to scan peptides acquired from phage display technology .
it is capable of finding , reporting , and precluding possible target - unrelated peptides , which is very helpful for the development of mimotope - based diagnostics , therapeutics , and vaccines .
the power and efficiency of sarotup was also demonstrated by preliminary tests in the present study .
recently , menendez and scott reviewed a collection of target - unrelated peptides recovered in the screening of phage - displayed random peptide libraries with antibodies .
they divided their collection into several categories according to the component of the screening system to which target - unrelated peptides bind .
very recently , brammer et al . reported a completely new type of target - unrelated peptides . in the review of menendez and scott
, target - unrelated selection is due to the binding to contaminants or components other than target ; however , in the report of brammer et al .
, target - unrelated selection is due to a coincident point mutation in the phage library [ 12 , 13 ] .
we compiled a set of 23 tup motifs from the above two references [ 12 , 13 ] , including 12 motifs specific for the capturing agents , 5 motifs specific for the constant region of antibody , 3 motifs specific for the screening solid phase , 2 motifs specific for the contaminants in the target sample , and 1 motif for a mutation in phage library ( table 1 ) .
the sarotup was implemented as a free online service , powered by apache and perl .
three pages are designed and integrated into a tabbed web interface with cascading style sheets codes .
the core program of sarotup was sar.pl , a cgi script coded with perl . in this script , the 23 tup motifs were converted to regular expressions , which were then used to match each input peptide sequence .
we constructed two - test data sets from [ 12 , 13 , 1523 , 25 , 26 ] .
the first data set contains 8 cases ; 6 of them are sourced from test cases used in extant programs for mimotope - based epitope mapping [ 1523 ] ; the left 2 are cases studies published recently [ 25 , 26 ] .
as shown in table 2 , the target of each case in the first data set is monoclonal antibody and the structure of corresponding antigen - antibody complex has been resolved , which is used to derive its structural epitope as the golden standard for evaluation . for each case
if target - unrelated peptides were found , a new panel of peptides excluding tup was produced .
the old and the new panel of peptides were then used to predict epitope using mapitope or pepsurf [ 15 , 21 , 22 ] . finally , the results were compared to show if sarotup could improve the performance of mimotope - based epitope mapping . the second data set is composed of 100 peptides in raw sequence format .
the first group has 77 sequences compiled from the first data set without any known tup motifs ; the second group has 23 sequences sourced from [ 12 , 13 ] with various tup motifs . the mixture of the two groups of sequences made the second data set , which was then used as the sample input and can be used to evaluate the efficiency of sarotup .
as a free online service , the web interface of sarotup has successfully been implemented as a tabbed web page .
the left tab is the default page , providing a brief introduction to this web service .
the users can either paste a set of peptide sequences in the text box or upload a sequence file to the sarotup server for scanning .
as shown in figure 1 , a panel of peptides in raw sequence format taken from the b12 test case was pasted in the text box . besides the raw sequences
however , only the standard iupac one - letter amino acid codes are accepted at present .
the lower section of the form has a series of options ( figure 2 ) .
it includes three drop lists for the screening target , screened library , and screening solid phase , respectively .
it also has two groups of check boxes for the capturing reagents and contaminants in the target sample or screening system . by default
however , the users can customize their scan according to their experiment at this section .
after the users submit their request , the scanning results of sarotup will be displayed on the middle tabbed page .
if any target - unrelated peptides are found , they will be reported in a table . at the same time , a new panel of peptides excluding target - unrelated peptides is produced and can be downloaded from the hyperlink created by the sarotup server ( figure 3 ) .
the file of the new panel of peptides will be stored on the server for a month and then automatically deleted .
we have tested sarotup on the internet explorer ( version 6.0 ) , mozilla firefox ( version 3.5.2 ) , and google chrome ( version 3.0 ) .
although sarotup looks a little bit different among different browsers , it works normally on all browsers tested . as shown in table 2 , the first test data set has 11 panels of peptides acquired from phage display libraries screened with 8 targets . in
the 11 panels of peptides sarotup scanned , there were target - unrelated peptides in 3 panels from cetuximab , 80r , and b12 test case , respectively ( table 3 ) .
this result suggested it was not rare that target - unrelated peptides sneaked into biopanning results and then were taken as mimotopes in study .
in all , 7 target - unrelated peptides were found ; 4 of them were due to binding to plastic ; the left 3 were due to binding to the fc fragment ( table 3 ) .
for the above 3 cases , the genuine epitopes recognized by cetuximab , 80r , and b12 monoclonal antibodies are compiled according to the ced records and pdbsum entries .
mapitope or pepsurf [ 15 , 21 , 22 ] were used to perform mimotope - based epitope prediction with or without sarotup procedure . for mapitope and pepsurf algorithm ,
; the stop codon modification was set to none ; and all other options were in default .
the cluster with best score was taken as the predicted epitope . in the cetuximab case ,
pepsurf was used because there are only four or three peptides in the panel , statistically too few for mapitope . in the case of 80r and b12 ,
mapitope was used because many peptides in the two cases exceeding the length limit of pepsurf , that is , 14 amino acids .
if a predicted residue is identical with a residue in the true epitope , it is underlined ( table 4 ) . as shown in table 4 ,
the number of true positives improved from zero to four in the cetuximab case with sarotup procedure . when it came to the b12 case
sarotup did not improve the number of true positives in the 80r case when the parameters are same to the cetuximab and b12 cases .
however , when the distance parameter was adjusted from default ( i.e. , 9 ) to 10 , sarotup did increase the number of true positive residues from eight to eleven .
these results indicate : ( 1 ) epitope prediction based on mimotope will be interfered if target - unrelated peptides are taken as mimotopes ; ( 2 ) sarotup can improve the performance of mimotope based epitope mapping through cleaning the input data .
the second data set has 100 peptides , varying from 6 to 22 residues long .
suppose that matching each pattern to each peptide manually costs 10 seconds , then it would take a researcher more than 6 hours ( 23,000 seconds ) to look through the second data set for target - unrelated peptides , even if he is as prompt during the whole period .
besides , a table of target - unrelated peptides and a new panel of peptides excluding tup was produced at the same time by sarotup .
it is true that some target - unrelated peptides can be identified through control and binding competition experiments .
however , using sarotup first will certainly save a lot of labor , money , and time for researchers in this area .
although the target of all tests described previously were monoclonal antibodies , sarotup can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors .
for the same reason , we can also expect that sarotup will extend its use to other similar in vitro evolution techniques , such as ribosome display [ 2931 ] , yeast display , and bacterial display [ 3335 ] .
furthermore , sarotup will not only benefit the mimotope - based epitope mapping , but also the development of new diagnostics , therapeutics , and vaccines .
target - unrelated peptides are not appropriate candidates for mimotope based diagnostics , therapeutics , and vaccines , since they are mimics to components or contaminants of the screening system rather than target .
therefore , it is reasonable to find and exclude possible target - unrelated peptides from the candidate list of new diagnostics , therapeutics , and vaccines .
screened a phage - displayed random peptides library with the cetuximab and got four different peptides , that is , qfdlstrrlk , qynlssralk , vwqrwqksyv , and mwdrfsrwyk .
as described previously , we scanned the four mimotopes with sarotup and the result suggested that the peptide vwqrwqksyv might be a tup .
indeed , the dot blot analysis of riemer et al . showed that qynlssralk bound the cetuximab with high affinity but vwqrwqksyv was less reactive with the cetuximab . trying to develop a mimotope vaccine , riemer et al .
after immunization mice with these constructs , they found that either the cetuximab or the antibodies induced by the qynlssralk vaccine construct inhibited the growth of a431 cancer cells significantly .
the inhibition of the antibodies induced by the vwqrwqksyv vaccine construct however , was not statistically significant when compared with the inhibition caused by the isotype control antibody .
sarotup must be used with caution since it is a tool only based on pattern matching at present .
as these tups are not embedded in sarotup at present , it is possible that a true tup can not be detected by sarotup . to reduce this kind of false negatives
besides the motif - based search , the database - based search can find out the known tup without known motifs .
it is also possible that a sarotup predicted target unrelated peptide is actually target - specific . to decrease this kind of false positives
, the users should customize the scan according to their experiment at the section of advance options .
for example , the user should select antibody without fc fragment as the target if fab was used in biopanning ; this will prevent sarotup from reporting peptides bearing the fc - binding motifs as tup .
as described above , sarotup in future will also provide an exact match tool based on database search . in this way
, a match might mean that different research groups have isolated the same peptide with a variety of targets .
thus , the false positive rate of sarotup can be decreased further when its new feature become available . at last , we must point out that the controlled experiment is still the gold standard to distinguish tups from the specific mimotopes .
sarotup , a web application for scanning , reporting and excluding target - unrelated peptides has been coded with perl .
it is also useful in the development of diagnostics , therapeutics , and vaccines . to our knowledge | as epitope mimics , mimotopes have been widely utilized in the study of epitope prediction and the development of new diagnostics , therapeutics , and vaccines . screening the random peptide libraries constructed with phage display or any other surface display technologies
provides an efficient and convenient approach to acquire mimotopes . however , target - unrelated peptides creep into mimotopes from time to time through binding to contaminants or other components of the screening system . in this study , we present sarotup , a free web tool for scanning , reporting and excluding possible target - unrelated peptides from real mimotopes .
preliminary tests show that sarotup is efficient and capable of improving the accuracy of mimotope - based epitope mapping .
it is also helpful for the development of mimotope - based diagnostics , therapeutics , and vaccines . |
reversible focal lesions in the splenium of the corpus callosum ( scc ) have been found in patients with various conditions including seizures , antiepileptic drug toxicity or withdrawal , viral encephalitis , hypoglycemia , wernicke 's encephalopathy , marchiafava - bignami disease , sympathomimetic - induced kaleidoscopic visual illusion syndrome , hemolytic uremic syndrome , altitude brain injury , and acute axonal injury .
analysis of the current literature revealed that the most frequent cause of reversible focal lesions of the scc is influenza virus - associated encephalitis / encephalopathy ( iaee ) .
the abnormality has been reported in association with various viruses ( e.g. , epstein - barr virus , rotavirus , influenza virus ) ( 1 - 3 ) .
the brain lesions described in patients with iaee include restricted diffusion involving the cerebral cortex and subcortical white matter in various locations , symmetric lesions in the brainstem , basal ganglia , thalamus , and cerebellar white matter with or without brain edema , and mild brain atrophy .
transient restricted diffusion of the scc in patients with iaee has also been described ( 4 ) . here ,
we report an adult who presented with non - specific dizziness and lesions of the scc on magnetic resonance imaging ( mri ) who was diagnosed with hemorrhagic fever with renal failure syndrome ( hfrs ) with serologically proven hantaan virus infection .
a previously healthy 53-yr - old man presented with a 6 - 7-day history of fever up to 40 and dizziness .
the initial diffusion - weighted images revealed high signal intensity in the corpus callosum with a low signal on the apparent diffusion coefficient ( adc ) map ( fig .
1 ) . the next day , he developed thrombocytopenia ( 4010/l ) and was transferred to our emergency room for further evaluation .
he stated that he had been tying rice straw into sheaves several days earlier , but not using any drugs , like metronidazole , antiepileptic drug or alcohol , and had not been exposed to toxins or experienced weight loss . on neurologic examination , he was slightly confused , and scored 24/30 on the mini mental status exam ( mmse ) .
the cranial nerve , motor , and sensory examinations , deep tendon reflexes , and cerebellar function tests were normal .
his blood tests showed marked thrombocytopenia ( 2110/l ) and azotemia ( bun / cr 23 mg / dl/1.5 mg / dl ) .
he was started on cefotaxime and clindamycin for a suspected infection and transfused with fresh frozen plasma , but his fever ( > 38.0 ) was sustained .
two days after admission , he was alert and his fever had subsided , but the oliguria , azotemia ( bun / cr 49 mg / dl/3.0 mg / dl ) , and thrombocytopenia ( 610/l ) had worsened ( figs . 2 , 3 ) .
four days later , the thrombocytopenia had recovered , although the azotemia ( bun / cr 82 mg / dl/7.1 mg / dl ) had not improved .
he was diagnosed with hfrs based on the detection of hantaan virus antibody in his serum .
six days later , he was alert , and his electroencephalograph ( eeg ) was normal . his platelet count had stabilized ( 31110/l ) , and a lumbar puncture was performed .
the cerebrospinal fluid ( csf ) analysis revealed no cells , a nor mal protein level of 26.0 mg / l , a normal glucose level of 80 mg / dl , and no organisms on gram staining .
the mri findings had resolved completely in follow - up studies conducted on day 14 ( fig .
hemorrhagic fever with renal failure syndrome occurs mainly in europe and asia and is characterized by a fever and renal failure associated with hemorrhagic manifestations .
hfrs is caused by airborne contact with secretions from rodent hosts infected with viruses belonging to the genus hantavirus in the family bunyaviridae .
the clinical features consist of a triad of fever , hemorrhage , and renal insufficiency ( 5 ) .
other common symptoms during the initial phase of the illness include , myalgia , abdominal and back pain , and diarrhea . in most cases , mild neurological symptoms such as headache , vertigo , and nausea
the pathogenesis is largely unknown , but findings from several studies have suggested that immune mechanisms play an important role .
after the infection , marked cytokine production , kallikrein - kinin activation , complement pathway activation , and increased levels of circulating immune complexes occur .
damage to the vascular endothelium , capillary dilatation , and leakage are clinically significant features of the disease .
myelin sheaths in the scc have a relatively high water component , and the scc is more susceptible to cytotoxic edema than are other brain areas ( 2 ) .
reversible brain lesions have been attributed to the transient development of intramyelinic edema due to the separation of myelin layers , which is a possible mechanism for the transiently decreased diffusion of the scc lesion ( 2 , 11 ) .
the influx of inflammatory cells and molecules , possibly combined with related cytotoxic edema , might have decreased the adc ( 2 , 12 , 14 ) .
the most common causes of reversible focal lesions of the scc are viral encephalitis , antiepileptic drug toxicity / withdrawal , and hypoglycemic encephalopathy .
they are not specific to iaee and have been reported secondary to various infectious agents , including rotavirus , measles , herpesvirus , salmonella organisms , mumps , varicella - zoster virus , adenovirus , o157 escherichia coli - associated hemolytic - uremic syndrome , legionnaires ' disease , and unknown pathogens ( 4 , 9 , 14 ) .
the similar clinical and imaging features suggest a common mechanism induced by different pathological events leading to the same results ( 1 ) .
the most likely causes of these transient lesions of the scc have been explained as a rapidly resolving intramyelinic infiltrate or the influx of inflammatory cells and macromolecules , combined with related cytotoxic edema , which is very similar to the pathogenesis of hfrs ( 2 , 7 , 9 , 10 , 13 ) .
this argues for a non - specific cause related to vasogenic edema , probably secondary to the inflammatory response ( 2 , 7 ) our patient was diagnosed with hfrs , and his mri showed scc lesions .
we report a reversible scc lesion associated with hfrs . to our knowledge , this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was a hantaan virus .
although it is not certain whether the neurologic symptoms in this patient might have been caused by fever , or scc lesions , or both , we suggest that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies . | this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was hantaan virus .
a 53-yr - old man presented fever , renal failure and a hemorrhagic tendency and he was diagnosed with hemorrhagic fever with renal failure syndrome ( hfrs ) . in the course of his illness , mild neurologic symptoms such as dizziness and confusion developed and magnetic resonance images revealed a reversible lesion in the splenium of the corpus callosum .
this case suggests that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies . |
as is the most common cause of left ventricular outflow obstruction in children and adults . it is most common type of valvular heart disease in europe and north america , occurring in 27% of the population over 65 years of age .
medical therapy alone is not effective for the long - term management of aortic valve disease , thus valve replacement remains the standard of care in patients with an acceptable risk profile .
we present a case of critical as with significant symptom manifested as chest pain that was managed successfully without surgical avr or tavr .
we will also briefly review incidence , etiology , grading of as and current guidelines for avr .
a 91-year - old gentleman with history of hypertension , dyslipidemia and as presented to our office on 7/2015 with complaints of new onset sub sternal burning pain of 6 weeks duration .
prior to 6 weeks patient could walk 2 blocks on level ground without shortness of breath or chest pain .
blood pressure was 140/90 mm hg and pulse was regular at 60 beats per minute .
cardiovascular exam revealed a grade 4/6 ejection systolic murmur best heard in the right second intercostal region radiating bilaterally to the neck .
ecg revealed left ventricular hypertrophy ( lvh ) with non specific st - t wave changes in inferior leads ( figure 1 ) .
echocardiography ( echo ) done in may of 2014 had revealed critical as with aortic valve area of 0.6 - 0.7 centimeter square and ejection fraction of 65 percent . the aortic valve ( av ) was heavily calcified .
aortic valve peak velocity ( av vmax ) 4.66 m / s ( figure 2 ) .
the patient was directly referred for a cardiac catheterization to evaluate his coronary anatomy prior to avr .
hemodynamic measurement revealed left ventricle ( lv ) pressure 240/0 with left ventricular end diastolic pressure ( lvedp ) of 10 .
pulmonary artery wedge pressure ( pawp ) , mean of 24 , right atrium ( ra ) mean of 8 , right ventricle ( rv ) 60/10 with an right ventricular end diastolic pressure ( rvedp ) of 14 , pulmonary artery ( pa ) 60/17 .
the aortic valve area was calculated at 0.6 centimeter square , which was unchanged from echo done may 1 , 2014 .
the left ventriculogram in the right anterior oblique ( rao ) view revealed normal lv systolic motion and ejection fraction of 55 - 60 percent . left main coronary artery revealed a common ostium - giving rise to the left anterior descending ( lad ) and the circumflex .
right coronary artery ( rca ) revealed a 99 % ostial narrowing ( figure 3 ) .
lad revealed a 60 % narrowing in its proximal segment and a 90% ostial diagonal narrowing ( figure 4 ) .
after a detailed discussion involving the patient , his son , interventional cardiologist and the cardio thoracic surgeon , patient requested that only pci of the rca be considered , as the chest pain was of recent duration with no change in aortic valve finding .
thus , a successful pci was done in the proximal rca with a bare metal stent ( figure 6 ) .
patient did extremely well after pci of the rca and the patient has remained asymptomatic to date .
thus , this new onset chest pain in patient with critical as was due to concomitant coronary artery lesion and was amenable to stenting and thus spared this elderly patient from the aortic valve replacement which would have been a high risk surgery for him .
as is the most common valvular heart condition in the developed world , affecting 3% of people between ages 75 and 85 and 4% of people over age 85 .
congenitally unicuspid , bicuspid , tricuspid , or even quadricuspid valves may be the cause of as . in adults who develop symptoms from congenital as
the main causes of acquired as include degenerative calcification and , less commonly , rheumatic heart disease .
other , infrequent causes of as include obstructive vegetations , homozygous type ii hypercholesterolemia , paget disease , fabry disease , ochronosis , and irradiation . based upon a variety of hemodynamic and natural history data
, clinicians generally grade the severity of stenosis as mild , moderate , severe , or critical .
grading of as are as follows : i ) mild : valve area exceeds 1.5 cm ; transvalvular velocity 2.0 to 2.9 m / s ; mean gradient < 20 mmhg ; ii ) moderate : valve area of 1.0 to 1.5 cm ; transvalvular velocity 3.0 to 3.9 m / s ; mean gradient 20 to 39 mmhg ; iii ) severe : valve area is less than 1.0 cm ; transvalvular velocity 4 m / s ; mean gradient 40 mmhg .
the term critical stenosis was defined based upon theoretical considerations showing that the aortic valve area must be reduced to one - fourth of its natural size before significant changes in circulation occur . as a result , since the triangular orifice area of the normal ( adult ) aortic valve is approximately 3.0 cm , an area exceeding 0.75 cm would not be defined as critical .
avr and tavr remain the only treatment proven to reduce the rates of mortality and morbidity in this condition . under current guidelines , the onset of symptoms of exertional angina , syncope and dyspnea in a patient who has severe as is a class i indication for surgery .
the annual rate of sudden death in patients with this condition is estimated at 1% to 3% but the surgical mortality rate in avr has been as high as 6% . with improvements in surgical techniques and prostheses , mortality rates have been reduced to 2.42% making a case for earlier intervention .
tavr has become widely available , but further investigation into its use in this patient cohort is warranted . while assessing the cases of asymptomatic as we have both traditional as well as novel markers at our disposal now . left ventricular ejection fraction ( lvef ) < 50 percent , peak aortic jet velocity > 4.0 m / s , valve area < 1 cm and mean pressure gradient > 40 mm hg are the traditional markers to denote severe as in asymptomatic patients . while , indexed left atrial size > 12.2 cm / m , lvh with wall thickness > 15 mm , global left ventricular longitudinal strain < 15.9 , bnp ( b - natriuretic peptide ) level > 130 pg / ml and increase in mean pressure gradient of > 20 mm hg during exercise testing are the novel markers of asymptomatic severe as .
bnp level does not appear to be significantly associated with the degree of as severity but does reflect heart failure status .
the american college of cardiology and american heart association ( acc / aha ) have issued the following recommendations for avr , based on the severity of stenosis and on whether the patient has symptoms : i ) severe stenosis , with symptoms : class i recommendation ( surgery should be done ) . without surgery ,
these patients have a very poor prognosis , with an overall mortality rate of 75% at 3 years ; ii ) severe stenosis , no symptoms , in patients undergoing cardiac surgery for another indication ( example coronary artery bypass grafting , ascending aortic surgery , or surgery on other valves ) : class i recommendation for concomitant aortic valve replacement ; iii ) moderate stenosis , no symptoms , in patients undergoing cardiac surgery for another indication : class iia recommendation ( i.e. , aortic valve replacement is reasonable ) ; iv ) very severe stenosis ( aortic peak velocity > 5.0 m / s or mean pressure gradient 60 mm hg ) , no symptoms , and low risk of death during surgery : class iia recommendation ; v ) severe stenosis , no symptoms , and an increase in transaortic velocity of 0.3 m / s or more per year on serial testing or in patients considered to be at high risk for rapid disease progression , such as elderly patients with severe calcification : class iib recommendation ( surgery can be considered ) .
on revisiting the above case description we realize that the patient did have critical as but was asymptomatic .
his chest pain was only due to concomitant coronary artery disease but was not due to as per se .
age and comorbidity of the patient posed a high risk for coronary artery bypass graft ( cabg ) with avr .
besides , the patient s symptoms were of new onset and subsequent cardiac catheterization revealing critical rca stenosis and the patient s preference for treating the cause of his recent symptoms , encouraged us to think otherwise . the decision to perform pci alone with the belief that this chest pain and cad would be amenable to the minimal risk procedure paid dividends .
in addition , patient received a bare metal stent with the option of undergoing surgical avr if symptoms were not relieved .
awareness amongst physicians about the fact that all critical aortic stenosis with chest pain may not require aortic valve replacement is important .
this can lead to less invasive treatment tailored to the need of the patient especially in those with advanced age , significant comorbidities and an extremely high risk for cabg with avr and can also result in decreased cost of care .
pharmacological nuclear stress testing may be another modality that can be used to differentiate etiology of the symptoms . | aortic valve replacement ( avr ) remains the cornerstone of treatment for symptomatic critical aortic stenosis ( as ) .
it is a class i indication that symptomatic patients with critical as undergo either surgical or transcatheter aortic valve replacement ( tavr ) .
we present a patient with critical as and new angina that was managed successfully with percutaneous coronary intervention ( pci ) of the right coronary artery .
physicians should consider that not all patients with critical as and angina necessarily require avr .
concomitant pathology leading to the symptoms should be carefully ruled out .
this leads to a less invasive , cost effective care plan especially in patients with advanced age and comorbidities for which any type of surgical valvular intervention may pose high risk . |
the various blood cell lineages in mammals arise from a multipotent haematopoietic stem cell via particular differentiation pathways .
one of these pathways , erythropoiesis , leads to the production of red blood cells ( rbcs ) , which transport oxygen and carbon dioxide around the body by means of the intracellular metalloprotein haemoglobin ( hb ) .
hb is a tetramer , consisting of two -globin and two -globin subunits . in mammals ,
these globin chains are encoded by two gene loci : the -globin locus and the -globin locus . in humans ,
the -globin locus consists of the embryonic - and adult -globin genes , and the -globin locus comprises the embryonic - , foetal - and - , and adult - and -globin genes [ 1 , 2 ] .
the globin genes expressed from these loci differ from embryonic to adult erythropoiesis in order to meet varying oxygen demands and facilitate placental transfer of oxygen from mother to embryo .
there are a number of severe diseases caused by the disruption of adult globin genes , including thalassaemias and certain types of anaemia . according to the world health organisation ,
approximately 5% of the world 's population carry genes involved in hb disorders , and as such , they present an enormous health burden .
thalassaemia is caused by a reduction or abolition of the expression of one or more globin genes , resulting in an imbalance of - and -globin chains in red blood cells and consequent anaemia [ 1 , 4 ] .
sickle cell anaemia is another prevalent haemoglobinopathy and is caused by a mutation in the adult -globin gene which generates a single glutamic acid to valine amino acid substitution .
this mutation leads to the polymerisation of globins in venous circulation [ 5 , 6 ] , which can trigger a rigid and sickled cell phenotype [ 7 , 8 ] and results in a number of acute conditions such as vaso - occlusion , splenic sequestration , and haemolytic anaemia .
there are currently a number of treatments available for patients suffering from thalassaemia and sickle cell anaemia .
the most common is packed red blood cell transfusion , but this is associated with a number of problems , such as sufficiency of supply , bacterial and viral infection , biochemical and biomechanical changes during storage ( red blood cell storage lesions ) , and the risk of immune rejection from the patient [ 10 , 11 ] .
furthermore , blood transfusions are ongoing throughout a patient 's life and often lead to a potentially fatal buildup of iron and associated reduction in organ activity .
residual production of foetal -globin persists naturally throughout life , and levels vary between individuals [ 12 , 13 ] .
this persistent expression allows two -globin chains to combine with two adult -globin chains to form what is known as foetal hb ( hbf ) . as only the adult -globin gene
is mutated in sickle cell anaemia , affected infants are protected from severe symptoms until they reach several months of age , due to the large amount of hbf still in circulation at birth .
furthermore , patients who have inherited alleles associated with increased levels of hbf , known as hereditary persistence of foetal hb ( hpfh ) , are protected into adulthood . similarly , a more asymptomatic disease phenotype has also been shown in patients with -thalassaemia who exhibit higher levels of hbf .
together , these observations indicate that increased foetal -globin is able to compensate in part for the loss of adult -globin function and thus ameliorates the symptoms of certain adult haemoglobinopathies .
accordingly , a number of drug treatments for -thalassaemia and sickle cell disease , for instance , 5-azacitidine , hydroxyurea , and butyrate , all act by nonspecifically reactivating foetal -globin gene expression by various mechanisms .
there is evidence that long - term administration of these drugs has chronic side effects , consistent with their lack of specificity [ 8 , 17 ] . as the existing methods of treatment for these haemoglobinopathies remain inadequate ,
alternative forms of therapy are currently being sought , and stem cell therapies should be considered .
this paper will discuss progress in utilising novel cellular reprogramming techniques to treat rbc diseases .
stem cells , both embryonic and adult , have the ability to differentiate into various cell types , making them a potentially attractive treatment option . embryonic stem cells ( escs ) and adult stem cells ( ascs ) each have their own strengths and disadvantages in these strategies .
escs are more easily grown in culture and are pluripotent , meaning that they are able to differentiate into any cell of the body .
the practicality of widespread esc use for therapeutic purposes , however , has been questioned due to issues of supply and ethical and legal considerations . moreover
ascs , on the other hand , overcome some of these problems as they can be harvested from each individual patient .
they are not abundant and are difficult to obtain , often being harboured in internal organs such as the gut and bone marrow .
they have proven difficult to culture in vitro , and furthermore , they are widely believed to be multipotent rather than pluripotent and are thus only able to differentiate into certain cell types .
cellular reprogramming potentially overcomes these issues and may offer new treatment methods for a range of diseases , including those of rbcs . takahashi and yamanaka were the first to show that it is possible to take differentiated somatic cells and transform them into cells with pluripotent potential .
they began by identifying a pool of 24 transcription factors which are important in maintaining stem cell traits and used retroviral transduction to express these factors in murine embryonic and adult fibroblasts .
they found that these cells then displayed characteristics and properties comparable to those of pluripotent escs .
they were able to further refine the required transcription factors by setting up numerous combinations to determine which were essential to this process and identified four factors , oct4 , sox2 , klf4 , and c - myc , needed to transform a somatic cell to an induced pluripotent stem cell ( ipsc ) .
takahashi et al . then applied these four factors to human cells and transformed neonatal and adult human fibroblasts into human ipscs ( hipscs ) .
this was a valuable subsequent step as it showed that this process can be reproduced with human cells and could thus potentially be utilised for stem cell - based therapies of human disease .
several other transcription factor combinations have since been shown to be sufficient to reprogram somatic cells [ 2224 ] and one particular combination , involving oct4 , sox2 , nanog , and lin28 , is particularly efficient and is now widely utilised .
for example , a number of the transcription factors used to generate these cells , including c - myc , klf4 , and lin28 , are oncogenes , and their misexpression can lead to cancer [ 2628 ] . in order for ipscs to be differentiated into a cell type of choice ,
the retrovirally transmitted genes need to be switched off or removed to reduce the possibility of their inducing tumours [ 22 , 29 ] .
another potential issue is that the transplantation of any cells that have not been fully differentiated from the ipsc state could lead to the formation of cancerous teratomas .
any cells remaining which are still in a pluripotent state could multiply and , without the appropriate growth controls , would have the potential to result in tumours in transplant patients . to potentially avoid the issue of tumour formation and uncontrolled proliferation of ipscs , an alternative methodology known as transdifferentiation is also being considered as a potential treatment strategy .
transdifferentiation is achieved by introducing various exogenous factors into a differentiated cell , such as a fibroblast , to directly convert it into another type of differentiated cell , thereby bypassing the pluripotent state ( figure 1(b ) ) . as early as 1990 , choi et al . were able to convert various cells types , including dermal fibroblasts and chondroblasts , into mononucleated , striated myoblasts that were indistinguishable from normal myoblasts in vivo
this was achieved through the expression of the myogenic regulatory factor myod , a transcription factor known to be involved in the determination of muscle cells . shortly after this
, another group showed that it was possible to turn myeloid 416b cells into mast cells through the forced expression of gata-2 and gata-3 , two transcription factors which play important roles in haematopoiesis .
investigated the effects of ectopically expressing the gene eyeless ( ey ) , an orthologue of the mammalian pax6 gene , in drosophila and found that eye structures formed in places such as the wings and legs .
since takahashi and yamanaka 's pioneering studies in cellular reprogramming , the field of transdifferentiation has advanced considerably .
zhou et al . investigated the effects of expressing ngn3 , pdx1 , and mafa , transcription factors involved in -cell differentiation , on exocrine cells of the adult pancreas .
they found that the coexpression of these factors was able to convert the exocrine cells into -cells .
the induced -cells were identical to endogenous -cells in morphology and showed similar expression of genes associated with -cell function .
these cells can also rescue the phenotype of hyperglycaemia , as they are able to secrete insulin and remodel surrounding vasculature . in other work ,
utilised the neural - specific transcription factors , ascl1 , brn2 , and myt1l , to rapidly convert both murine embryonic fibroblasts and adult fibroblasts directly into functional neurons .
these induced neuronal cells express neuron - specific proteins , generate action potentials , and form functional synapses .
a further advance has shown that it is also possible to transdifferentiate fibroblasts into functional neural progenitor cells by transient induction of oct4 , sox2 , klf4 , and c - myc in cells cultured in a defined neural reprogramming medium . this process bypassed the generation of ipscs and gave rise to multipotent progenitors with the capacity to expand and differentiate into a number of neural lineages .
all of these experiments indicate that it is possible to direct a differentiated cell to another cell fate through the application of extragenic factors .
advances in cellular reprogramming have raised the attractive possibility that this technology could be utilised to generate a limitless source of immune - matched , pathogen - free erythrocytes for transfusion .
they found that hipscs are capable of generating haematopoietic cells with phenotypic and morphological characteristics similar to those derived from hescs ; however , these hipsc - derived cells exhibited a dramatically reduced capacity ( by greater than 1000-fold ) to generate erythroid cells . a subsequent study by lapillone et al .
, however , showed that it was indeed possible to produce significant numbers of mature erythroid cells from hipscs in vitro .
this group employed the methods outlined by thomson 's group using oct4 , sox2 , nanog , and lin28 to convert fibroblasts to hipscs .
they then cultured these cells in medium containing the cytokines scf , tpo , flt3 ligand , rhu bmp4 , rhu vegf - a165 , il-3 , il-6 , and erythropoietin ( epo ) .
they analysed the expression profiles of these cells over 20 days of culture and found that pluripotent stem cell markers decreased whilst the erythroid markers cd36 , cd235a , and cd71 increased .
cells at day 20 were found to have a high erythroid potential and were plated in sequential cocktails of cytokines comprising scf , il-3 , and/or epo .
erythroid maturation was achieved after another 25 days and was confirmed by morphological examination and by flow cytometric analysis of erythroid markers ( cd235a , cd71 , and cd36 ) .
furthermore , these erythroid cells were able to enucleate , albeit with reduced capacity compared to hesc - derived erythroid cells , and were found to express functional hb ( predominantly hbf ) .
these hipsc - derived erythroid cells were compared to those differentiated from hescs , and no significant differences were detected in terms of erythroid commitment , expression of erythroid markers , and type and functionality of hb .
revealed that while functional rbcs could be generated from hipscs , when compared to hescs , hipscs were shown to have reduced ( approximately 8-fold ) amplification potential in producing mature erythroid cells .
a recent study has set out to determine whether this reduced efficiency is an intrinsic property of hipscs , or whether it can be increased by altered culture conditions .
this study showed not only that it is possible to generate rbcs from hipscs with similar efficiency to hescs , but also that these cells can be differentiated from hipscs generated using episomal vectors , obviating the need for potentially deleterious retroviral transfection .
this group cultured hipscs derived from human fibroblasts with the op9 bone marrow stroma line to induce hematopoietic differentiation and followed this with selective expansion of erythroid cells in serum - free media with cytokines supporting rbc differentiation .
the erythroid cultures established in this study consisted of a pure population of cd235a+ cd45 leukocyte - free rbcs which had robust expansion capabilities , as well as a long lifespan of up to 90 days .
these hipsc - derived cells can enucleate and were shown to express foetal - and embryonic -globin demonstrating successful reprogramming of the -globin locus .
the results from this study show that it is possible to produce significant numbers of erythroid cells from fibroblast - derived hipscs , and that thus cellular reprogramming could contribute to the treatment of haemoglobinopathies . furthermore , since these rbcs are generated from transgene - free hipscs , they circumvent problems associated with genomic integration and undesired reactivation of reprogramming factors . another recent study has shown that it is possible to use transdifferentiation to produce haematopoietic cell lineages , including mature erythrocytes . in this work , szabo et al .
transduced human dermal fibroblasts with oct4 alone and showed that these cells then express the panhaematopoietic marker cd45 but lack the pluripotency marker tra-1 - 60 .
they then cultured these cells in a haematopoietic cytokine cocktail containing scf , g - csf , flt3lg , il-3 , il-6 , and bmp-4 supplemented with epo and observed an emergence of cells expressing adult -globin protein and the red blood cell marker glycophorin - a .
in addition , the authors noted that these erythroid cells express adult rather than embryonic globins , unlike cells derived from human pluripotent stem cells [ 39 , 40 ] , perhaps suggesting that they have indeed been transdifferentiated from adult fibroblasts , bypassing the hipsc stage .
as the techniques used in this study avoid the pluripotent state as well as have a high yield , expansion capacity , and clinical feasibility , this strategy could provide a reasonable basis for autologous cell replacement therapies .
these studies demonstrate that it is possible to differentiate hipscs into mature erythrocytes in vitro for transfusions . in order to utilise these methods to treat rbc diseases successfully
, fibroblasts could potentially be taken from either a healthy immunomatched individual , or a universal donor , and be converted to erythrocytes via the hipsc state .
a study has already developed hipscs from an individual with a bombay phenotype of the abo blood group system , where the abh antigen is not expressed on rbcs , and blood can be donated to anyone .
this group has further demonstrated that it is possible to differentiate haematopoietic lineages from these cells although they have not explicitly shown mature erythrocyte differentiation .
this possible treatment method circumvents many of the problems associated with current transfusions , such as questionable quality , lack of supply , and immune - rejection .
however , issues still remain , such as cost and the risk of iron overloading .
in order to cure , rather than treat , rbc diseases , healthy progenitor cells must be transplanted into patients and subsequently repopulate the haematopoietic system .
a promising study has already shown that it is possible to use reprogrammed cells to treat sickle cell anaemia in mice .
hanna et al . took cells from the tail tips of a humanized sickle cell anaemic mouse model , in which the mouse -globin genes have been replaced with human -globin , and the mouse -globin genes have been replaced with human and ( sickle ) globin genes , and through adaptation of the protocol developed by takahashi and yamanaka , who reprogrammed these cells into ipscs .
once this was accomplished , they were able to correct the mutant -globin gene by replacing it with a copy of the wildtype human -globin gene through homologous recombination . following this ,
the ipscs were differentiated into haematopoietic progenitors through the ectopic expression of hoxb4 , and by growing in haematopoietic cytokines on an op9 bone marrow stroma cell line .
this group then carried out extensive testing to determine whether the treated mice displayed any further signs of the disease .
firstly , functional correction was evaluated by electrophoresis for the human -globin proteins and , and they found a significant increase in - and a decrease in -globin .
blood counts were also performed 12 weeks after transplant , which showed that treated mice had increased rbcs , hb , and packed cell volume as well as reduced reticulocytes , a common indicator of sickle cell disease and severity .
lastly , they examined symptomatic indicators of sickle cell anaemia such as urine concentration , body weight , and breathing rate and found that all three of these parameters were ameliorated in the treated mice .
this study thus provides an important proof of principle that cellular reprogramming can be employed to correct erythroid disorders , albeit in this case , in conjunction with gene therapy .
normal erythropoiesis is dependent upon the correct expression of globin genes . where globin genes are incorrectly expressed , or are mutated , anaemia or thalassemia results .
current therapy for these disorders involves packed red blood cell transfusions , which are limited by supply , risk of infection , expense , and patient rejection .
drug - based therapies involve the nonspecific reactivation of foetal globins and have long - term side effects . in seeking alternative strategies
, recent advances have shown that cellular reprogramming can now generate large quantities of red blood cells in culture , potentially for use in transfusions .
furthermore , these strategies have been successfully combined with gene therapy to treat a sickle cell anaemia mouse model , suggesting that cellular reprogramming will provide a realistic future alternative to conventional treatment of haemoglobinopathies . | haemoglobinopathies such as thalassaemia and sickle cell disease present a major health burden .
currently , the main forms of treatment for these diseases are packed red blood cell transfusions and the administration of drugs which act to nonspecifically reactivate the production of foetal haemoglobin .
these treatments are ongoing throughout the life of the patient and are associated with a number of risks , such as limitations in available blood for transfusion , infections , iron overload , immune rejection , and side effects associated with the drug treatments .
the field of cellular reprogramming has advanced significantly in the last few years and has recently culminated in the successful production of erythrocytes in culture .
this paper will discuss cellular reprogramming and its potential relevance to the treatment of haemoglobinopathies . |
he had a left ventricular ejection fraction of 42% and regional wall motion abnormalities compatible with multi - vessel territories .
coronary angiography findings revealed total occlusion at the proximal left anterior descending artery ( lad ) , 50 - 75% stenosis at the mid to distal left circumflex artery , and 90% stenosis at the mid right coronary artery ( rca ) .
cardiac surgery was performed through a median sternotomy , and the heart was displaced using a deep pericardial retraction suture , gauze swab , and tissue stabilizer . after confirming the lack of atheroma burden in the ascending aorta by epiaortic ultrasound examination , the left radial artery was connected to the ascending aorta . after completing the lad grafting using the left internal thoracic artery ,
the patient was in the trendelenburg position , and norepinephrine was infused at a dose of 0.15 g / kg / min . after stabilizer application , there was a temporary deterioration in the patient 's hemodynamic status , which was stabilized without the aid of incremental doses of norepinephrine dosage .
after opening the anastomotic site at the om branch , the surgeon informed us the patient was bleeding from the arteriotomy site despite placement of a proximal snare .
the co2 blower was more aggressively used to aid grafting at a flow rate of 3 - 5 l / min .
bleeding was replaced by colloid solution , and all hemodynamic variables were stable : systemic blood pressure 121/58 mmhg , heart rate 59 beats / min , pulmonary arterial pressure 24/11 mmhg , cardiac index 2.6 l / min / m and mixed venous oxygen saturation 82% . approximately 5 minutes after the commencement of grafting , the surgeon decided to insert an intracoronary shunt for better visualization .
suddenly , the patient 's systemic blood pressure decreased to 64/56 mmhg despite increasing the norepinephrine dose from 0.05 g / kg / min to 0.30 g / kg / min .
the pulmonary arterial pressure increased to 34/20 mmhg , and the mixed venous oxygen saturation decreased to 66% .
vasopressin 0.6 iu was quickly administered intravenously , and the transesophageal echocardiography ( tee ) revealed a massive gas floating at the orifice of the rca ( fig .
although the patient remained in normal sinus rhythm , complete heart block occurred within minutes , and ventricular pacing was commenced . over the next several minutes , ventricular fibrillation developed ; despite aggressive treatment , including defibrillation , epinephrine , and direct manual cardiac massage , adequate circulation could not be restored , and cardiac massage was continued . upon preparing for emergent conversion to cardiopulmonary bypass ( cpb ) , epinephrine
300 g was injected directly to the aortic root by the surgeon using a 26-gauge needle .
subsequently , manual cardiac massage was continued for several cycles , and spontaneous recovery of unspecified cardiac rhythm was observed .
then the patient 's systemic mean arterial pressure gradually rose from 38 to 103 mmhg , and recovery of normal sinus rhythm and contractile performance followed immediately .
the remaining grafts were performed successfully without conversion to cpb , and no sign of bleeding was observed at the puncture site in the aorta .
the patient had an unremarkable postoperative course without neurologic deficits and was discharged 10 days after the surgery .
despite using a proximal coronary snare , this method of hemostasis can be incomplete in the presence of severe atheroma or calcification , causing bleeding through the arteriotomy site .
cardiac displacement invariably causes hemodynamic deterioration , and when the diastolic systemic blood pressure decreases below the pressure generated by the co2 gas blower , gas may migrate in a retrograde fashion to the aortic root . even in the presence of a proximal snare
, the proximal coronary lumen may not be completely obstructed due to the presence of calcified atheroma , as evidenced by continued bleeding from the arteriotomy site after application of the snare . as the rca ostium is in the highest position of the sinus of valsalva
, gaseous materials would be prone to migrate into the rca , causing embolic infarction .
a minor gas embolism may pass unnoticed or be overcome with supportive measures to aid emboli clearance .
however , in the case of a massive embolism with the possible introduction of room air due to venturi effect , hemodynamic collapse can occur and may require aggressive treatment , such as cardiopulmonary resuscitation ( cpr ) with aspiration of the air by direct puncture of the aortic root .
it may even require emergent conversion to cpb , which is known to be associated with increased morbidity and mortality .
therefore , when bleeding from the arteriotomy site persists despite the use of a proximal snare or intracoronary shunt , anesthesiologists should be aware of the possibility of a gas embolism and advise the surgical team to stop or limit the use of the co2 blower .
when a gas embolism results in hemodynamic deterioration , supportive measures should be immediately undertaken to increase the coronary perfusion pressure and facilitate emboli clearance .
gas embolism should also be confirmed with tee , and a pacemaker should be at hand given that complete heart block due to atrioventricular nodal ischemia may occur in the case of an embolism involving the rca .
finally , in the case of failure of supportive measures such as drug administration to the venous side , direct aortic injection of the drugs can be attemped before initiating cpb . in coronary artery bypass graft surgery
, direct cardiac massage may not be aggressively and effectively performed due to the fear of disrupting the previously grafted conduits , limiting the drug delivery from the venous side to the systemic circulation .
prior to the 1960s , intracardiac injection of medications was used blindly or directly , but this approach was abandoned due to the devastating complication of lacerating the epicardial coronary arteries and cardiac tamponade .
however , direct injection to the aortic root following a sternotomy appears to be feasible and may be life - saving , as severe right ventricular dysfunction may limit systemic drug delivery injected through the venous side despite the performance of direct cardiac massage . in the current case , intra - aortic injection of epinephrine may have resulted in a more effective increase in the systemic vascular resistance , thereby increasing the coronary perfusion pressure and thus facilitating emboli clearance . in conclusion , in the rare case of a gas embolism occurring during opcab occurs , the potential for catastrophic results necessitates immediate and appropriate managements including supportive measures , needle aspiration of gas , and cpr with direct cardiac massage . in refractory cases , while considering emergent conversion to cpb , direct injection of drugs to the aortic root can be attempted before initiating cpb .
this may be a simple , relatively safe , and life - saving approach for the management of a gas embolism during opcab because a sternotomy has already been conducted . | although compressed gas ( co2 ) blowers have been used safely to aid accurate grafting during off - pump coronary bypass surgery , hemodynamic collapse due to gas embolism into the right coronary artery may occur .
supportive measures to facilitate gas clearance by increasing the coronary perfusion pressure have been reported to be successful in restoring hemodynamic stability . however , right ventricular dysfunction and atrioventricular nodal ischemia may hinder effective systemic delivery of the vasoactive medications , even when performing resuscitative measures such as direct cardiac massage .
we herein report a case of cardiac arrest that was caused by a right coronary gas embolism and that could not be restored by cardiac resuscitation .
when supportive measures fail , direct aortic injection of epinephrine to increase the coronary perfusion pressure can be attempted before initiating cardiopulmonary bypass , and this approach may be life - saving in situations that limit systemic drug delivery from the venous side despite the performance of direct cardiac massage . |
multiple sclerosis ( ms ) is a chronic progressive inflammatory and neurodegenerative disease of human central nervous system ( cns ) which usually affects young adults , with a reported median age of onset of approximately 30 years.1 in approximately 85% of patients with ms , the disease is initially recognized by a relapsing - remitting course which may eventually lead to a progressive deterioration of neurological status along with significant loss of neurological function.2 neuropathologically , ms presents with various patterns , however , inflammatory demyelination and oligodendrocyte loss and neuronal and axonal degeneration3 constitute salient features of microscopic examination of ms lesions .
currently , the cause and cure for ms remain elusive and in many cases ms ends with significant neurological disabilities . while massive activation of the inflammatory arm of the immune system against putative cns antigen(s ) such as myelin basis protein plays a major role in the pathogenesis of the demyelinating process of ms , the neurodegenerative process prevails in the long run and causes significant cognitive and functional deterioration in affected patients .
electrophysiologic examination of demyelinated lesions indicates conduction block as the predominant feature of these lesions .
the pathophysiologic basis of such conduction block in ms rests on segmental demyelination with loss of whole internodes of myelin . in normal individuals , blocking the voltage - gated potassium channels does not have any significant impact on conduction of the action potential since these channels are enclosed by layers of myelin sheath .
however , loss of myelin during pathogenesis of ms , exposes the potassium channels and interferes with production and conduction of the action potential .
in fact , the impulse conduction fails specifically at the site of demyelination , while the normal segments of the axons on both sides continue their impulse conduction normally .
certain features which determine the impulse conduction block in demyelinated axon consist of the size of demyelinated area , extent of myelin loss along the internode , and the time elapsed since demyelination happened .
the duration of demyelination is an important factor since myelin loss sets a number of adaptive axonal responses in motion .
for example , development of a revised group of ion channels along the demyelinated membrane may result in restoration of impulse conduction .
the size of the demyelinated lesion is another significant factor since axons repair centripetally inside the lesion and extensive lengths of demyelination are repaired slowly which in turn reflects the fact that the chance of functional recovery correlated inversely with the lesion size .
it appears that myelin loss involving the paranodal area is stronger factor in blocking conduction than a comparable loss of distributed along the internode .
apart from the segmental demyelination which is associated with conduction block , histopathologic examination of ms tissue has shown that some node are inappropriately wide in ms lesions due to paranodal demyelination or retraction of the paranodes.4 another salient feature of demyelinated axons is abnormal reorganization of the ion channels . the speedy conduction velocity and uninterrupted transmission of electric signals in mammalian myelinated axons rest on the appropriate spatial distribution ofion - selective channels . na channels are gathered at nodes of ranvier in concentrations which are ~25 times that of internodal regions.5 these clusters are essential for efflux of ion currents at nodes and allow fast saltatory conduction .
on the contrary , other studies have demonstrated that k currents appear only after loosening of the myelin sheath from the axonal membrane.6 k channels are located only in paranodal and internodal areas and normally they do not contribute to generation of the action potential.7 scientific studies which have focused on action potential propagation in normal and demyelinated axons have revealed a reorganization of axolemmal ion channels , which in turn leads to conduction impairments . loss or decrease of sodium channels from the nodes of ranvier8 causes the nodes to be inexcitable and leads to conduction block .
sherratt et al9 demonstrated that in demyelinated regions sodium channel concentrations are reduced , while voltage - sensitive potassium channels ( which are not active on normal myelinated axons ) become detectable .
this exposure of k channels at juxtaparanodal region leads to efflux through fast k channels and causes axonal conduction block by preventing sufficient depolarization and initiation of the action potential at the node of ranvier.10 demyelination also exposes slow k channels , further interrupting normal hyperpolarization and blunting normal repetitive impulse release from the presynaptic area terminals.11 in 1981 , bostock et al12 demonstrated that potassium channel blockers dendrotoxin and 4-aminopyridine improved conduction impairments in experimentally demyelinated axons and laid the scientific rationale for clinical studies of potassium channel blockers in ms patients .
fampridine ( or 4-aminopyridine [ 4-ap ] ) is a broad - spectrum blocker of voltage - sensitive potassium channels , which has been demonstrated to improve conduction and propagation of the action potential along the demyelinated axons .
in addition , fampridine increases the duration of the pre - synaptic action potential and amplitude which in turn leads to increased transmitter release.13 apart from being a voltage - activated potassium channel blocker , 4-aminopyridine potentiates synaptic and neuromuscular transmission by affecting the beta subunit of voltage - activated calcium channels.14 this novel mode of action of 4-aminopyridine which occurs independent from its blocking effect on the potassium channels , provides another rationale for its positive effect on the neuromuscular function in patients with spinal cord injury , myasthenia gravis and ms .
hawthorne , ny , usa ) is one of the three isomeric amines of pyridine with chemical formula h2nc5h4n , which acts a selective potassium - channel blocker .
dalfampridine , which is a sustained - release oral form of fampridine , is rapidly and completely absorbed from the gastrointestinal tract , however , its absolute bioavailability has not been determined .
compared to an aqueous oral solution , the extended release tablet shows a relative bioavailability of 96% , with a delayed absorption pattern which provides a less rapid rise to a lower maximum plasma concentration . in healthy volunteers who took a single 10 mg dose of dalfampridine
, peak serum concentrations were reached 3 to 4 hours following oral administration and it was almost completely and rapidly eliminated by urinary excretion .
the elimination half - life of dalfampridine is 6.4 hours in healthy individuals . as a lipid - soluble agent
, dalfampridine passes through the blood brain barrier and blocks potassium channels in both the central and peripheral nervous systems.15 daldampridine binds reversibly to the cytoplasmic side of potassium channels , blocking the ion conductance pathway which in turn prolongs the action potentials in unmyelinated nerve fibers and enhances neurotransmitter release at synaptic endings.16 dalfampridine has been studied extensively in symptomatic treatment of ms patients with walking impairment .
impairment of ambulation is a major neurological deficit in ms patients and significantly interferes with their quality of life .
it is hypothesized that dalfampridine improves clinical symptoms of ms by restoring conduction in demyelinated axons through voltage - dependent potassium channel blockade.15 in addition , dalfampridine has been used for treatment of lambert - eaton myasthenic syndrome,17 where it prolongs neurotransmitter release at the neuromuscular junction .
studies involving animal models have demonstrated that can reverse tetrodotoxin toxicity.18 pharmacokinetic assessmentsof both immediate - release and sustained release formulations of fampridine have been done in normal individuals as well as those patients with ms and spinal cord damage .
uges et al19 performed a pharmacokinetic study involving 9 healthy individuals ( 7 men and 2 women ) to assess immediate - release fampridine in intravenous , entric - coated , and uncoated oral formulations .
the bioavailability of the enteric coated tablets was calculated from the area under the serum concentration curve 95% 29% ( mean sd ) , which was not much different from that calculated from urinary excretion 98% 8% . there was no evidence that metabolism of fampridine involved glucoronidation , sulfonation , or n - acetylation .
investigators reported that 89.6% 7.5% of the medication was excreted in the urine unchanged after 24 hours of the intravenous formulation and 86.1% 2.7% with the entric coated formulation .
one dose - ranging pharmacokinetic study included 12 ms patients who received increasing doses of sustained - release fampridine from 7.5 to 25 mg every 12 hours.20 the investigators reported a mean time to peak concentration of 5 hours and mean serum half - life of 5.2 hours .
vollmer et al21 studied the pharmacokinetics of dalfampridine in clinical trials of ms patients and found that multiple dosing of this medication was associated with its accumulation .
in addition , the investigators noted that the steady - state pharmacokinetic profile of fampridine sustained - release 20 mg bid administered for 2 weeks appeared to support the administration of twice - daily dosing in this population .
dalfampridine ( ampyra ) is available as 10 mg extended release tablets with relative bioavailability of 96% , peak of plasma concentration of 34 hours and half life of 5.26.5 hours .
the major goals of treating ms patients consist of prevention of exacerbations , limiting disability progression , and improving cognitive decline . the available treatments for ms fall under two categories : disease modifying agents such as beta - interferons and glatiramer acetate and medications for symptomatic treatment such as baclofen for treatment of spasticity and dalfampridine which is used to restore the impulse conduction along the demyelinated axons and improve muscle strength and walking ability . in january 2010
, dalfampridine was approved by the fda for symptomatic treatment of ms patients with specific indication for improving walking . in the past
4-aminopyridine has been used as an experimental agent which presumably enhances nerve conduction of demyelinated axons through its effects on potassium channels for treatment of fatigue in ms .
sheean et al22 reported that use of fampridine in ms patients was associated with improvements in fatigue as well motor and visual symptoms .
in addition , dalfampridine has been assessed and studied in other cohorts of ms patients in the context of various clinical trials to determine its efficacy in improving ms patients ambulation . in this manuscript only some of the more significant clinical trials have been cited and discussed .
using quantitative measures of motor function , schwid et al23 performed one of the earlier clinical trials of sustained - release fampridine - sr in ms patients .
this was a randomized , double - blind , placebo - controlled , crossover clinical trial included 10 patients with clinically definite ms , limb weakness and stable motor deficits ( edss 6.07.5 ) .
study subjects were randomized to be treated with sustain - release fampridine 17.5 mg orally twice daily for a period of one week or placebo for a period of one week .
study outcomes included time to walk 8 meters , time to climb four stairs , maximum voluntary isometric contraction measured quiantitatively , manual muscle testing , grip strength , edss , and the patient s global impression .
of these outcomes , only timed gait progressed on sustain - release fampridine compared with placebo in 9 of the study participants .
seven participants preferred the medication over placebo based on the findings from the patient s global impression .
goodman et al24 reported the results of a four - center randomized , double - blind placebo - controlled study of controlled release aminopyridine in 31 ms patients .
the study participants were treated with increasing doses of 20 to 80 mg of aminopyridine daily in divided doses .
a total of 25 participants received the active medication and 11 participants were treated with placebo .
outcome measures of this clinical trial consisted of timed ambulation , manual testing of leg strength , paced auditory serial addition task , 9-hole peg test , and a fatigue diary .
the results of this clinical trial indicated a statistically significant and dose - related enhancement of timed ambulation in ap - treated patients compared to those who received placebo .
a significant enhancement of leg strength was also observed in approximately 11% of ap - treated participants compared with a 4% worsening of placebo - treated participants , p = 0.01 .
the reported side effects consisted of dizziness , insomnia , paresthesia , nausea , headache , tremor , pain and anxiety . on daily doses of 60 and 70 mg
a phase 2 , multi - center , randomized , double - blind , placebo - controlled , parallel - group , dose - comparison ( 10 , 15 , or 20 mg twice daily orally ) clinical trial of sustained - release fampridine in ms patients demonstrated significantly more consistent responders as determined by improvement in ambulation as compared to the placebo - group : 36.7% versus 8.5%.25 fampridine was generally well tolerated and serious and severe adverse effects were more common in those patients who received the highest dose . a multi - center randomized , double - blinded , placebo - controlled phase 2 dose - raning clinical trial was conducted by goodman et al which assessed the concept that whether the dose of fampridine - sr could be safely elevated to 80 mg / daily in patients with ms.26 a cohort of 36 ms patients were randomized in a 2 to 1 ratio to be treated with an increasing dose of fampridine - sr for a period of 8 weeks . the dosage of fampridine - sr was escalated from 10 to 40 mg twice daily by increments of 5 mg twice daily on a weekly schedule .
a group of evaluations were done once a week which included ms functional composite ( msfc ) , fatigue questionnaires , and lower extremity manual muscle testing .
the objectives of this clinical trial included assessment of the safety of higher doses of fampridine - sr as well as the efficacy and dose - response to this drug by ms patients .
five study participants were terminated from fampridine - sr due to adverse events at doses larger than 25 mg and these events consisted of seizures in two individuals at doses of 30 and 35 mg twice daily .
no subjects in the placebo arm of the study dropped out due to the adverse events . in the group which was treated with fampridine - sr
compared to the group which was treated with placebo showed improvement in the lower extremity muscle strength ( prospective analysis ) and walking speed ( post - hoc analysis ) .
the investigators concluded that future clinical trials should restrict the dose of fampridine - sr to 20 mg twice daily and walking speed and lower extremity muscle strength tests are potential efficacy measurements .
another phase 3 multicenter clinical trial of dafampridine which was controlled and double - blind included 301 ms patients of different types ( 27% with relapsing - remitting ms and 73% with progressive ms ) . during the 14 weeks duration of this trial
, participants were assigned in a ratio of 3 to 1 to be treated with dalfampridine 10 mg ( n = 229 ) or placebo ( n = 72 ) orally twice daily for a period of 14 weeks.27 the primary aim of this study included steady progress on timed 25-foot walk to define responses with proportion of timed walk responders in each arm .
the investigators reported a higher proportion of timed walk responders in the fampridine - treated arm ( 78/224 or 35% ) compared to those who were treated with placebo ( 6/72 or 8% ) .
based on the results of this study , treatment of ms patients with fampridine was associated with gait improvement with a decrease in patient s gait impairment .
a more recent executed phase 3 multi - center double - blind clinical trial of extended - release dalfampridine in patients with definite ms of any clinical course assessed the efficacy , safety , and pharmacodynamics of extended release oral dalfampridine in these patients.28 the length of this study was 9 weeks and the participants were randomized to be treated with dalfampridine ( 10 mg twice daily ; n = 120 ) or placebo ( n = 119 ) for a period of 9 weeks .
the primary aim of this study consisted of constant progress on the timed 25-foot walk , with percentage of time walk responders ( twrs ) in each treatment arm .
the last on - treatment assessment collected information from 8 to 12 hours postdose , to assess and determine the persistence of the impact of the medication .
the results of this clinical trial indicated that the quantity of twrs was higher in the dalfampridine - treated group ( 51/119 or 42.9% ) compared to the placebo - treated group ( 11/118 or 9.3% .
the average enhancement of walking speed among the dalfampridine - treated twrs during the 8-week efficacy assessment period was 24.7% compared to baseline ; the mean enhancement during the finalon - treatment visit was 25.7% , reflecting continuation of the impact of medication over the interdosing phase .
the authors concluded that this clinical trial generated class 1 evidence that treatment of patients with definite ms with extended - release dalfampridinetablets caused meaningful enhancement of walking ability in and the effect of the medication continued persisted between doses .
based on the animal studies , dalfampridine demonstrates properties of a powerful convulsant stemming from its broad - spectrum suppressing activity on the potassium channels in the peripheral and central nervous systems.11 based on a number of clinical trials of 4-ap side effects consist of dizziness , insomnia , paresthesia , nausea of mild to moderate intensity , tremor , pain , anxiety and seizures.2934 dalfampridine is neither a substrate for nor an inhibitor of the p - glycoprotein transporter , therefore its pharmacokinetics is not affected by the medications which suppress this transporter .
it does not interact with the medications which are substrates for the p - glycoprotein transporter .
dalfampridine is contraindicated in patients with history of epilepsy or history of severe renal insufficiency.35 the reason that dalfampridine use is not recommended in epileptic patients is that epilepsy is characterized by abnormal neuronal depolarization with excessive inhibition on potassium channels , hence a further reduction of potassium channel activity by dalfampridine is not advised .
pathogenesis of ms involves loss of oligodendrocyte / myelin complex which turn results in re - organization of axolemmal ion channels which leads to conduction abnormalities including conduction delay or block .
pharmacological agents which block potassium channels have been investigated in the context of clinical trials with positive impact on impulse conduction in experimentally - induced demyelination as well as in patients with ms .
dalfampridine ( which is marketed as ampyra ) , is an extended release form of fampridine ( 4-aminopyridine ) , which has been recently approved by the us fda for symptomatic treatment of ms patients .
while this new oral blocker of voltage - gated potassium channels does not have any impact on the underlying pathology of ms , it has been demonstrated to improve fatigue and walking ability in these patients . | multiple sclerosis ( ms ) is a progressive immune - mediated neurodegenerative disease of human central nervous system ( cns ) , which causes irreversible disability in young adults .
the cause and cure for ms remain unknown .
pathophysiology of ms includes two arms : inflammatory demyelination and neurodegeneration .
the inflammatory demyelination of ms which is mainly promoted by a massive activation of the immune system against putative cns antigen(s ) leads to loss of oligodendrocyte / myelin complex which slows down or halts impulse conduction in denuded axons .
practically , loss of myelin significantly reduces signal conduction along the demyelinated axons through alterations in the distribution of axonal ion channels .
dalfampridine ( 4-aminopyridine or 4-ap ) is an oral potassium channel blocker , which was recently approved by fda for symptomatic treatment of ms .
dalfampridine , which acts at the central and peripheral nervous systems , enhances conduction in demyelinated axons and improves walking ability of ms patients .
a number of clinical trials have evaluated the safety and efficacy of fampridine in ms patients with the degree of gait improvement as the main outcome .
the objective of this manuscript is to provide an overview of the pharmacology , pharmacokinetics , clinical trials , side effects and interactions of dalfampridine used in treatment of ms patients . |
several imaging modalities including pa , occlusal and panoramic radiography , cone beam computed tomography ( cbct ) and ct are used before and after implant treatment as well as in the maintenance sessions . after successful implant placement ,
periapical radiography is usually a suitable imaging modality for long - term follow - ups .
radiographically , a thin radiolucent margin around the implant indicates implant mobility and is an important sign of failed osseointegration [ 13 ] .
the popularity of digital radiography in dentistry is mainly due to the adjustability of digital images by using image inversion , embossed tools , brightness , contrast and magnification enhancement filters [ 49 ] . by applying image inversion to digital images ,
inverted digital images are used for various purposes in dentistry such as the measurement of bone loss due to periodontal disease and localization of the mandibular canal and mental foramen [ 10,1117 ] .
several previous studies have compared the diagnostic accuracy of unprocessed digital images ( psp and charge - coupled device or ccd ) , film - based radiographs and inverted images for detection of peri - implant bone defects and simulated periodontal lesions using image tool and adobe photoshop software programs [ 1214 ] .
this study aimed to compare the diagnostic value of unprocessed ( psp ) and inverted digital images for detection of peri - implant defects using scanora software .
soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss .
osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .
a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .
the sicace screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .
osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,
the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .
the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space . to stabilize fixtures in bone , drilling
was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap .
all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . ) were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness
periapical radiographs were obtained from the bone segments at the same day the fixtures were placed .
all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )
the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed .
after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .
four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale :
definite defectprobable defectnot sureprobably no defectdefinitely no defect
examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .
the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) .
study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .
the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .
independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities .
soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss .
osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .
a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .
the sicace screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .
osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,
the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .
the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space .
to stabilize fixtures in bone , drilling was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap . all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . )
were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness
periapical radiographs were obtained from the bone segments at the same day the fixtures were placed .
all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )
the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed .
after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .
four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale :
definite defectprobable defectnot sureprobably no defectdefinitely no defect
examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .
the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) .
in this study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .
the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .
independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities .
a total of 60 digital pa radiographs including 30 unprocessed and 30 inverted radiographs were obtained of 30 implants with the same diameter and length : 10 implants with no defects , 10 with small ( 0.425 mm ) diameter peri - implant defects in the coronal 8 mm and 10 with large ( 0.725 mm ) diameter peri - implant defects in the coronal 8 mm .
based on the results , unprocessed images had a higher diagnostic value than inverted images . according to the t - test , in the equality of the means , no significant difference was detected in the sensitivity of unprocessed and inverted images for detection of peri - implant defects ( absolute positive predictive value ) and for detection of a probable lesion ( complete positive predictive value)(p>0.05 ) .
considering the equality of variances , the t - test showed that unprocessed images had higher absolute negative predictive value ( p=0.049 ) and complete negative predictive value ( p=0.017 ) than inverted images ( table 1 ) .
the mean standard deviation of absolute and complete negative and positive predictive values of unprocessed and inverted images the mean standard deviation of sensitivity and specificity of unprocessed and inverted images for detection of small and large defects significantly different from zero ( p<0.05 ) considering the equality of variances , the t - test showed that unprocessed images had a higher absolute sensitivity for detection of small defects than inverted images ( p=0.48 ) .
however , the mean complete sensitivity of the two modalities for detection of small defects despite the non - equality of variances ( p=0.001 ) and equality of the means , was not significantly different ( p=0.81 ) ( table 2 ) . considering the equality of variances ( p=0.055 ) , the t - test failed to find a significant difference in the absolute sensitivity for detection of large defects between the unprocessed and inverted images ( p=1.0 ) .
however , despite the equality of variances ( p=0.003 ) , the t - test found a statistically significant difference in complete sensitivity for detection of large defects and unprocessed images had a higher mean value in this respect ( p=0.019 ) ( table 2 ) . in the non - equality of the means ,
the mean absolute specificity of unprocessed and inverted images was significantly different based on the t - test ( p<0.05 ) and unprocessed images had a higher diagnostic value .
however , in the equality of the means , the complete specificity of unprocessed and inverted images was not significantly different ( p>0.05 ) ( table 2 ) .
periapical radiography is conventionally used as the standard follow up radiography after implant placement to assess the peri - implant tissue status .
the digital imaging techniques and different enhancement filters have enabled more accurate diagnoses in dentistry .
this study showed that unprocessed images had a higher diagnostic value than inverted images for detection of small and large peri - implant defects .
this finding may be due to the unfamiliarity of the inverted images to the eyes of the observers .
our obtained results were in accord with those of kavadella et al , de molon et al , and jorgenson et al , despite the differences in the type of receptors ( film and digital sensor ) , type of defects and method of creating the defects .
however , our results were in contrast to those of scaf et al , in 2007 .
compared unprocessed and inverted digitized images for detection of bone loss due to periodontal disease and found no significant difference in the diagnostic value of unprocessed and inverted images .
scaf et al , in their study used pa radiographs available in the records of patients with periodontal disease .
the difference between our results and those of scaf et al . may be attributed to the different methodology and the software programs used .
kavadella et al , in their in - vitro study compared film - based conventional radiography with unprocessed and inverted digital radiography with ccd sensors for detection of peri - implant lesions .
our results had some differences with those of kavadella et al . in our study ,
the sensitivity and specificity of unprocessed images were higher than those of inverted digital images .
the complete sensitivity for detection of small defects was 0.90 for unprocessed and 0.62 for inverted images .
the complete sensitivity for large defects was 1 for unprocessed and 0.85 for inverted images .
. however , kavadella et al . showed high specificity and low sensitivity values .
such differences may be due to the methodology of studies i.e. method of defect formation , using fresh bone in our study and use of magnification enhancement ( x2 ) by the observers in the study by kavadella et al .
however , our results regarding the lower accuracy of inverted digital images compared to unprocessed images were similar to those of kavadella et al .
molon et al , in their study compared the diagnostic accuracy of unprocessed and converted ( cmos sensor ) images with film - based conventional radiographs for detection of bone loss due to simulated periodontal disease .
the results showed that inverted digital images had lower accuracy than film - based radiographs . despite the difference between the type of sensors used in our study and the study by molon et al ,
similar results were obtained indicating the higher diagnostic accuracy of unprocessed digital images and film - based radiographs than inverted images .
jorgenson et al , in their comparative study evaluated the diagnostic accuracy of conventional f - speed film radiographs , unprocessed digital radiographs and inverted digital images ( psp , digora ) in patients with vertical bone defects .
the digital sensor used in our study was similar to that used by jorgenson et al , and our results were in accord with their findings .
based on the results , we found that unprocessed digital images had higher diagnostic accuracy than inverted images for detection of small and large peri - implant defects . | objectives : this study aimed to compare the diagnostic accuracy of inverted and unprocessed digitized periapical radiographs for detection of peri - implant defects.materials and methods : a total of 30 osteotomy sites were prepared in three groups of control , study group 1 with 0.425 mm defects and study group 2 with 0.725 mm defects using the sic and astra tech drill systems with 4.25 mm and 4.85 mm diameters .
small and large defects were randomly created in the coronal 8 mm of 20 implant sites ; implants ( 3.4 mm diameter , 14.5 mm length ) were then placed .
thirty periapical ( pa ) radiographs were obtained using digora imaging system ( soredex corporation , helsinki , finland ) , size 2 photostimulable storage phosphor ( psp ) plate sensors ( 40.0mm30.0 mm ) and scanora software .
unprocessed images were inverted using scanora software by applying image inversion and a total of 60 images were obtained and randomly evaluated by four oral and maxillofacial radiologists .
data were analyzed using the t-test.results:significant differences were observed in absolute and complete sensitivity and specificity of the two imaging modalities for detection of small and large defects ( p<0.05 ) .
unprocessed digital images had a higher mean in terms of absolute sensitivity for detection of small defects , complete sensitivity for detection of large peri - implant defects and definite rule out of defects compared with inverted images.conclusion:unprocessed digital images have a higher diagnostic value for detection of small and large peri - implant defects and also for definite rule out of defects compared with inverted images . |
upper gastrointestinal bleeding ( ugib ) is a major cause of mortality and morbidity accounting for about 56.5/100,000 hospitalization with a mortality rate of 8.2% .
aorto - esophageal fistula ( aef ) , an abnormal anatomical communication between the aorta and esophagus is a rare cause of ugib .
as the thoracic aneurysm grows in size , the increasing tension on its wall will erode the aorta and rupture into the surrounding esophagus leading to uncontrolled bleeding .
as ulcer related or variceal bleeding constitutes for most of the ugib bleeding , even in elderly who are at risk of aef and the inability of the esophagogastroduodenoscopy ( egd ) to detect most cases , aef is often not diagnosed timely .
all these factors make the timely diagnosis of aef challenging , and at times missed altogether leading to catastrophic consequences .
we present a case of aef which was initially treated as variceal bleeding and later promptly diagnosed with computed tomography ( ct ) .
a 70-year - old male patient with hypertension with regular ethanol use , presented to a local hospital with complaints of hematemesis and melena from the past 1-day .
he was hemodynamically stable on presentation with initial hemoglobin level of 7.2 g / dl .
he underwent timely egd , which showed grade 1 mid and distal esophageal varices without any active bleeding .
subsequently , his condition deteriorated with the development of hypotension and transferred to our medical center for further management .
weiss tear without active bleeding along with the presence of large blood clots in the gastric fundus .
as the source of bleeding was not clear despite two egd and clinical evidence of gi bleeding , he underwent emergent colonoscopy which once again failed to detect any source lower gi bleeding .
however , later in the day , he developed massive hematemesis and hematochezia leading to persistent hemorrhagic shock .
emergent intubation was done for airway protection , massive transfusion protocol was initiated , and a ct angiogram was performed .
it revealed type b aortic dissection with rupture and the active extravasation of blood into the esophagus , confirming the diagnosis of aef [ figure 1 ] .
he underwent emergency percutaneous thoracic endovascular aortic repair with aortic stent placement [ figure 2 ] .
his shock resolved hemoglobin stabilized and did not have any more episodes of bleeding thereafter .
repeat egd showed a 3-cm opening in the midesophagus with ragged and irregular borders , not seen earlier egds .
his hospital course was complicated by the infection of the endograft which was replaced and fortified with an omental flap and being is presently being , followed by for reconstruction surgeries .
( a ) communication and extravasation between aortia and esophagus ( blue arrow head ) in the arterial phase .
( b ) accumulation of blood in lower esophagus in venous phase ( yellow arrow head ) .
( c ) significant accumulation of blood in the stomach in the kidney delay images ( red arrow head ) .
( d ) sagittal reconstruction images shows commination ( blue arrow ) and extravasation ( yellow arrow ) of blood between aorta and esophagus endovascular graft in the thoracic aorta
aorto - esophageal fistula is an abnormal anatomical communication between the aorta and esophagus . in 1818
, dubrueil described this condition for the 1 time in a french soldier who ingested a beef rib , leading to massive hematemesis .
almost a century later , chiari described the triad of midthoracic pain , herald hemorrhage , and fatal hematemesis seen in these patients .
the exact incidence of this condition is not known , as the vast majority of these patients die before a definitive diagnosis can be made . in the past , infections such as tuberculosis and syphilis , leading to aortitis were the common cause of this condition . in this antibiotics era , thoracic aortic aneurysm constitutes for more than half of these cases .
other causes described in the literature are cancer , acid reflux disease , trauma , caustic ingestion , and esophageal biopsies .
aefs are classified as primary if they are caused by the spontaneous erosion of the aortic wall into the esophagus and as secondary if they occur as a complication following aortic or esophageal surgeries .
owing to the increasing number of interventions of the aorta , secondary aef is 10 times more common than primary .
the herald bleeding described by chiari is only seen in half of these patients . once the bleeding starts , rapid extravasation from aorta will cause a sudden drop in blood pressure .
, the blood pressure will raise again dislodging the fresh and loose clot around the aef leading to terminal bleeding .
commonly used resuscitation fluids including pseudo random binary sequence lack adequate clotting factors add to the compromise of clot integrity . unlike in other structures ,
the lax mediastinum around the aorta and esophagus prevent autotamponade , to prevent persistent extravasation of blood .
these factors can contribute to torrential bleeding , the final hematemesis as described by chiari .
the risk of aortic aneurysm increases with age , with an incidence of 12.5% in elderly americans .
the sensitivity of egd to identify the source of bleeding in other causes of ugib is 90% , but for aef it is < 10% .
it may show pulsatile mass , bluish discoloration , or simply blood clotting in the esophagus in one - third of cases .
a ct arteriogram is diagnostic in most cases , which show active extravasation of blood from the aorta to esophagus .
once diagnosed , aef should be treated with emergent aortic stenting to control the bleeding .
owing to contamination from the gi tract , one - quarter of stents become infected . apart from broad - spectrum antibiotics and antifungals ,
once the infection is controlled , the complex aortic and esophageal reconstruction is done in multiples stages .
aef , once considered to be uniformly fatal , is now a treatable condition with timely intervention .
aorto - esophageal fistula is a rare , but life - threatening condition presenting as ugib .
a high level of suspicion is needed , as routine tests performed for ugib typically only detect one - third of cases . avoiding over - resuscitation can be helpful in preventing subsequent hemorrhages . | aorto - esophageal fistula is a rare cause of upper gastrointestinal bleeding .
thoracic aneurysm , the most common cause of this condition , will slowly increase over time and can erode the wall of the aorta creating a fistula and leading to torrential bleeding .
high clinical suspicion is required for timely diagnosis as common investigations routinely done for gastrointestinal ( gi ) bleeding , including esophagogastroduodenoscopy , fails to detect most cases .
the classical triad of midthoracic pain , herald bleeding and fatal hematemesis described in this condition is seen in only one - third of cases .
physician should be wary of this condition , especially in elderly patients with uncontrolled gi bleeding and who are also at risk of thoracic aneurysm .
computed tomography angiogram detects most cases and emergent endovascular repair with stents controls the initial bleeding .
later , both the aorta and the esophagus are repaired and reconstructed in staged procedures . |
the discovery of the silicon pn junction is a landmark in the development of modern microelectronics .
silicon and other inorganic semiconductors are the predominant materials in electronic manufacturing today ; however , the development of organic - based electronic components has been a focus of intensive research and development .
organic electronics have become an active research area in recent years because they are an alternative to the traditional semiconductor technology and challenges in design on a smaller scale from microscale to nanoscale .
moreover , organic materials have vast potential for integration in low - cost microelectronic devices .
organic - based semiconductors , for example polymeric gels , may have superior properties due to their flexibility against to regulate some physical properties of the materials even after the production , e.g. , by changing the volume of the gel .
circuits and displays based on organic electronics may also be flexible , low - weight , and environment friendly .
due to great diversity and functionality of the polymers , they may also be produced for specific requirements , like biocompatible semiconductors .
last , some water - tolerant organic devices may be biocompatible , that is , they may be used in circuits that directly reside on or are implanted inside animal and human tissue and could perform various sensing , interfacing , and controlling functions for drug delivery , prosthetics , and neural - electronic integration .
insulating polymers have been made conductive so far by using various methods such as ion implantation [ 1 - 4 ] , press contacting , and photochemical doping .
the impressive properties of these materials that have been taken advantage are the ability to manipulate electronic properties by changing their molecular structure .
differently current rectifying in semiconductor diodes via diffusion of ionic charges across a pn junction sets up a built in potential ; there is unidirectional current is observed across a membrane where a built in potential arises from diffusion of anions and cations in organic junctions .
we considered that the principle of the electrical conductivity in polymeric gels can be changed by doping static ( inactive ) ions having counter ions as charge carriers and these gels can be used to form pn junctions that work similar to inorganic semiconductors .
these semiconductor polymeric gels can be doped with both negative ( n - type ) and positive ( p - type ) counter ions as charge carriers .
we have shown that the pn junction formed by combining p - type and n - type gels together in much closer contact rectifies the current .
p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .
the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .
the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1 1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .
after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .
thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .
after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements .
n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3 10 , 3 10 , 3 10 mol / l [ 13 - 15 ] .
the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .
aam , nipa , and doping concentrations of the samples were summarized in table 1 .
properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples .
the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .
the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels .
p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .
the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .
the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1 1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .
after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .
thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .
after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements .
n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3 10 , 3 10 , 3 10 mol / l [ 13 - 15 ] .
the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .
aam , nipa , and doping concentrations of the samples were summarized in table 1 .
properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples .
the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .
the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels .
the pyranine binds to the polymer chains , over its oh group , chemically during the polymerization as it is discussed in detail in references [ 10 - 12 ] , and they form stable charged sites doped with positive counter ions . thus , the polyacrylamide gel is doped with pyranine having ions as side groups and na as counter ions , so - called p - type semiconducting gel .
similarly , nipa gel was doped with maptac , having cl as counter ions , so - called n - type semiconducting gel .
the maptac binds to the polymer chains chemically during the polymerization , thus they form stable charged sites doped with negative counter ions as represented in scheme 1together with the p - type gel .
this doping process was also discussed in detail in references , . when these gels are in the collapsed state , no current is observed .
when the gels start to swell , the current starts to flow through the gels .
schematic representation of the p - type ( a ) and n - type ( b ) gels doped with pyranine and maptac the currents decrease with time for a constant applied voltage as shown in fig .
as the charge carriers ( the ions in water phase of the gel ) were gathered on the electrodes , the density of the charge carriers decrease in time , and thus the current . as seen , the initial values of the currents measured for the doped gels are considerably bigger than that of the neat gel ( the gel that was not doped with ions ) at a certain swelling ratio , v / v0 = 1.28 and under constant applied voltage , u = 5 v. furthermore , the initial amplitude of the currents increases with increasing doping concentration .
this clearly shows that the net charge in the gel increases with increasing doping concentration .
aj / mgel vs time plots for the doped ( p2 , p3 , p4 ) and neat ( p1 ) gels under constant applied voltage , 5 v , and swelling ratio , v / v0 ~ 1,28 .
bj / mgel versus time plots upon short - cut of the circuit for samples p4 , p3 , p2 , p1 .
the vertical bar represents the standard error for each plot examples for the short - cut experiments -the source of the voltage was removed and the current was measured by means of an ammeter without changing its polarity- were also indicated in ( fig .
1b ) where all the current takes first a negative value , as it is expected , and then goes to zero with time .
figure 1 clearly indicates that the free charges leading the current are accumulated on the surfaces of the gel on which the voltage is applied ( similar to charging a parallel plate capacitor ) and the gels have ionic charge carriers .
figure 2a shows typical current - time plots of the slab gels for increasing pyranine content .
the gels were swollen in pure water up to certain swelling degree ( v / v0 = 1,3 ) , under a fixed applied voltage , 1 v. as seen , the currents for all samples decrease with time ; however , the initial values of them increase with increasing doping agent ( pyranine content ) .
aj / mgel versus time plots of the samples p6 , p7 , and p8 , b the ratios of j / mgel for the doped and neat gels ( p8/p5 ) as function of the applied voltage for varying swelling ratio , v / v0 figure 2b shows the ratio of the normalized current densities ( j / mgel ) for doped ( p8 ) to neat ( p5 ) gels as function of the applied voltage for varying swelling ratio .
the currents were measured 5 s later just after applying voltage . as seen , the doping effect on the current decreases when the swelling ratio is increased .
this may be due to the vast increase in the free ions coming from the water which causes screening .
this picture may indicate that doping effect ( p - type behavior ) can be fulfilled up to a certain swelling degree of the above - mentioned gel which semiconducting behavior will be lost .
the instantaneous currents the currents that were measured 5 s later just after changing the voltage- as function of the volume of the gels for varying voltages were also measured , and the results were indicated in fig .
as seen from this figure , distinct peaks appear as the solvent uptake , v / v0 , increases .
the peaks almost disappear for the gels doped with pyranine due to increasing homogeneity of the gels in the presence of the ions .
the results coincide with the recent literature where three dominant peaks , each corresponding to a different swelling stage in the conductivity , were observed .
figure 3 indicates that the volume of the gel is one of the main parameters determining the current .
variation in j / mgel against v / v0 for ( a ) the neat gel , p1 , and for ( b ) the doped gel , p2 these peaks were related to the distribution of dense polymer regions ; they are defined as the blobs appearing in a microstructure of the given paam gel having at least some average sizes .
this special behavior in these gels may result specific features related to the electrical features .
the heterogeneity of the gel may decrease essentially in case when it is prepared with the charged groups .
the internal electric field due to the charged sites could disturb the tendency between the polymer units ( affinity of the aam momoners to the cross - linker molecules ) because the charge excess has a tendency to form denser polymer regions especially in mesoscale regions .
the formation of the rigid network in the charged gel makes it more homogenous in comparison with the neutral gel .
this could cause the decreasing amplitude of the peaks , and overall decrease in the conductivity as it is confirmed by fig .
3 ( see also fig . 2 of reference ) . therefore , in the charged gel ,
the penetration of neutral water molecules through the system of large channels located in the vicinity of small blobs is facilitated .
figure 4 represents the normalized current density per unit mass as function of the voltage for the gels ( sample p3 ) swollen with water ( fig .
dmso is not a polar molecule ; therefore , the ions like h or oh are absent in it . as seen from this figure , the current density decreases fairly for the gels swollen with dmso since no contribution to the current occurs via h and oh ions .
the current occurs mainly due to doped ions ( the counter ions of the doping agent ) .
this observation clearly indicates that the possible negative effects of the water upon the pnjunctions that may be formed by means of our gels can be eliminated .
j / mgel versus applied voltage , u , for the doped gel ( p3 ) swollen in ( a ) pure water , b and in dmso all the above - mentioned measurements were repeated for n - type gels , and the similar results were reproduced . here the results are not given to avoid the repetition . in fig .
5 , representative examples of i - u(current voltage ) character of polymeric diode are given for the neat and the doped gels .
figure 5a shows the current voltage character that was made by means of close connection of p-(sample p4 ) and n - type ( sample n4 ) slab gels- putting one of the gel on the top of the other for each gel .
the swelling ratio of each gel was kept the same , v / v0 = 1,2 . when the neat gels are used for both the p - and n - type sides ,
figure 5a clearly shows that the current of the interface between the cationic and anionic gels , which is passing through only one direction , originates directly from the doping effect of the gels .
a i versus u plot for pn junction formed with p - and n - type gels ( p4-n4 ) , b i versus u plot for the junction formed with neat gels ( p1-n1 )
in this study , we have shown that p- and n - type doped hydrogels can be synthesized and be used to built pn junction .
the synthesis of these gels and making junctions are more simple , inexpensive , and scalable than traditional pn junctions .
the current voltage characteristic of the polymeric pn junction can be changed by changing doping concentration , swelling ratio etc that will be discussed in our feature works .
the percentage of the standard deviations changes around 20% as can be seen from fig .
the reasons for this high standard deviations can be explained by the following arguments : ( 1 ) the electrical futures of these gels depend strictly on the swelling ratio of the gels as can be seen from fig . 3 and from the detailed discussion given in a recent work and ( 2 ) on the internal morphology ( nonergodicity or random distribution of the heterogeneous regions in the gels ) .
this strict volume dependence and the randomness of the internal distributions of the heterogeneous regions set a barrier for comparison of the samples from the point of the current amplitudes due to the difficulties to bring the gels to exactly the same swelling ratios and to repeat the synthesis with exactly the same internal morphology .
therefore , the stability and reversibility problems need detailed experiments and probably more controllable polymer synthesis .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | in this study , we synthesized the new kinds of semiconducting polymeric gels having negative ( n - type ) and positive ( p - type ) counter ions as charge carriers .
the polyacrylamide gel was doped with pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) , havingions as side groups and na+ as counter ions , so - called p - type semiconducting gel .
the doping process was performed during the polymerization where the pyranine binds to the polymer strands over oh group chemically via radical addition . in a similar way ,
n - isopropylacrylamide ( nipa ) gel was doped with methacrylamidopropyltrimethyl ammonium chloride ( maptac ) , having cl as counter ions , so - called n - type semiconducting gel . here
maptac was embedded by copolymerization within the polymer network ( nipa ) .
these semiconducting gels can show different electrical properties by changing the concentration of the doping agents , swelling ratio etc .
we have shown that the pn junction , formed by combining p - type and n - type gels together in close contact , rectifies the current similar to the conventional si and ge diodes . |
in june 2014 , a 61-year - old female was admitted to keimyung university dongsan medical center because of a severe dry cough that had lasted for over 20 days . according to her medical history ,
she had undergone a hysterectomy in 2005 , and the pathologic diagnosis was leiomyoma of the uterus . in 2006 ,
chest computed tomography ( ct ) findings demonstrated multiple nodules in both lungs ( fig .
microscopic examination revealed a proliferating spindle cell tumor showing relatively bland nuclei , no pleomorphism , and no mitoses .
in addition , immunohistochemical staining analysis was positive for smooth muscle actin , desmin , vimentin , estrogen receptor ( er ) , and progesterone receptor ( pr ) , and negative for cd34 , pan - ck , and s100 proteins . the result of the pathologic study was benign leiomyoma , and the nodules were diagnosed as pulmonary benign metastasizing leiomyoma ( pbml ) .
, the patient did not return to our outpatient clinic for follow - up . upon the patient s return ,
a posteroanterior chest x - ray showed a very large mass on the right cardiac border ( fig .
chest ct demonstrated a 101-mm mass on the right lower lobe that compressed the posterobasal segmental bronchus ( fig .
a ct - guided needle biopsy of the huge mass revealed it to be leiomyoma .
after right thoracotomy , a very large mass , 100 mm in size , originating from the visceral pleura of the right lower lobe was observed ( fig .
the mass was in contact with the parietal and diaphragmatic pleura , but no invasion was noted . near the mass , multiple fine ,
scattered , whitish nodules measuring less than 1 cm were found on the diaphragmatic , parietal , and visceral pleura .
we performed an excision of the mass in the right lower lobe and excised fine nodules from the parietal and diaphragmatic pleura for biopsy .
intrapulmonary nodules found in the middle lobe and upper lobe were not excised , because they had not increased in size .
the necrotic lesion showed severe cellular atypia , frequent mitoses ( more than 15 per 10 high power fields [ hpfs ] ) , and continuity of the leiomyoma and leiomyosarcoma ( fig . 2c ) .
immunohistochemical staining revealed that the tumor was positive for smooth muscle actin ( sma ) , er , and pr , but negative for cd34 , pan - ck , s100 , and hmb45 ( fig .
the nodular lesions taken from the parietal and diaphragmatic pleura showed no malignant cells . the patient was discharged 8 days after the operation without complications .
large heterogeneous hypermetabolic masses were found from the left para - aortic area to the left pelvic cavity ( fig .
3 ) , and we diagnosed them as metastatic leiomyosarcoma . additionally , increased fluorodeoxyglucose ( fdg ) uptake was observed in the peribronchial lymph nodes , the mediastinum , and the supraclavicular lymph nodes .
two months later , en - bloc resection of the retroperitoneal masses with left nephrectomy was performed , and the pathologic diagnosis was metastatic leiomyosarcoma .
we decided to continue observing fdg uptake in the mediastinal and peribronchial lymph nodes rather than prescribing chemotherapy , because metastasis of leiomyosarcoma to the lymph nodes is rare . a chest ct scan done 1 year after surgery demonstrated no changes in the pulmonary nodules and no recurrence of the mass .
benign metastasizing leiomyoma ( bml ) results from metastasis of a uterine leiomyoma ; thus , bml patients have a history of uterine leiomyoma
. the condition may occur at any age , but occurs most often in women from 30 to 74 years old .
bml can occur in a number of different areas of the body , including but not limited to the lungs , para - aortic lymph nodes , abdominal lymph nodes , heart , breast , liver , and esophagus .
the most common site of occurrence is in the lungs , in which case it is known as pbml .
the pathogenesis of pbml is unknown , but the most common theory is hematogenous spreading of leiomyoma during uterine surgery .
immunohistochemical staining of affected tissue is positive for desmin , sma , pr , and er . most cases of pbml do not require treatment , but pbml that metastasizes or increases in size does need to be treated , as it can cause respiratory failure , chest pain , or hemoptysis .
surgical excision is the treatment of choice , but this is only possible in cases of resectable pbml .
in some cases of pbml that a repositive for er or pr , reducing hormone release via hysterectomy , bilateral adnexectomy , and/or hormone therapy may decrease the tumor s size .
the incidence of the sarcomatous transformation of benign uterine leiomyomas is only 0.1%0.8% , and is most often seen in women in their 50s .
pbml can also undergo malignant transformation ; excluding our case , just 1 case of the malignant transformation of pbml has been reported .
radiologically , it is difficult to differentiate between normal pbml and pbml that has become malignant .
on fdg pet - ct , heterogeneous fdg uptake of the tumor may reflect necrosis within the leiomyosarcoma . in our case ,
pathologically , the current criteria used to differentiate a malignant smooth muscle tumor from a benign mass are the presence of necrosis , a high mitotic index ( > 5 mitoses seen in 50 hpfs ) , nuclear atypia , cellularity , and an ill - defined tumor border .
however , in primary pulmonary leiomyosarcoma ( ppl ) , progress is slow , and metastasis of ppl occurs late in the disease process .
if the condition is diagnosed early , wide excision with negative margins ( r0 ) is the standard treatment .
the 5-year survival rate in ppl patients who undergo complete resection is over 50% , and recurrence is rare .
lymph node resections are generally not necessary because ppl rarely shows lymph node involvement . for advanced lesions , depending on the size and grade of the tumor , radiation therapy , chemotherapy , or both may be indicated as an adjuvant therapy . however , the median survival of patients with an unresectable sarcoma or metastatic disease is about 12 months .
overall , early diagnosis and complete mass resection are vital in cases of the malignant transformation of pbml .
however , preventive resection or diagnostic excisional biopsy is not recommended for all pbml patients , since the occurrence of a malignant change is extremely rare
if there is a very large , newly developed mass that is increasing in size rapidly , or if a malignant mass is found on pet - ct , diagnostic mass resection should be considered . | pulmonary benign metastasizing leiomyoma ( pbml ) is defined as metastasis of a leiomyoma to lung tissue .
it was first reported in 1937 .
p bml is known as a benign disease , but can undergo malignant transformation . only 1 case of the malignant transformation of pbml to leiomyosarcoma has been reported previously . in this report , we present a case of malignant transformation of pbml . |
copd is a common , usually progressive disease characterized by chronic inflammation of the airways and persistent airflow limitation .
its prevalence is increasing worldwide , and it is expected to be the third largest global cause of mortality by the year 2030.1 in japan , a large epidemiological study ( the nippon copd epidemiological study ) reported that the prevalence of airflow limitation was 10.6% , and at least 8.6% of subjects were sought to have copd.2 several studies have shown that the prevalence of potentially undiagnosed airflow limitation in both western35 and asian6 countries is ~ 3%15% .
many patients with copd continue to be underdiagnosed and untreated.2,7 since copd is a preventable and treatable disease , the importance of early detection has been emphasized.8 the use of simple copd screening questionnaires to detect persistent airflow limitation may help in the early diagnosis of copd .
these tools reliably detect airflow limitation in the general population and may facilitate the early , accurate diagnosis of copd in general practice settings.9,10 some copd diagnostic questionnaires have already been reported.1114 the copd population screener ( copd - ps ) , which was developed by a clinician working group in the united states , is a five - item , self - administered questionnaire that was validated for screening individuals in the general population who are at high risk of copd .
it is composed of three copd - related items ( breathlessness , productive cough , and activity limitation ) and one question , each regarding smoking history and age.15 in a previous population - based study , we verified the validity of the japanese version of the copd - ps questionnaire for the identification of individuals at increased risk of airflow limitation.16 this instrument is easy to score and would be suitable for large - scale screening of possible airflow obstruction ( ao ) .
another copd screening tool , the international primary care airway group ( ipag ) questionnaire,11 is in use worldwide .
it consists of eight items and takes more time to administer than the copd - ps questionnaire .
the purpose of this study was to compare these two questionnaires in the general japanese population , as no previous research has done so , as well as to assess the ability of both instruments to discriminate between subjects with and without persistent ao .
this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .
the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .
the population of the town is ~8,000 and has been stable for over 50 years .
according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 ,
registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . of the 2,643 subjects who were enrolled between june 2012 and october 2012 ,
307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .
the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .
the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .
each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .
the results were assessed by two pulmonary physicians , who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests .
the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .
the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 . the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1
< 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .
the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .
it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .
these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire , a cutoff point of 4
has been found to be useful for copd screening.16 the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .
each question is scored individually , with a summed total score ranging from 0 to 38 .
a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .
baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal
spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,
another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .
likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .
receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .
the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .
we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .
the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .
in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .
we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .
all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .
this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .
the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .
the population of the town is ~8,000 and has been stable for over 50 years .
according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17
this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 , registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry .
of the 2,643 subjects who were enrolled between june 2012 and october 2012 , 307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .
the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .
the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .
each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .
the results were assessed by two pulmonary physicians , who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests .
the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .
the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 .
the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1 < 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .
the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .
it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .
these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire ,
the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .
each question is scored individually , with a summed total score ranging from 0 to 38 .
a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan .
however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .
baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal
spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,
another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .
likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .
receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .
the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .
we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .
the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .
in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .
we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .
all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .
the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .
the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,
almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .
the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and
for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .
the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) . the previously identified cutoff point of 4 was used for the copd - ps questionnaire .
the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% .
in this population - based study that included never - smokers , a cutoff point of 20 on the ipag questionnaire would be adequate for screening for ao ( table 3 ) .
the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .
if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .
if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .
roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .
the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .
there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,
no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .
however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) .
reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,
classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao
, classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .
thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .
the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .
the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .
the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .
the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,
almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .
the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and
for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .
the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) .
the previously identified cutoff point of 4 was used for the copd - ps questionnaire .
the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . in this population - based study that included never - smokers , a cutoff point of 20 on
the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .
if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .
if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .
roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .
the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .
there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,
no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .
however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) . reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,
classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao , classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .
thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .
the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .
the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .
in the present population - based study , the japanese version of the copd - ps questionnaire was compared with the ipag questionnaire in a general japanese population at the age of 40 years or older .
the ipag questionnaire had superior likelihood ratios of both positive and negative tests compared with those of the copd - ps questionnaire .
however , in comparison with two questionnaires , no significant differences were founded in the auc values obtained from the roc curves discriminating between subjects with and without ao and in the sensitivity , specificity , or positive and negative predictive values .
the two questionnaires were correlated with each other , fev1/fvc , and for both of them , the ors were significantly greater than 1.0 .
these findings suggest that the copd - ps and the ipag questionnaires are useful screening tools for detecting persistent ao . in addition , the subjects with a 4 point on the copd - ps questionnaire and those with a 20 point on the ipag questionnaire are at increased risk for ao .
overall , the categorical and continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire were 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) , 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) , and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively .
reclassifications showed the ipag questionnaire to be superior to the copd - ps questionnaire ; however , there was no significant difference between the two questionnaires .
a cutoff point of 17 on the ipag questionnaire is used in general health checkup settings and general practices in japan.24 in this study , we found that a cutoff point of 20 was superior .
the reason for this discrepancy is not clear , although it may be due at least in part to the backgrounds of the study subjects .
the present study was population based and included never - smokers . in contrast , in a previous study that enrolled subjects in general health checkup settings , a cutoff point of 17 resulted in a higher sensitivity of 86.0% and a smaller auc of 0.66 ( lower than 0.70 ) than a cutoff point of 20.24 the present study was implemented in the town of hisayama , in which the age and occupational distributions of the population have been almost identical to those of japan as a whole , and thus , we recommend a cutoff point of 20 for the general japanese population .
there were only marginal differences between the two questionnaires in terms of ability to discriminate between subjects with and without ao .
moreover , the copd - ps questionnaire consists of fewer items than the ipag questionnaire and requires less time to complete .
it takes ~5 minutes to fill out copd - ps questionnaire , however , the completion of the ipag questionnaire takes ~510 minutes . in this regard ,
the japanese version of the copd - ps questionnaire should be an adequate measure for large - scale screening for possible ao . | backgroundthe incidence of chronic obstructive pulmonary disease ( copd ) is increasing worldwide . in japan and other countries ,
epidemiological studies have found that many patients with copd are underdiagnosed and untreated , and thus , early detection and treatment of copd has been emphasized .
screening questionnaires may have utility in the initial detection of copd.objectivethis study aimed to validate and compare the copd population screener ( copd - ps ) and the international primary care airway group ( ipag ) questionnaires in a general japanese population.patients and methodseligible subjects 40 years of age and older living in the town of hisayama were solicited to participate in a health checkup in 2012 .
all subjects 4079 years of age without physician - diagnosed asthma or lung resection were recruited , and 2,336 subjects who fully completed both questionnaires and who had valid spirometry measurements were analyzed .
persistent airflow obstruction ( ao ) was defined by a postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 0.70 .
receiver operating characteristic curves , net reclassification improvement , and integrated discrimination improvement were used to examine the ability of the copd - ps and ipag questionnaires to discriminate between subjects with and without ao.resultsthe overall area under the receiver operating characteristic curve for the copd - ps questionnaire was 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) and for the ipag was 0.775 ( 95% ci , 0.7350.816 ) , with no significant difference ( p=0.09 ) .
the net reclassification improvement and integrated discrimination improvement were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively.conclusionthe five - item copd - ps questionnaire was comparable to the eight - item ipag for discriminating between subjects with and without ao . the copd - ps is a simple and useful screening questionnaire for persistent ao . |
the human cytomegalovirus ( cmv ) or human herpesvirus 5 is one of the major causes of congenital infections .
its clinical manifestations range from asymptomatic forms ( 90% of cases ) to severe fetal damage and , in rare cases , death due to abortion . furthermore
, 10%15% of the children who are asymptomatic at birth may develop late sequelae , especially hearing defects , after a period of months or even years .
latency following a primary infection ( first contact with the virus ) may be punctuated by periodic reactivations that give rise to recurrent infections , and in utero transmission may occur during either primary or recurrent infections .
actually recurrent infections may be due to reinfection with a new strain or to reactivation , but it is likely that most recurrent infections are due to reinfection .
the risk of congenital infection is much higher during primary infection [ 25 ] , when the rate of transmission from mother to fetus is 30%40% [ 1 , 6 ] , as against 0.15%2.2% during reactivations or reinfections [ 1 , 69 ] when , furthermore , most of the newborns are asymptomatic .
symptomatic cases are due more to reinfection than reactivation [ 2 , 10 ] .
it has been reported that the risk of fetal damage is greater if the primary infection occurs during the first trimester of pregnancy [ 1113 ] .
the prevalence of congenital infection ranges from 0.2% to 2.5% in different populations [ 1420 ] , in which the risk factors include particular races or ethnic groups , a low socioeconomic status , premature birth , and admission to an intensive care unit [ 6 , 17 ] .
furthermore , the prevalence of congenital infection varies with the prevalence of the infection in the population .
the seroprevalence of cmv among women of childbearing age ranges from 35% to 95% in different countries [ 12 , 2124 ] and , as well as increasing with age , may also depend on sexual activity and occupation , particularly occupations involving close contacts with children in a community setting . in the case of parents ,
contact with the urine or saliva of their children is a major source of infection [ 2527 ] .
the incidence of primary infection among pregnant women ranges from 0.5% and 4% [ 28 , 29 ] ; the rate of seroconversion during pregnancy ranges from 0.4% to 2% [ 12 , 13 , 30 , 31 ] and depends on the seroprevalence of the infection in the population , being 3.7% among women belonging to populations with a low seroprevalence ( 55% ) and 1.0%1.6% among those belonging to populations with a high seroprevalence ( 85% ) .
the risk of acquiring infection during pregnancy is 0.71.38 100 pregnancies [ 23 , 29 ] among seronegative women , and 0.20.8 100 pregnancies among women as a whole .
as far as prevention is concerned , in addition to health education campaigns , the serological screening of pregnant women has been proposed . however , there is no consensus in the scientific community concerning the implementation of screening , and it is not recommended by any public health system because of its cost / benefit ratio , although many doctors in israel , belgium , and france do test their pregnant patients .
furthermore , although the current public health legislation in italy ( law decree 245 of 10 september 1998 ) does not include free cmv antibody screening during pregnancy , it is prescribed by many general practitioners .
the aim of this study was to assess the incidence and risk of acquiring cmv infection in pregnant women in an urban area in northern italy in the period 20052007 .
during the three years 20052007 , the microbiology unit of hospital of legnano , received samples for the detection of cmv antibodies from 2817 pregnant women ( mean age 32 years , range 1546 ; 2522 ( 89.5% ) were italian and 295 ( 10.5% ) of foreign origin ) .
forty - eight women ( 1.7% ) were 20 or less than 20 years of age , 928 ( 32.9% ) women were aged between 21 and 30 , 1750 ( 62.1% ) aged between 31 and 40 , and 91 ( 3.2% ) between 41 and 50 .
2318 women ( 82.3% ) underwent their first screening in the first trimester ( group a ) , 316 ( 11.2% ) in the second trimester ( group b ) , and 183 ( 6.5% ) in the third ( group c ) .
the requests were made by the general practitioners as part of the routine screening required during pregnancy .
all of the samples were analyzed for the presence of anti - cmv igg and igm antibodies by means of an enzyme - linked immunosorbent assay ( elisa ) ( eti - cytok - g - plus , eti - cytok - m reverse plus , diasorin , saluggia , italy ) .
the cutoff value used to determine igg was 0.4 iu / ml , whereas the samples were considered igm - positive when their absorbence was equal to , or greater than the control cutoff value .
the igm - positive samples were confirmed using an enzyme - linked fluorescent assay ( elfa ) ( vidas cmv igm , biomrieux , lyon , france ) and were considered positive when their index was 0.90 , borderline when their index was between 0.70 and 0.90 , and negative when their index was > 0.70 . as igm anti
- cmv antibodies may test positive for more than 12 months and may be produced during reactivation or reinfection , the samples that were igm - positive at elisa were also tested for igg avidity ( liaison cmv igg avidity , diasorin saluggia , italy ) , which was considered low if the index was < 0.2 , moderate if it was between 0.2 and 0.3 , and high if it was 0.3 .
low igg avidity levels strongly suggest an infection contracted less than three months before , whereas a high avidity tends to exclude this .the elisa igm - positive samples were also tested for the presence of rheumatoid factor ( arthri - slidex , biomrieux , lyon , france ) . in the case of positivity for igm ,
the patients ' general practitioners were contacted and advised to evaluate the case and refer patients to a reference center .
at the first screening , the elisas showed that 1925 women ( 68.3% ; 95% ci : 66.6%70.0% ) were positive for anti - cmv igg ( positive or negative for igm ) and 26 ( 0.9% ; 95% ci : 0.55%1.25% ) were positive for igm antibodies ( 25 in the first trimester , and one in the second trimester for whom no previous data were available as she did not undergo screening during the first trimester ) .
table 1 shows the results of igm and igg elisa by trimester of first screening ( groups a
elfa of the 26 elisa igm - positive samples showed that 17 ( 65.4% ) were positive or borderline , and nine were negative ( 34.6% ) , including the sample that was igm - positive at elisa screening in the second trimester .
none of the differences in the prevalence of igg and igm between contiguous age classes was statistically significant .
of the 892 women who were anti - cmv antibody negative at first screening , 687 ( 77.0% ) were in the first trimester of pregnancy ( group a ) , 131 ( 14.7% ) in the second trimester ( group b ) , and 74 ( 8.3% ) in the third ( group c ) .
three hundred and seventy - four of the women of group a ( 54.4% ) were also screened in the second trimester , and 258 ( 37.6% ) were also screened in the third ( table 2 ) .
of these , two became positive for igm ( confirmed by elfa ) and igg , one in the second trimester ( 0.3% ) , and one in the third ( 0.4% ) , for a mean seroconversion rate of 0.32% .
of the 131 women of group b , 64 ( 48.9% ) were also screened in the third trimester ; there were no cases of seroconversion ( table 2 ) . of the 1899 women who were anti -
cmv igg antibody positive ( and negative for igm ) at first screening , 1606 ( 84.6% ) were in the first trimester of pregnancy ( group a ) , 184 ( 9.7% ) in the second trimester ( group b ) , and 109 ( 5.7% ) in the third ( group c ) .
one hundred and fifteen of the women of group a ( 7.2% ) were also screened in the second trimester , and 66 ( 4.1% ) were also screened in the third .
of the 184 women of group b , 20 ( 10.9% ) were also screened in the third trimester ( table 2 ) .
nineteen of the 28 igm - positive samples at elisa ( 67.9% ) were confirmed as being igm - positive by means of elfa , and 13 ( 46.4% ) showed low or moderate igg avidity .
the 19 elfa - confirmed cases ( 17 first screened in the first trimester , and the two seroconversions ) included six ( 31.6% ) with a high degree of igg avidity , five ( 26.3% ) with moderate avidity , and eight ( 42.1% ) with a low degree of avidity ( table 3 ) .
table 4 shows the results of the confirmatory igm elfa and igg avidity tests by trimester of pregnancy .
in particular , of the five cases showing moderate avidity , one was recorded in our files as having been seronegative for both igg and igm four and a half months before ( i.e. , about two months before conception ) , and another was the case of seroconversion in the third trimester after being seronegative in the first and second . in the remaining three cases ,
the only data available were those of the initial positive sample , but the general practitioner of one of these women , who was contacted after the detection of igm positivity , reported symptoms compatible with ongoing cmv infection .
no symptoms were reported by the general practitioners in any of the other two cases nor additional information was available .
all of the 13 cases with low or moderate igg avidity were therefore considered as having primary infection : 11 ( 84.6% ) occurring in the first trimester , one ( 7.7% ) in the second , and one ( 7.7% ) in the third ( both seroconversions ) .
unfortunately there were no data regarding the transmission of infection to the fetus because the igm - positive women were all referred for further investigations to reference centers throughout the area .
the cumulative incidence of cmv infection ( new cases observed during pregnancy ) calculated on the basis of the 13 cases with low or moderate igg avidity was 1.4% ( 95% ci : 0.971.83 ) , and the risk of infection during pregnancy , calculated on the basis of all of the women ( seronegative and seropositive ) , was 0.5 100 pregnancies ( 95% ci : 0.240.76 ) . the differences in incidence and risk by age was not statistically significant . to take into account the loss to follow - up within the groups and different admission to the study of women during different trimester ,
there were also calculated the density incidence as cases / pregnant woman - trimester and the correlated risk for all women ( seronegative and seropositive ) : they were , respectively , 0.8% ( 95% ci : 0.471.13 ) and 0.4% ( 95% ci : 0.170.63 ) .
the overall prevalence of anti - cmv igg antibodies in our pregnant women was 68.3% ( 95% ci : 66.670.0 ) , without any significant differences between age classes .
as first pregnancies in italy generally occur later than they did in the past , the majority of women have already recovered from primary infection by the time they reach childbearing age and almost certainly by the time of their first pregnancy . moreover , in this study 95% of the women had an age between 21 and 40 years while age classes of 20 or less than 20 and over 40 years were under - represented ; so this could be a further cause of the lack of difference in seroprevalence .
on the basis of the results of the igg avidity test , the cumulative incidence of cmv infection was 1.4% ( 95% ci : 0.971.83% ) , the density incidence was 0.8% ( 95% ci : 0.471.13 ) , and the risk of infection was 0.5% ( 95% ci : 0.240.76% ) without any significant differences by age .
seroconversion or clinical data indicating acute infection were available for three of the five cases with moderate avidity in this study , thus moderate avidity was considered as a potential marker of acute infection .
moderate and low igg avidity were considered together , and both were included in the calculation of incidence .
however , the incidence may be an underestimate because only about half of the seronegative women underwent further screening in the second trimester and about one - third in the third , and so some cases of seroconversion may have been missed .
for the same reason , the proportion of primary infections ( 84.6% ) occurring in the first trimester may be overestimated ; however , assuming the same rate of seroconversion among the seronegative women who did not undergo further screening , the majority of primary infections occurred in the first trimester . the fact that 84.6% of the primary infections occurred in the first trimester may have been due to different behaviors before the pregnancy was recognized , whereas greater care during pregnancy may lead to less exposure .
the fact that there were no differences related to the age of the women indicates the same type of behavior at different ages .
it is therefore important to start screening in the first trimester of pregnancy , when there is a greater risk of infection and in order to have initial findings to compare with subsequent follow - up . in the absence of baseline data ,
the presence of igg without igm in women undergoing their first screening in the third trimester raises doubts as it may be the result of a previous infection occurring at any time in life before the pregnancy ; however , although this is statistically the most probable situation , the possibility of an infection occurring in the first trimester with the subsequent loss of igm can not be excluded .
finally some limitations of the study must be taken into account as no outcome data for newborns , substantial loss to follow - up , and limited testing of igg positive women for reinfections or reactivations .
however , for the latter two cases , as there are no official recommendations , the follow - up was performed at the discretion of the general practitioner with compliance of pregnant woman who , above all , must pay for cmv antibody screening . in conclusion , although screening is not recommended by any public health system ( including italy 's ) because of its cost / benefit ratio , it is actually adopted by many general practitioners in our area . such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent infection , especially in relation to their behavior with children , who are a major source of infection .
furthermore , the screening identified primary infections in pregnant women who could be referred to reference centers to check for prenatal infection .
amniocentesis , funicolocentesis , ultrasonography , and magnetic resonance imaging can all be used to detect infection and allow the planning of appropriate interventions ( e.g. , antiviral therapy , termination of pregnancy ) . although some authors consider that screening is not justified on the grounds of its economic cost , the imperfect nature of congenital infection prognostic criteria , the risk of spontaneous abortions induced by invasive tests such as amniocentesis , and the few data concerning effective treatments during pregnancy , it is unthinkable to deny pregnant women appropriate information concerning the health of their unborn child as this raises a number of ethical and legal questions .
the incidence and risk of cmv infection in pregnancy found in our area , therefore , support the use of serological screening , certainly in the first trimester when the risk of infection is higher and , in the case of seronegative women , possibly also one screening in the second trimester and one in the third . | the fetal consequences of cmv infection make it one of the most serious infections contracted during pregnancy , but the scientific community is divided over the proposed implementation of preventive screening for anti - cmv antibodies .
the aim of this study was to assess the incidence and risk of infection during pregnancy in 2817 women who underwent anti - cmv igg and igm antibody screening during the period 20052007 .
the prevalence of anti - cmv igg antibodies was 68.3% ( 95% ci : 66.670.0 ) ; the seroconversion rate in the 892 seronegative women was 0.32% ; the results of igg avidity testing revealed an cumulative incidence of 1.4% ( 95% ci : 0.971.83 ) , density incidence of 0.8% ( as cases / pregnant woman - trimester ) ( 95% ci : 0.471.13 ) , and a risk of infection of 0.5% ( 95% ci : 0.240.76 ) . the screening identified 13 cases of primary infection ( 84.6% of which occurred in the first trimester of pregnancy ) .
the possibility to identify these cases and consequently to plan appropriate interventions , supports the use of screening during pregnancy , especially in the first trimester when the risk of infection is greater . |
bladder cancer is the second most commonly diagnosed genitourinary malignancy in the usa , with an estimated 74,000 newly diagnosed cases and 16,000 deaths in 2015 .
the incidence of bladder cancer rises with age , peaking between age 50 and 70 years , and is three times more common in men than in women .
commonly accepted risk factors for bladder cancer include cigarette smoking , occupational exposure to aniline dyes , benzidene compounds , analgesic abuse ( phenacetin ) and chronic irritation , such as indwelling catheters . of all newly diagnosed cases of bladder cancer , about 70% present as non - muscle invasive bladder cancer ( nmibc ) ,
it comprises a heterogeneous group of patients and includes pathological stage ta ( confinement to the epithelium or mucosa ) , t1 ( invasion of the subepithelial connective tissue or lamina propria ) and cis ( tis : flat , high - grade , non - papillary carcinomas confined to the urothelium ) . of all newly diagnosed nmibc , 70% present as stage ta , 20% as t1 and 10% as cis .
approximately 50 - 70% of nmibc will recur and roughly 1020% will progress to muscle ( i.e. , muscularis propria ) invasive disease . in patients with low - grade ta disease ,
patients with high - grade ta tumors had a progression - free survival of 61% and a disease - specific survival of 74% , whereas patients with t1 disease had a progression - free survival of 44% and a disease - specific survival of 62% , providing support to the view that grouping all patients with nmibc as one disease is misleading as one patient 's prognosis can be quite different from that of another patient .
when considering a patient 's prognosis with nmibc , it is necessary to consider not only clinical and pathological factors but also take into account the potential effect of the intravesical treatment received and molecular alterations present in the tumors .
table 1 summarizes the available predictive models to predict recurrence and progression in patients with nmibc .
the most important risk factor for progression is grade , not stage , because patients with high - grade tumors progress with similar frequency regardless of whether they were invasive ( t1 ) or non - invasive ( ta ) .
millan - rodriguez et al . evaluated a cohort of 1529 primary nmibc patients treated with transurethral resection ( tur ) and random bladder biopsy and identified prognostic factors for recurrence , progression and disease - specific mortality ( median follow - up : 40 months ) .
multivariate analysis demonstrated that the main prognostic factors of recurrence were multiplicity , tumor size > 3 cm , presence of cis and treatment with bacillus calmette guerin ( bcg ) . the prognostic factors for progression were grade 3 disease , multiplicity , tumor size > 3 cm , cis and treatment with bcg .
furthermore , the prognostic factors for mortality were presence of grade 3 disease and cis .
prediction of disease recurrence and progression in patients with non - muscle invasive bladder cancer in another study , millan - rodriguez et al .
risk groups were classified as :
low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor)intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor)high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) .
low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor ) intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor ) high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) .
the rates of recurrence , progression and mortality were 37% , 0% and 0% in the low - risk group , 45% , 1.8% and 0.73% in the intermediate - risk group and 54% , 15% and 9.5% in the high - risk group .
the relative risks of recurrence , progression and mortality in the low - risk versus intermediate - risk and high - risk groups were 1.37 , 2.84 and 1 , and 1.87 , 24.76 and 14.69 , respectively .
the european organization for research and treatment of cancer ( eortc ) developed risk tables to predict recurrence and progression in patients with nmibc using clinical and pathologic information from 2596 patients enrolled in seven clinical trials that utilized prophylactic treatments after tur .
the median follow - up was 3.9 years and 78% of patients had received intravesical treatment , mostly with chemotherapy . in their study cohort , 80% of patients had a maximum tumor diameter < 3 cm and 56% of patients had pta tumors .
the eortc risk tables [ tables 2 and 3 ] for recurrence and progression were based on the scoring system derived from the following six clinical and pathological factors :
number of tumorstumor sizet categorytumor grade ( who 1973)prior recurrence ratepresence of concurrent cis .
weights used to calculate eortc risk tables recurrence and progression scores probability of recurrence and progression according to the eortc risk tables total score based on these eortc risk tables ( electronic calculator is available at http://www.eortc.be/tools/bladdercalculator/ ) , the probability of recurrence varied from 15% to 61% at 1 year and from 31% to 78% at 5 years . the probability of progression varied from 0.2% to 17% at 1 year and from 0.8% to 45% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.66 and 0.74 , respectively .
of note , only 6.6% ( 171 of 2596 ) patients received bcg for six induction instillations .
in addition , patients with high - grade disease did not have a second tur or receive maintenance bcg therapy .
bcg is currently the most effective intravesical therapy for nmibc , especially in intermediate- and high - risk tumors . a meta - analysis of 24 randomized trials ( n = 4863 ) showed that bcg significantly reduces the risk of progression to muscle - invasive disease after tur in patients with nmibc who receive maintenance bcg .
the patient 's response to bcg at 3 or 6 months is an important prognostic factor to predict subsequent tumor recurrence and progression .
because the eortc risk tables were generated using nmibc patient 's clinical and pathological information , where majority of the patients did not receive bcg induction and/or bcg maintenance therapy , the eortc risk tables tend to overestimate patient 's risk for recurrence and progression after bcg therapy . the spanish club urolgicoespaol de tratamientooncolgico ( cueto ) group developed a scoring model using information from 1062 patients treated with intravesical adjuvant bcg therapy to predict risk of recurrence and progression .
patients had bcg induction as weekly instillations for 6 weeks and six additional instillations repeated at 2-week intervals as a maintenance therapy . by the end of the study ,
4.2% of patients received fewer than six instillations , 22.5% of patients received six to nine instillations and 73.3% of patients received more than 10 instillations .
the risk tables for recurrence and progression were based on the scoring systems derived from seven clinical and pathological factors :
agegendernumber of tumorsrecurrent tumort categorytumor grade ( who 1973)presence of concurrent cis .
tumor grade ( who 1973 ) presence of concurrent cis . based on the cueto risk tables , the probability of recurrence varied from 8.24% to 41.79% at 1 year to 20.98% to 67.61% at 5 years .
the probability of progression varied from 1.17% to 13.97% at 1 year to 3.76% to 33.57% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.636 and 0.687 , respectively . using the cueto risk tables ,
the calculated risk of recurrence were lower than that obtained by the eortc risk tables .
the lower risks in the cueto tables is likely attributable to using bcg , which is a more effective intravesical therapy .
although the eortc and cueto risk tables are the two best - established predictive multivariate models for recurrence and progression risk calculation in patients with nmibc , limitations include retrospective analysis , use of the 1987 tnm classification and use of the who 1973 grading system .
in addition , repeat tur for high - grade cancer and immediate instillation of chemotherapy , such as mitomycin , was not routinely performed after tur ; hence , both models are prone to overestimate the risk of recurrence and progression in patients treated with current standard of care .
a restaging tur is routinely performed these days in patients with high - grade nmibc , especially for high - grade t1 , as the rate of residual tumor is high and 30% of tumors are upstaged when muscle is absent in the first obtained specimen .
photodynamic diagnosis with blue light and narrow - band imaging have been shown to reduce residual tumor rates by roughly 20% and to improve recurrence - free survival of nmibc patients compared with white light cystoscopy .
a meta - analysis of seven randomized trials ( n = 1476 ) confirmed the effectiveness of single immediate intravesical instillation of chemotherapy in treated patients compared with tur alone , reporting a 39% decrease in the odds of recurrence .
the efficacy of intravesical mitomycin c can be optimized by administering a dose of 40 mg at a concentration of 2 mg / ml in water , complete bladder emptying just before dose administration , fluid restriction and oral bicarbonate to alkalinize the urine .
this approach improved the recurrence - free rate at 5 years from 24.6% to 41% and increased the interval to tumor recurrence from 11.9 to 29 months . using a multi - institutional cohort of 4689 patients with nmibc , xylinas et al .
showed that both the eortc and cueto risk tables exhibit a poor discrimination for disease recurrence and progression .
these models overestimated the risk of disease recurrence and progression in high - risk patients .
additional factors not included in the eortc or the cueto models could be added to new prognostic models to enhance their usefulness .
bladder neck involvement , prostatic urethra involvement , lymphovascular invasion and depth of lamina propria invasion , i.e. , whether the tumor is superficial to , into or beyond the muscularis mucosae ( t1a , t1b , t1c ) , have been identified as risk factors for progression in patients with nmibc . in the prognostic factor meta - analysis of 33 studies of high - grade t1 bladder cancer patients (
n = 8880 ) , the highest impact risk factor for progression and cancer - specific survival was depth of invasion ( t1b / c ) into the lamina propria .
in this meta - analysis , several other previously proposed factors , i.e. lymphovascular invasion , associated cis , non - use of bcg , tumor size > 3 cm , gender , multiple tumors and older age , also predicted progression .
nmibc patients with progression to muscle - invasive disease tend to have worse prognosis compared with patients with primary muscle - invasive disease , and it underscores the need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc . risk groupings and risk tables provide average predictions , which may or may not apply to the patient interested in knowing individualized risk .
nomograms have been proposed as a method that avoids the arbitrary division of patients into risk groups .
developed nomograms to estimate the risk of disease recurrence and progression in patients with nmibc using a large international cohort .
nomograms based on age , gender , urine cytology and dichotomized nmp22 had accuracy of 0.811 for recurrence of any bladder cancer ; 0.772 for recurrence of grade 3 ta or t1 or cis and 0.774 for recurrence of stage t2 bladder cancer after bootstrap validation . however , this nomogram had several limitations , including failure to incorporate established predictors , such as pathologic grade and stage , number and pattern of previous recurrences , time since the original diagnosis and prior use of intravesical therapy .
furthermore , the performance of the nomograms varied significantly among the institutions , emphasizing the need for external validation prior to its clinical use . artificial neural networks ( ann ) are algorithms that can be trained to identify complex patterns between input variables and outcomes in the data sets and then apply these patterns to new cases .
they have a theoretical advantage over conventional statistics as they are not limited by predefined mathematical relationships between input variables and outcomes ; thus , they are able to model complex non - linear parameters .
qureshi et al . used ann to predict bladder cancer recurrence and progression within 6 months of diagnosis in a small cohort of about 100 patients with nmibc .
the accuracy of ann in predicting stage progression and recurrence was 80% and 75% , respectively .
however , there was no significant difference between the ann and the clinicians predictions of progression and recurrence in the patients with nmibc .
on restricting the validation subset to patients with t1g3 tumors in relation to stage progression , ann accuracy was better than predictions of clinicians .
several investigators have compared ann and other machine learning techniques with standard statistical approaches to predict outcomes and did not find improvement in predictive accuracy with ann and other machine learning techniques over standard statistical approaches .
most of the currently available predictive tools assume that nmibc is pure urothelial carcinoma and does not consider the impact of variant histology on prognosis prediction .
variant histology such as micropapillary , sarcomatoid and plasmacytoid has been shown to be associated with poor prognosis , and early cystectomy is generally advocated in such cases .
small cell carcinoma of the bladder is considered a systemic disease and chemotherapy followed by tailored local therapy is recommended for patients with non - muscle invasive small cell carcinoma of the bladder .
however , spaliviero et al . reported not significantly worse outcomes in conservatively managed t1 micropapillary bladder cancer patients compared with patients treated with early radical cystectomy . given the conflicting findings on the impact of variant histology in smaller studies , the association of variant histology with prognosis deserves further evaluation in larger studies .
for molecular biomarkers to be of clinical use , they should be able to increase the predictive accuracy beyond the standard clinical and pathological parameter models .
several investigators have attempted to use molecular biomarkers as prognostic factors to predict outcomes in patients with nmibc .
however , molecular biomarkers have shown mixed results so far and are not sufficiently validated to be used in clinical practice at this time .
it is becoming clear that superficial low - grade cancers and invasive or high - grade cancers harbor distinctive genetic defects : the low - grade , non - invasive papillary tumors are characterized by activating mutations in the h - ras gene and fibroblast growth factor receptor 3 ( fgfr3 ) gene and the high - grade invasive tumors are characterized by structural and functional defects in the p53 and retinoblastoma protein ( rb ) tumor - suppressor pathways .
the deletion of both arms of chromosome 9 occurs frequently in bladder cancer during the earliest stages of tumorigenesis .
however , these chromosomal aberrations do not seem to distinguish between the two tumor development pathways .
tumor invasion and progression in the bladder seems to be a multifactorial process , promoted by micro - environmental changes that include the up - regulation of n - cadherin , the down - regulation of e - cadherin , the overexpression of matrix metalloproteinases 2 and 9 , an imbalance between angiogenic and anti - angiogenic factors and increased synthesis of prostaglandin . in patients with t1 bladder cancer , nuclear overexpression of p53 protein
has been reported to have a higher probability of disease progression . in a meta - analysis , p53 was a predictor for recurrence , progression and mortality in patients with bladder cancer .
however , investigators of this meta - analysis had concerns of overestimating findings because of publication and reporting bias , and suggested that current evidence is not sufficient to conclude whether changes in p53 act as markers of outcome in patients with bladder cancer .
hernandez et al . , in a prospective cohort of 772 patients with nmibc , showed that fgfr3 mutations are prevalent in low - grade ta and that these mutations are independent predictors of recurrence in patients with low - grade ta tumors .
van rhijn et al . showed that molecular grading , the grading system based on fgfr3 and mib-1 ( ki-67 ) status , is an independent predictor for recurrence , progression and disease - specific survival .
van rhijn et al . validated the utility of molecular grading as a prognostic factor to predict outcomes in patients with nmibc .
the addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% .
evaluated nmp22 for detecting recurrence and progression in patients with nmibc in a large multi - institutional international cohort .
there was no clearly defined nmp22 cut - off that could indicate higher risk disease , but there was a continuum of risk for recurrence and progression .
the international consensus panel on cytology and bladder tumor markers evaluated the prognostic utility of molecular markers for bladder cancer .
molecular markers were classified into six groups , i.e. microsatellite - associated markers ( e.g. fish , loh ) , proto - oncogenes / oncogenes ( e.g. her-2/neu , h - ras , bcl-2 , mdm-2 , fgfr-3 , c - myc ) tumor suppressor genes ( e.g. , p53 , rb ) , cell cycle regulators ( e.g. , p21 , p27 , ki-67 , cyclin - d1 , cyclin - e ) , angiogenesis - related factors ( vegf , cox-2 , tsp-1 ) and extracellular matrix adhesion molecules ( e.g. e - cadherin , mmps , timps , cd44 , u - pa ) . the panel concluded that although certain biomarkers , such as ki-67 and p53 , appear to be promising in predicting recurrence and progression in patients with bladder cancer , the data are still heterogeneous .
the panel recommended that identifying definitive criteria for test positivity , a clearly defined patient population , standardization of techniques used to evaluate markers and clearly specified endpoints and statistical methods will help to bring accurate independent prognostic indicators into the clinical management of patients with bladder cancer .
nmibc comprises of a heterogeneous group of patients and includes pathological stage ta , t1 and cis .
patients with low - grade ta disease have very low risk of progression while patients with t1 disease with concurrent cis have a much higher risk of progression , approaching 50% .
the eortc and cueto risk tables are the two best - established predictive models for recurrence and progression risk calculation in patients with nmibc . however
, both the eortc and the cueto risk tables exhibit a poor discrimination for prognostic outcomes and overestimate the risk of disease recurrence and progression in high - risk patients in external validation .
additional prognostic factors such as depth of lamina propria invasion should be added to new prognostic models to enhance their usefulness .
molecular biomarkers such as ki-67 , fgfr3 and p53 appear to be promising in predicting recurrence and progression , but need further validation prior to using them in clinical practice .
future research should focus to enhance the predictive accuracy of the risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts .
| introduction : non - muscle invasive bladder cancer ( nmibc ) comprises about 70% of all newly diagnosed bladder cancer , and includes tumors with stage ta , t1 and carcinoma in situ ( cis . ) since , nmibc patients with progression to muscle - invasive disease tend to have worse prognosis than with patients with primary muscle - invasive disease , there is a need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc.materials and methods : a detailed medline search was performed to identify all publications on the topic of prognostic factors and risk predictions for superficial bladder cancer / nmibc .
the manuscripts were reviewed to identify variables that could predict recurrence and progression.results:the most important prognostic factor for progression is grade of tumor .
t category , tumor size , number of tumors , concurrent cis , intravesical therapy , response to bacillus calmette
guerin at 3- or 6-month follow - up , prior recurrence rate , age , gender , lymphovascular invasion and depth of lamina propria invasion are other important clinical and pathological parameters to predict recurrence and progression in patients with nmibc .
the european organization for research and treatment of cancer ( eortc ) and the spanish club urolgicoespaol de tratamiento oncolgico ( cueto ) risk tables are the two best - established predictive models for recurrence and progression risk calculation , although they tend to overestimate risk and have poor discrimination for prognostic outcomes in external validation .
molecular biomarkers such as ki-67 , fgfr3 and p53 appear to be promising in predicting recurrence and progression but need further validation prior to using them in clinical practice.conclusion:eortc and cueto risk tables are the two best - established models to predict recurrence and progression in patients with nmibc though they tend to overestimate risk and have poor discrimination for prognostic outcomes in external validation .
future research should focus on enhancing the predictive accuracy of risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts . |
personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies in order to achieve the best medical outcomes for individual patients , thus improving public health . despite the variety of clinical , morphological , and molecular parameters
used to classify human malignancies , patients receiving the same diagnosis can have markedly different clinical courses and treatment responses . since there is no simple way to determine who will have an adverse reaction ,
the current system of one - size - fits - all- diagnoses is simply not good enough .
an increasing number of studies have demonstrated sex differences in drug reactions to the same drug treatment .
migeon implied that males and females responded differently to drug treatments and that sex plays a key role in cancer .
in addition , females are historically less studied subjects due to the complication of estrous cycle , and therefore such studies would further benefit women 's health and promote public health .
recent advancements in biotechnology have accelerated the search for molecular biomarkers useful in the diagnosis and treatment of disease .
molecular biomarkers of disease risk and status are critical to an accurate treatment by identifying patients most likely to benefit from particular drugs or experience adverse reactions . because medicine is always practiced on individuals rather than populations , the goal is to change the assignment of therapies from a population - based approach to an individualized approach .
gene - expression data can be used to identify patients with a good disease prognosis , thereby preventing some patients from unnecessary therapies and toxicity .
for example , gene - expression profiling was used to predict clinical outcomes in pediatric patients with acute myeloid leukemia and to find genes whose aberrant expression leads to a poor prognosis .
thus , accurate classification of prognosis of patients leads to efficient cancer treatment and prolonged survival of patients .
classification algorithms are needed for biomedical decision making in clinical assignment of patients to treatment therapies based on individual risk factors and disease characteristics . since many of those genes are not relevant , feature selection is a commonly addressed problem in classification [ 3 , 4 ] .
the goal of gene selection is to identify a set of genes which plays an important role in the classification .
a common approach is to select a fixed number of the highest ranked genes based on t - test - like statistics , some discrimination measures [ 6 , 7 ] , or classification algorithms including support vector machines ( svm ) [ 8 , 9 ] and random forest ( rf ) [ 10 , 11 ] .
development of a biomarker classifier involves two distinct components : ( 1 ) a procedure for building a classifier and ( 2 ) validation / evaluation procedure for estimating the error rate of biomarker .
the most important consideration is the validation / evaluation of a biomarker classifier to assess whether it can accurately and unbiasedly predict new samples based on a set of selected features .
two methods are commonly used to develop and assess performance of a classifier : the split - sample procedure and cross - validation procedure . in the split - sample procedure , the sample dataset
is randomly split into two subsets : a training set for model building and a test set for performance assessment .
that is , the training dataset is used for building the biomarker classifiers and the test set is used for evaluating the classifier .
however , samples are often insufficient to split into two sample sets of approximately equal size for model building and model testing , and a cross - validation ( cv ) procedure is commonly used for performance assessment [ 12 , 13 ]
. a cv can be regarded as a generalization of the split - sample method .
it involves repeatedly splitting the data into a training set containing most of the samples and applying the prediction rule to the test set of the remaining samples to estimate the prediction accuracy rate .
the prediction accuracy is the average accuracy of the numerous training - test partitions . for model building ,
given that differences in the biology of lung cancer and other diseases exist between men and women [ 14 , 15 ] , investigations to identify genomic biomarkers for clinical assignment of therapies on an individual patient basis are crucial . in this paper , the ratio of between - group to within - group sums of squares ( bw ratio , ) gene selection algorithm is used to obtain a feasible set of influential genes via variable importance ranking procedure in the training phases of 20 trials of 10-fold cv within leave - one - out cross - validation ( loocv ) procedure as illustrated below .
the genes with largest bw ratios are ranked high as significant genes . in the development of biomarker classifier
, the predictive accuracy of the classifier must be evaluated on a separate set of data . to derive an unbiased accuracy estimate , a nested cross - validation procedure , 20 trials of 10-fold cv within loocv ,
is used in this paper . in other words , in each loocv , 90% of the
wherefore genes are selected only using learning data sets of size ( n 1 ) each , and in the evaluation / validation step a never - touched and left - out single observation in loocv is used to assess the predictive performance of selected genes . in this way , each test case is never used for gene selection . to avoid a bias due to partitions of data ,
if the performance is assessed using the very same data that are used for developing the classifier , this obviously leads to a biased down estimate of classification error . in other words , if one applies a method to the original data with 20 trials of 10-fold cv only as a way of building the classifier and selects a set of genes and then on that very same data calculates the classification error , it clearly leads to a biased upward estimate of classification accuracy .
three publicly available data sets of interest in this paper are pediatric acute myeloid leukemia ( aml ) , b - cell chronic lymphocytic leukemia ( b - cll ) , and primary cutaneous melanoma .
the data sets are downloaded from the brb - arraytools data archive for human cancer gene expression located at the website http://linus.nci.nih.gov/~brb/dataarchive_new.html .
sex differences in disease rates or in rates of adverse reactions to treatment are common , which we intend to exploit to obtain sex - specific biomarkers from gene - expression data .
we hypothesize that genomic biomarkers developed from the sex - specific application of classification algorithms will further improve class prediction accuracy .
the summary of our algorithm to find sex - specific predictive / prognostic genomic biomarkers for efficacy or toxicity in individualized treatment of patients for serious diseases is as follows . in each loocv trial , firstly , the data is partitioned into a test data set with one observation and the remaining data as the learning data set .
the learning data set is further separated into male and female patients ' learning data sets .
thus , this process will be applied n times , where n represents the total number of patients .
secondly , within each trial of loocv , twenty trials of 10-fold cv are conducted for each set of male and female patients in the learning data set . at each 10-fold cv step ,
two sets of top - ranked genes , one for male patients ( smi ) and one for female patients ( sfi ) , are obtained via the bw ratio to differentiate types of patients such as diseased versus nondiseased .
this can be done by applying variable importance ranking approach in order to extract most influential genes and by combining a measure of importance in each gene .
a score taking the average of the measure in cross - validation ( cv ) is prioritized in the list of genes according to variable importance .
thirdly , the mutually exclusive sets of male - specific genes and female - specific genes are obtained .
fourthly , within each loocv trial , after the sets of potential sex - specific genes are determined in the second and the third steps , they are fitted to a model with diagonal linear discriminant analysis ( dlda ; ) using each learning data set for male and female patients .
finally , the performance is measured with the test data with one observation in each loocv trial .
this method is implemented in r. the r code is available upon request . within each trial of loocv , each set of top - ranked genes for male and female patients is obtained in the second and the third steps as follows .
first , in order to build genomic biomarkers , 20 trials of 10-fold cv are applied to each learning data set for males and females , where 90% were randomly selected without replacement as a set for each trial of cv .
next , for each trial of 10-fold cv , the bw ratio was applied to this 90 percent learning set and the top 25 ranked genes were selected in each process with the target endpoint of a dataset .
the bw ratio for a gene j is defined as
( 1)bwj=iki(yi = k)(xkjxj)2iki(yi = k)(xijxkj)2 ,
where x-j=ixij / n indicates the average value of a gene j across all the training samples , x - kj=i(yi = k)xij / nk indicates the average value of a gene j for a class k , and i indicates an observation . here
this criterion has been shown to be reliable for variable selection from high - dimensional data sets [ 6 , 12 ] .
next , to avoid selection bias from a pattern of selection of learning samples , we repeat the entire process 20 times by shuffling samples at every 10-fold cv . in order to obtain the variable importance ranking , 200 sets of top 25 ranked genes were combined so that the maximum possible rank score of a gene would be 200 .
a set of genes that has been selected at least once ( rank score > 0 ) was obtained separately for males and females .
these most influential genes used in the classification process are identified in order to extract a feasible set of sex - specific genes . for both male and female learning data sets ,
the final product of the 20 trials of 10-fold cv described in each loocv is the sets of genes selected for male and female patients in the learning data set smj = { g1 , , gxmj } and sfj = { g1 , , gxfj } , j = 1 , 2 , , n , respectively .
the sets smj and sfj contain prognostic / predictive genes for male and female patients , respectively
. there would be n sets of selected genes . within each trial of loocv , genes that are commonly identified between male - specific and female - specific genes
finally , the test sample with one observation is tested using these sex - specific genes in each loocv . at the end of the entire loocv
, each selected gene has a combined variable importance score that is less than or equal to n. to verify sex - specific biomarkers , we consider the following four different cases : we classify the outcome of ( 1 ) male patients with a set of male - specific genes smj,(2 ) female patients with a set of male - specific genes smj,(3 ) male patients with a set of female - specific genes sfj , and ( 4 ) female patients with a set of female - specific genes sfj .
we anticipate that data with a set of male - specific genes have higher predictable power to predict male patients than the data with female - specific genes .
similarly , data with female - specific genes have higher predictable power to predict female patients than the data with male - specific genes . upon completion of loocv
, the performance of sex - specific biomarkers is obtained . in order to validly evaluate the performance of a gene set selected by the proposed method ,
cv utilizes resampling without replacement of the entire data set to repeatedly develop classifiers on a training set and to evaluate these classifiers on a separate test set and then averages the results over the resamples .
in this section , we apply the proposed algorithm to the following genomic data sets to find the most meaningful sex - specific predictive / prognostic genomic biomarkers for improving individualized treatment of patients and for evaluating the biomarkers from the proposed algorithm . current chemotherapy enables a high percentage of pediatric patients with aml to enter complete remission ( cr ) , but a large number of them experience relapse ( r ) with resistant disease . because of the wide heterogeneity of aml , predicting a patient 's risk for treatment failure or relapse at the time of diagnosis is critical for the optimal treatment . this gene - expression data set consists of 54 aml pediatric patients ( < 15 years old ) with an oligonucleotide microarray containing 12,566 probe sets and it is also available at ftp://ftp.ncbi.nih.gov/pub/geo/data/soft/gds/gds1059.soft.gz .
patients with cr for more than 3 years are classified as having a good prognosis , while patients experienced relapse within 1 year of the first cr are considered as having a poor prognosis . in this data set , there are 28 patients with cr and 25 patients with r. since a five - month male patient experienced induction failure ( no cr achievement ) within 3 months of the start of treatment is considered neither cr nor r , we exclude this subject . therefore , there are 32 male patients and 21 female patients in this data set . with the prognostic endpoint ( r / cr ) ,
the average accuracy of 66% ( sd 3.0% ) for pediatric patient classification was obtained when no gene selection was introduced . when a set of 200 genes was selected in a learning phase of each cv ,
the average accuracy of 68.0% ( sd 3.0% ) was achieved . when a set of 20 genes was selected in a learning phase of each cv ,
the average accuracy of 71.0% ( sd 5.0% ) was obtained . since it appeared to be no substantial difference between accuracy and the number of genes selected , a feasible set of 25 genes in the learning phase of each cv
was collectively ranked by the bw ratio to find sex - specific biomarkers . at the end of loocv trials , a set of male - specific genes that were selected at least 75% of the time in the entire loocv were 1882_g_at ( mecom : mds1 and evi1 complex locus ) , 37902_at ( cryz : crystallin , zeta ( quinone reductase ) ) , 38789_at ( tkt : transketolase ( wernicke - korsakoff syndrome ) ) , 39105_at ( vasp : vasodilator - stimulated phosphoprotein ) , 40844_at ( ctr9 : ctr9 , paf1/rna polymerase ii complex component , homolog ( s. cerevisiae ) ) , 36981_at ( srp9 : signal recognition particle 9 kda ) , 36338_at ( luzp1 : leucine zipper protein 1 ) , 31870_at ( cd37 : cd37 antigen ) , 1624_at ( rap1gds1 : rap1 , gtp - gdp dissociation stimulator 1 ) , and 39142_at ( nudt21 : cleavage and polyadenylation specific factor 5 , 25 kda ) .
these ten top - ranked male - specific genes were selected as potential male - specific genomic biomarkers to classify male patients into r / cr . among them
38789_at ( tkt : transketolase ) and 36338_at ( est ) were also included in the thirty - five genes associated with prognosis of pediatric aml identified by yagi et al . .
in order to select a feasible set of sex - specific biomarkers a cut - off criterion of 75 percent is used . since
every dataset may have a different sample size , the number of genes selected is given by the percentage , which is a rank score of the selected genes greater than 75% of the sample size in our case .
for example , if a sample size is 100 , then genes that have rank scores greater than 75 have been selected .
similarly , nine top - ranked genes for classifying female patients into r / cr were 40601_at ( tm2d1 : tm2 domain containing 1 ) , 36330_at ( ccbl1 : cysteine conjugate beta- lyase ; cytoplasmic ( glutamine transaminase k , kynurenine aminotransferase ) ) , 40586_at ( eef1e1 : eukaryotic translation elongation factor 1 epsilon 1 ) , 36648_at ( crsp9 : cofactor required for sp1 transcriptional activation , subunit 9 , 33 kda ) , 32351_at ( gpr20 : g protein - coupled receptor 20 ) , 1718_at ( arpc2 : actin - related protein 2/3 complex , subunit 2 , 34 kda ) , 38622_at ( mtg1 : mitochondrial gtpase 1 homolog ( s. cerevisiae ) ) , 36496_at ( impa2 : inositol(myo)-1(or 4)-monophosphatase 2 ) , and 38337_at ( znf193 : zinc finger protein 193 ) .
these nine top - ranked genes which were selected at least 75% of the time in the entire loocv were considered as potential female - specific genomic biomarkers to classify female patients into r / cr .
data with male - specific genes showed higher prediction accuracy ( 71.9% ) to classify male patients than the accuracy ( 43.8% ) to classify male patients from data with female - specific genes .
similarly , data with female - specific genes showed higher prediction accuracy ( 76.2% ) to classify female patients than the accuracy ( 61.9% ) to classify female patients from data with male - specific genes .
as shown in table 1 , sensitivity and specificity were also higher in using sex - specific genes for each sex .
genomic aberrations and mutational status of the immunoglobulin variable heavy chain ( vh ) gene have been shown to be among the most important predictors for outcome in patients with b - cll .
b - cll is the most common leukemia in the western world , and due to its clinical heterogeneity ( wide range of life expectancy ) and correlations to genomic aberrations , it is important to predict a patient 's vh mutation status at the time of diagnosis for optimal treatment .
in addition , the study presented by haslinger et al . suggested that the genomic signature for vh mutational status might be sex related .
the gene - expression data consists of 100 b - cll patients with an oligonucleotide microarray containing around 12,000 probe sets , and it is available at http://linus.nci.nih.gov/~brb/dataarchive_new.html .
patients are classified as either vh - mutated or unmutated ( m / nm ) .
there are 62 males ( 33 m and 29 nm ) and 38 females ( 18 m and 20 nm ) , with a total of 51 mutated and 49 unmutated patients . in step 1 , for each data set with male only and female only patients using the target endpoint ( i.e. , vh - mutated ( m ) versus unmutated ( nm ) ) , we separately selected and ranked 25 potential prognostic genes for males and for females in every cv trial and separately combined ranks of these genes for males and females during the learning phase of 20 trials of 10-fold cv within each loocv trial . in every loocv trial , we prioritized and combined the final top - ranked 200 genes from the male patients result ( sm ) and 200 genes from the female patients result ( sf ) as explained in section 3.1 . at the end of the entire loocv trials , a set of potential sex - specific genes
are obtained for each sex by prioritizing and combining n sets of top - ranked 200 genes .
after deletion of overlapped genes in both males and females , eleven potential male - specific genes were obtained to classify male patients into m / nm . they were 41209_at ( lpl : lipoprotein lipase ) , 41755_at ( cobll1 : cobl - like 1 ) , 39878_at ( pcdh9 : protocadherin 9 ) , 38211_at ( zbtb20 : zinc finger and btb domain containing 20 ) , 39488_at ( pcdh9 : protocadherin 9 ) , 36886_f_at ( kir2dl3 : killer cell immunoglobulin - like receptor , two domains , long cytoplasmic tail , 3 ) , 32140_at ( sorl1 : sortilin - related receptor , l(dlr class ) a repeats - containing ) , 33535_at ( p2rx1 : purinergic receptor p2x , ligand - gated ion channel , 1 ) , 39967_at ( ldoc1 : leucine zipper , downregulated in cancer 1 ) , 32842_at ( bcl7a : b - cell cll / lymphoma 7a ) , and 36899_at ( satb1 : special at - rich sequence binding protein 1 ( binds to nuclear matrix / scaffold - associating dna 's ) ) . for female patients ,
only five genes were selected at least 75% of the time in the entire loocv trial as potential female - specific genomic biomarkers to classify female patients into m / nm . they were 33745_at ( phkg2 : phosphorylase kinase , gamma 2 ( testis ) ) , 38152_at ( loh11cr2a : loss of heterozygosity , 11 , chromosomal region 2 , gene
a ) , 34142_at ( pde8a : phosphodiesterase 8a ) , 39593_at ( fgl2 : fibrinogen - like 2 ) , and 217_at ( klk2 : kallikrein 2 , prostatic ) . to verify the sex - specific genomic biomarkers , we considered the performance of four different cases in the loocv trials as described in section 2 .
data with male - specific genomic biomarkers showed higher accuracy ( 67.7% ) to classify male patients into m / nm than the accuracy to classify male patients into m / nm from data with female - specific genomic biomarkers ( 40.3% ) .
however , female data with female - specific genes showed prediction accuracy similar to random guess close to 50% .
therefore , there is insufficient evidence to support that the selected female - specific genes were female - specific genes ( see table 2 ) with 38 female patients in this data set .
although cutaneous melanoma represents a small subset , it is the most life - threatening neoplasm of the skin , and its incidence and mortality have been increasing worldwide .
the key underlying molecular events have not been clearly elucidated , which may explain why no target has been developed and why almost no clinical benefits from new therapies have been clearly demonstrated in patients with melanoma since the late 1970s .
additionally , gene - expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors .
this gene - expression data set was collected from 83 patients corresponding to the training data set and 17 patients corresponding to the validation data set .
the probes are from tumor tissue and from reference tissue that are differentially labeled by the incorporation of cyanine 3 ( cy3 ) and cyanine 5 ( cy5 ) , respectively .
set , the endpoint was patient prognosis and survival along with tumor stages , defined as follows . in stage
i , cure rates are excellent with surgical removal , since they are the least likely to spread . in stage ii , melanomas can be cured , but the success rate lags behind that of stage i because a small number of cancer cells may have spread to distant sites . in stage iii , since the tumor has started to metastasize ( the spreading of a disease from one organ or part to another nonadjacent organ or part ) , the survival rate for these stages is lower than the earlier ones .
stage iv is associated with metastasis beyond the regional lymph nodes to distant sites in the body , such as the lung , liver , or brain , or to distant areas of the skin .
based on the tumor size , descriptions , and number of lymph nodes the stages are categorized in two classes .
a class of high survival and small tumor size ( hs / st ) is defined and composed of stages 1 and 2 .
the second is defined as low survival and non - small tumor size ( ls / nst ) , which is composed of stages 3 and 4 .
tables 3 and 4 show the distribution of the primary cutaneous melanoma data based on sex , the defined clinical endpoint hs / st and ls / nst for the training data and validation data . among 83 patients in the training dataset ,
there are 27 males ( 12 hs / st and 15 ls / nst ) and 56 females ( 30 hs / st and 26 ls / nst ) .
there are 42 cases of hs / st and 41 cases of ls / nst in total .
after preprocessing the data the final gene count is 4641 genes . among 17 patients in the validation data
set , there are 8 males ( 1 hs / st and 7 ls / nst ) and 9 females ( 1 hs / st and 8 ls / nst )
. there are 2 cases of hs / st and 15 cases of ls / nst in total .
since the validation set was separately provided , the sex - specific genes were selected via 20 trials of 10-fold cv in the learning set .
the following ten male - specific genes were selected at least 75% of the time during 20 trials of 10-fold cv : a_23_p128263 ( prb1 : proline - rich protein bstni subfamily 1 ) , a_23_p83838 ( ca8 : carbonic anhydrase viii ) , a_24_p212990 ( mgc70863 : similar to rpl23ap7 protein ) , a_23_p333650 ( rad9b : rad9 homolog b ( s. cerevisiae ) ) , a_32_p125251 ( n / a ) , a_23_p108835 ( ypel5 : yippee - like 5 ( drosophila ) ) , a_32_p150856 ( loc407835 : mitogen - activated protein kinase kinase 2 pseudogene ) , a_23_p11936 ( ubxn11 : ubx domain protein 11 ) , a_24_p169976 ( n / a ) , and a_32_p3998 ( znf600 : zinc finger protein 600 ) . for female patients ,
the following eight female - specific genes were selected from 20 trials of 10-fold cv : a_23_p69497 ( clec3b : c - type lectin domain family 3 , member b ) , a_24_p118884 ( n / a ) , a_23_p152420 ( kiaa0182 ) , a_24_p265177 ( phc3 : polyhomeotic - like 3 ( drosophila ) ) , a_23_p251421 ( cdca7 : cell division cycle associated 7 ) , a_23_p211738 ( ubp1 : upstream binding protein 1 ( lbp-1a ) ) , a_23_p47377 ( hsd17b12 : hydroxysteroid ( 17-beta ) dehydrogenase 12 ) , and a_32_p113646 ( cdna flj45341 fis , clone brhip3009672 ) . using a given validation set ,
the sex - specific genes were verified . as shown in the confusion matrix in table 5 , there was no difference in the performance from female data with female genes compared with the performance of female data with male genes .
however , there was only one misclassification for male patients with male genes as shown in table 6 , while there were four misclassifications for male patients with female genes as shown in table 7 .
therefore , we conclude that there exists evidence of male - specific genes in a classification of primary cutaneous melanoma .
large inter individual differences in benefit from chemotherapy highlight the need to develop predictive genomic biomarkers for selecting the right treatment for the right patient .
inappropriate chemotherapy can result in the selection of more resistant and aggressive tumor cells . to date
, no reliable genomic biomarkers have been developed to provide the physician with prechemotherapy information to accurately predict the efficacy of a specific therapy .
we proposed a procedure to find sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm using variable importance ranking via combination of 20 trials of 10-fold cv and loocv .
the proposed procedure was applied to data sets obtained from the brb arraytools data human cancer archive .
however , the issue arose out of there being not enough samples , and the data was unbalanced ( i.e. , more males than females or more positives ( disease patients ) than negatives ( nondisease patients ) ) . while patient 's sex information was not specified for many of the publicly available data sets adding to that the difficulty of searching for data , we found the following three genomic data sets that had sex information : ( 1 ) pediatric patients with aml , ( 2 ) b - cell chronic lymphocytic leukemia ( b - cll ) , and ( 3 ) primary cutaneous melanoma . in one application , pediatric patients with aml were classified by the algorithms as having either a good or poor prognosis , in terms of the likelihood of induction failure or relapse within one year of the first complete remission , based on gene - expression profiles .
if this were brought into clinical application , a patient with a confidently predicted good prognosis might want to elect out of adjuvant chemotherapy and its associated debilitating side effects . with current rule - based decisions ,
the overall average accuracy of this data set with a variable selection from pooled patients ( males and females ) was about 71.0% .
however , using male - specific genes found by the proposed procedure , the accuracy was improved to about 72% as we found in the model validation studies . similarly , using female - specific genes found by the proposed procedure ,
the average accuracy was improved to about 76% ( see table 1 ) . in the b - cell chronic lymphocytic leukemia ( b - cll )
dataset we found male - specific prognostic genomic biomarkers associated with b - cell chronic lymphocytic leukemia and its average classification accuracy was improved to about 68% .
there was no substantial evidence to find female - specific prognostic genomic biomarkers in this data set .
similarly , male - specific predictive genomic biomarkers associated with a classification of primary cutaneous melanoma were found with the classification accuracy of about 88% .
the scope of our paper was to find sex - specific genomic biomarkers , if any , imbedded in the data instead of finding genomic biomarkers from the data .
if commonly identified genes were kept in the proposed procedure , our procedure was not sex - specific genomic biomarker classifier involving two populations ( males and females ) any more but rather it became genomic biomarker classifier involving one combined population .
in fact , even though commonly identified genes were kept , it did not necessarily improve the classification accuracy .
for a counterexample , for the pediatric aml data of yagi et al . , the accuracy for female patients with female - specific genes by keeping the commonly identified genes was 62% ; however , the accuracy without commonly identified genes was 76% .
for the male patients with male - specific genes by keeping the commonly identified genes the accuracy was 59% , but the accuracy without the commonly identified genes was 72% .
for the related note , the accuracy of this data can be found anywhere between 59% and 66% with gene preprocessing and between 58% and 71% with gene selection .
it is not an easy task to find sex - specific genes , let alone verifying and proving that they are indeed sexspecific .
we have presented a procedure for finding sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm .
the procedure is shown to have good sensitivity and specificity in the sense that the sex - specific genes obtained can improve prediction accuracy in classification of individual patient 's prognosis .
the proposed procedure to discover predictive and prognostic sex - specific genomic biomarkers for individualized treatment of diseases can play a critical role in developing safer and more effective therapies that replace one - size - fits - all drugs with treatments that focus on specific patient needs . | numerous studies have demonstrated sex differences in drug reactions to the same drug treatment , steering away from the traditional view of one - size - fits - all medicine .
a premise of this study is that the sex of a patient influences difference in disease characteristics and risk factors . in this study , we intend to exploit and to obtain better sex - specific biomarkers from gene - expression data .
we propose a procedure to isolate a set of important genes as sex - specific genomic biomarkers , which may enable more effective patient treatment .
a set of sex - specific genes is obtained by a variable importance ranking using a combination of cross - validation methods .
the proposed procedure is applied to three gene - expression datasets . |
in the last decade , preimplantation genetic diagnosis ( pgd ) has become an important alternative to prenatal diagnosis for couples at high risk of transmitting inherited disorders to their children .
fluorescence in situ hybridization ( fish ) has been offered for gender selection in x - linked diseases ( 1 , 2 ) , chromosomal translocations ( 3 - 5 ) , aneuploidy , and recurrent implantation failure ( 6 , 7 ) .
procedures using polymerase chain reaction ( pcr ) has been applied for single gene defects including x - linked diseases ( 8 - 11 ) .
ornithine transcarbamylase ( otc ; ec 2.1.3.3 ) is placed in the mitochondrial matrix where it contributes to the detoxification of nitrogenous wastes through the urea cycle , and catalyzes the synthesis of citruline from carbamyl phosphate and ornithine .
the otc gene is located on the short arm of the x chromosome within band xp21.1 ( 12 ) .
it spans 73 kb , has an open reading frame of 1,062 nucleotides , and contains 10 exons and 9 introns ( 13 , 14 ) .
otc deficiency ( mim-#311250 ) is an x - linked and co - dominant metabolic disorder with partial penetrance in females ( 15 ) .
the phenotype of otc deficiency is extremely heterogeneous , ranging from asymptomatic adult hemizygous males to acute neonatal hyperammonemic coma in the first week of affected male babies .
approximately 80% of heterozygous females are asymptomatic and remaining 20% show clinical severity similar to males with partial deficiencies ( 16 , 17 ) .
there are more than 341 mutations known to cause otc deficiency , all of which are specific for the individual families .
mutations have been found in all exons and introns with a relative paucity of mutations in the sequence encoding the leader peptide ( exon 1 and a part of exon 2 ) and in exon 7 ( 17 , 18 ) . to our knowledge , only two cases have been reported where specific pgd for otc deficiency were carried out using multiplex or duplex - nested pcr assay and healthy babies devoid of the otc mutation were born ( 19 , 20 ) . in this study
, we describe the successful pregnancy and birth after pgd for otc deficiency with simultaneous analyses of fish and duplex - nested pcr in korea .
we have applied a duplex - nested pcr for the amplification of both the causative mutation and the y chromosome specific sry gene , and fish for aneuploidy screening of chromosome x , y and 18 .
a couple was referred to our center after therapeutic termination of the second pregnancy following prenatal diagnosis for otc deficiency . in the first pregnancy ,
a male baby was born but died at 10 days after birth due to liver dysfunction accompanying hyperammonemia .
molecular genetic analysis of the couple 's otc gene was performed by pcr and direct sequencing at seoul asan hospital and revealed a single base substitution ( r320x ) in the exon 9 of the female partner . in the second pregnancy , the fetus
was identified to be an affected boy by pcr and direct sequencing on a chorionic villus sample , and the pregnancy was therapeutically terminated . in order to avoid a pregnancy with an affected baby , the couple was counseled for pgd in cheil general hospital . in order to determine the efficiency of single cell pcr and allele drop - out ( ado ) rate , primers used for the detection of the causative mutation and y chromosome specific sry gene were tested using single lymphocytes ( table 1 ) .
ovarian stimulation , in vitro fertilization , and blastomere biopsy were done as previously described ( 4 , 5 ) .
after the blastomere biopsy procedure , the embryos were washed several times and transferred to the g2.2 medium ( vitrolife sweden ab , kungsbacka , sweden ) .
each blastomere was washed twice through two drops of g2.2 medium and transferred into sterile 0.2 ml pcr tubes containing 5 l of alkaline lysis buffer .
the nested - duplex pcr analysis was performed by previously described protocol ( 6 ) .
mutation analysis by restriction fragment length polynorphism ( rflp ) with bcli restriction enzyme was carried out only for positively amplified pcr products of blastomeres .
the fish analysis for chromosome x , y and 18 was done as previously described and the manufacture 's protocol ( 4 , 5 ) .
the embryos with the normal genotype were selected and transferred on day 4 after fertilization .
all 10 exons and exon - intron boundaries of the otc gene were screened by pcr followed by direct dna sequencing analysis .
the heterozygous otc gene with r320x nonsense mutation ( cgatga ) and wild type was detected in the exon 9 of the female partner by rflp ( fig . 1 ) and direct sequencing ( data not shown ) .
this r320x nonsense mutation was associated with acute neonatal hyperammonemia in their first pregnancy and reduced enzyme activity by 10 - 15% ( 21 ) .
the efficiency and accuracy of the duplex - nested pcr analysis were tested on single lymphocytes from the heterozygous female partner and normal male partner .
pcr on single lymphocytes resulted in 90.6% ( 58/64 ) amplification rate , whereas none of the negative controls showed a positive band .
after ovarian hyperstimulation , a total of 18 oocytes were retrieved and 17 oocytes were injected using intracytoplasmic sperm injection in order to prevent dna contamination from inseminated sperm . among a total of 11 embryos ,
two blastomeres per embryo from 9 embryos were biopsied and simultaneously analyzed by duplex - nested pcr and fish , and one blastomere per embryo from 2 embryos by only duplex - nested pcr . as a result of duplex - nested pcr and rflp analysis with the bcli restriction enzyme ,
ten showed successful amplification for the exon 9 of the otc gene , and the amplification rate was 90.9% , almost same as that of the single lymphocyte test .
there were two unaffected males , five affected males , two unaffected females , and one heterozygous female ( fig . 2 and table 2 ) .
on the other hand , out of nine blastomeres analyzed by fish , only six blastomeres were identified as chromosomally normal embryos ( fig .
however , among total four unaffected embryos ( embryo 1 , 2 , 3 , and 8) diagnosed by pcr , only two embryos ( embryo 3 and 8) were identified as chromosomally normal by fish .
the other two embryos ( embryo 1 and 2 ) were shown to have trisomy 18 and monosomy 18 , respectively . only one male embryo ( embryo 8) was transferred , and another female unaffected embryo ( embryo 3 ) was cryopreserved at blastocyst stage on day 5 after fertilization .
this pgd cycle resulted in a singleton pregnancy and the genotype of the fetus was confirmed to be a normal male by amniocentesis at 16 weeks of gestation .
the genotype of the baby for the exon 9 of otc gene and level of ammonia were confirmed as normal .
most pgd cycles for x - linked disorders such as duchenne muscular dystrophy and fragile x syndrome involved the selection of female embryos which were diagnosed as normal or heterozygous . these gender selections were supported by couples at risk of x - linked recessive conditions .
ado for affected allele could lead to yielding an affected heterozygous baby , even if the female embryo was diagnosed as normal by pgd for otc deficiency .
gender selection with fish for y chromosome - specific loci is more reliable because amplification failure could occasionally occur in single cell pcr .
thus , our protocol with pcr and fish in this study provides higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for x - linked co - dominant defects .
we applied duplex - nested pcr and fish analysis in two blastomeres from each embryo , which were more than 6-cell stage .
biopsy of more than a quarter of blastomeres from each embryo could interfere with developmental potential of embryos .
thus , single blastomere biopsy was applied to less than 6-cell stage embryos in this study .
sequential pcr and fish analysis in one blastomere ( cell recycling ) was reported and could be used to detect the aneuploidy , but the rate of ado after pcr seems to be higher using this method ( 22 , 23 ) .
a duplex - nested pcr analysis was developed for the simultaneous diagnosis of a r320x mutation and a gender selection , and fish analysis was used to detect aneuploidy for chromosome x , y and 18 . as a result of duplex nested pcr and fish analysis ,
concordant results have been obtained from two blastomeres at the sex locus , and the amplification efficiency was 100% ( 9/9 except two blastomeres , that were not analyzed by fish analysis ) for sry .
and the amplification rate was 90.9% ( 10/11 ) for exon 9 of the otc gene .
several strategies have been developed to decrease amplification failure and ado in single cell analysis of pgd .
these included the development of sequential first and second polar body analysis ( 24 ) , improvement of the pcr condition by increasing the denaturation temperature and time ( 19 ) , using a more powerful lysis method ( 25 - 28 ) and the use of fluorescent pcr for mutations or linked markers and multiplex pcr ( 29 - 32 ) .
as mentioned above , we have also endeavored to optimize single cell pcr conditions , which included longer initial denaturation time , higher denaturation temperature , and the selection of taq polymerase , the concentration of mgcl2 , and lysis methods .
the incidence of ado was 5 - 15% in many pgd laboratories ( 27 , 28 ) . at a preliminary experiment on single lymphocytes in this study ,
the ado rate for exon 9 of the otc gene was about 13.0% when using duplex - nested pcr .
we could not absolutely exclude the possibility of ado in the diagnosis of unaffected female embryo owing to the relatively higher ado .
thus , fish was simultaneously applied for the aneuploidy screening and sex selection to prevent the pregnancy of heterozygous female . in this study
, we report the successful pgd and delivery of a healthy boy in a couple at risk of transmitting the otc deficiency .
this was carried out by the simultaneous analysis of the duplex - nested pcr for a r320x mutation in the otc gene underlying the otc deficiency and y chromosome - specific loci , and fish for aneuploidy screening of chromosome x , y and 18 .
this strategy could provide higher accuracy for the selection of genetically and chromosomally normal embryos in pgd for single gene defects . | ornithine transcarbamylase ( otc ) deficiency is an x - linked co - dominant disorder .
a couple , with a previous history of a neonatal death and a therapeutical termination due to otc deficiency , was referred to our center for preimplantation genetic diagnosis ( pgd ) .
the female partner has a nonsense mutation in the exon 9 of the otc gene ( r320x ) .
we carried out nested polymerase chain reaction ( pcr ) for r320x mutation and fluorescence in situ hybridization ( fish ) for aneuploidy screening . among a total of 11 embryos , two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex - nested pcr and fish , and one blastomere per embryo from 2 embryos by only duplex - nested pcr . as a result of pcr and restriction fragment length polymorphism analysis , four embryos were diagnosed as unaffected embryos having the normal otc gene . among these embryos , only one embryo was confirmed as euploidy for chromosome x , y and 18 by fish analysis . a single normal embryo was transferred to the mother , yielding an unaffected pregnancy and birth of a healthy boy .
based on our results , pcr for mutation loci and fish for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for single gene defects . |
musculoskeletal stiffness and limited range of motion ( rom ) can restrict functional joint
movement , particularly during ankle supination following neurological diseases1 .
ankle dorsiflexion rom restrictions impair
dynamic balance and gait , which may contribute to secondary injuries ; reduced ankle rom and
tight calf muscles can cause poor gait , inefficient energy use , impaired balance , and an
increased risk of falls2 .
musculoskeletal
transformations manifesting as abnormal joint stiffness and limited rom result in numerous
restrictions in the function and joint movements of patients3 .
limited passive ankle dorsiflexion rom during knee extension can
alter foot positioning and result in compensatory foot movements , leading to an abnormal
gait , which can result in ankle sprains and lower extremity overuse injuries4 .
studies have reported that balance - training
programs that alter the somatosensory input , such as the use of an unstable surface to
induce equalized weight distribution , can be helpful in hemiplegic patients5 .
kim et al . used an ankle board with a slope
in their center to achieve efficient ankle stretching and increased dorsiflexion6 .
they suggested that stretching exercises
using their device resulted in a greater increase in foot pressure compared to that with
stretching exercises using existing inclined boards during ankle dorsiflexion
stretching6 .
therefore , we investigated
the effects of an unstable inclined board on the active and passive ankle rom in patients
with ankle stiffness .
the study included 10 female patients ( mean age , 22.8 5.3 years ; height , 158.9 3.0 cm ;
weight , 61.2 4.7 kg ) with ankle stiffness caused by diseases or medical conditions .
the
patients were selected based on ankle stiffness scores obtained using a symptom checker
tool .
the patients were assigned randomly to two groups ( both n = 5 ) and their height and
weight were measured .
the patients were not cognitively impaired and were capable of
independent walking for 20 m without difficulty .
patients were excluded if they had a
history of or current neurological conditions . before participating ,
the purpose and methods
of the study were explained to the patients , and all provided informed consent , according to
the principles of the declaration of helsinki .
active and passive ankle dorsiflexion were measured using a standard 31.75 4.45 cm clear
plastic goniometer , with one increment , a 360 scale , and a bubble level at each end .
group
1 performed the ankle dorsiflexion stretching exercise using a wooden inclined board , and
group 2 performed ankle dorsiflexion stretching exercises using an air - cushioned inclined
board that provided an unstable surface .
rubber sheets were attached to the inclined boards to prevent falls due to
sliding .
the patients performed ankle dorsiflexion stretching exercises for 5 min , with a
10-min rest between each set , five times a day for 1 week .
the active and passive ankle
dorsiflexion angles were measured bilaterally using the goniometer before and after the
study .
independent and paired t - tests were used to assess within- and between - group differences in
ankle dorsiflexion , with spss for windows ( spss , chicago , il , usa ) .
the amount of active and passive dorsiflexion did not differ between the two groups before
the exercises , with active dorsiflexion angles of 18.2 6.4 and 19.3 4.2 and passive
dorsiflexion angles of 23.4 5.5 and 22.6 5.7 in groups 1 and 2 , respectively ( both p
> 0.05 ) .
the stretching exercises significantly ( p < 0.05 ) increased the active and
passive ankle dorsiflexion angles in both groups compared to those at baseline .
the active
dorsiflexion angle was significantly increased ( p < 0.05 ) in group 2 compared to that in
group 1 after the exercises .
the active dorsiflexion angles before and after the exercises
were 18.2 6.4 and 22.1 5.0 in group 1 and 19.3 4.2 and 28.1 6.9 in group 2 ,
respectively .
the passive dorsiflexion angles did not differ significantly between the
groups after the exercises ( p>0.05 ) and were 23.4 5.5 and 32.2 5.3 in group 1 and
22.6 5.7 and 35.1 6.0 in group 2 before and after exercising , respectively .
the
air - cushioned inclined board provided an unstable surface for performing ankle dorsiflexion
stretching exercises .
the results showed that ankle stretching exercises using the unstable
inclined board significantly increased the active dorsiflexion angle compared to that with
exercises using a wooden inclined board .
we believe that the use of the air - cushioned
inclined board provides somatosensory input and subsequently stimulates active ankle
dorsiflexion by promoting automatic postural control .
sensory proprioception of the ankle
and sensory compression of the plantar are important in the control of sway7 .
an increase in the number of somatosensory
inputs affects the muscles , and the use of an unstable support surface result in many
different somatosensory inputs that limit compensatory action7 .
performing cognitive tasks in a standing posture reduces internal
focus and induces external focus , which promotes automatic postural control8 .
stretching improve the flexibility of
tendons , and changes in the mechanical condition of tendons depend on the type of stretching
performed9 .
, the passive dorsiflexion angles did not differ significantly between the two
groups after ankle stretching exercises .
however , stretching exercises significantly
increased the active and passive ankle dorsiflexion angles in both groups compared to those
at baseline .
these results suggest that passive ankle exercises using a wooden inclined
board only stretch muscle and tissue , while exercises using an unstable inclined board
stretch muscle and tissue , and stimulate activation of the ankle dorsiflexor .
active ankle
dorsiflexion was more effectively improved with ankle stretching exercises using an unstable
inclined board than with exercises using a wooden inclined board . | [ purpose ] the present study assessed the effects of using an unstable inclined board on
the active and passive ankle range of motion in patients with ankle stiffness . [ subjects ]
the study included 10 young female patients with ankle stiffness .
[ methods ] the patients
were divided into the following two groups : a group that performed ankle dorsiflexion
stretching exercises using a wooden inclined board and a group that performed stretching
exercises using an air - cushioned inclined board ( unstable inclined board ) .
active and
passive ankle dorsiflexion angles were measured bilaterally using a goniometer .
[ results ]
both inclined boards significantly increased active and passive ankle dorsiflexion .
after
performing ankle stretching exercises , active dorsiflexion significantly increased the
unstable inclined board compared to that using the wooden inclined board .
however , the
passive dorsiflexion angles did not differ significantly between the two groups after
ankle stretching exercises .
[ conclusion ] the use of an unstable inclined board might
stimulate activation of the ankle dorsiflexors in addition to stretching muscle or tissue .
active ankle dorsiflexion was more effectively improved with stretching exercises using an
unstable inclined board than with exercises using a wooden inclined board . |
renal tubular involvement in sjgren 's syndrome ( ss ) usually manifests with fanconi syndrome , distal ( type 1 ) renal tubular acidosis ( rta ) , nephrogenic diabetes insipidus , and hypokalemia .
, we describe a case a young female with ss who presented with features of gitelman syndrome and hypokalemic paralysis .
a 29-year - old female presented with weakness of both upper and lower limb weakness since 15 days .
she had a history of dry eyes and dry mouth for the past 6 months . on examination ,
laboratory evaluation showed hemoglobin - 9.5 g / dl , platelet 2.95 lakh / mm .
blood urea nitrogen was 7 mg / dl , serum creatinine was 0.65 mg / dl , serum sodium was 134 meq / l , serum potassium was 1.81 meq / l and serum chloride was 84.3
urine chloride ( spot ) was 36.7 meq / l , urine creatinine ( spot ) 23.5 mg / dl , and urine calcium ( spot ) 1.23 mg / dl .
this patient satisfied the american european consensus criteria for diagnosis of primary sjgren 's syndrome ( ss ) .
ten days after admission , she was discharged on spironolactone and prednisolone . on follow - up
this patient presented with muscle weakness and her laboratory reports showed hypokalemia and metabolic alkalosis with normal blood pressure .
her urinary chloride levels were above 20 meq / l , which ruled out extra - renal causes of metabolic alkalosis .
the presence of hypomagnesemia and absence of hypercalciuria ( urinary calcium creatinine ratio < 0.2 ) ruled out bartter syndrome .
hypokalemic alkalosis , high urinary chloride , hypomagnesemia , absence of hypercalciuria in background of no history of diuretic use favored the diagnosis of gitelman syndrome .
tubular involvement in ss is usually distal tubular dysfunction , type i ( distal ) renal tubular acidosis and nephrogenic diabetes insipidus .
proximal tubular abnormalities are less frequent , and rarely fanconi 's syndrome has been reported in patients with ss .
acquired gitelman syndrome is relatively rare . to the best of our knowledge , only five cases of acquired gitelman syndrome have been reported in english literature so far . among these five cases ,
four had ss , one was a case of chronic sialoadenitis . acquired gitelman syndrome secondary to ss presenting with hypokalemic weakness is very rare .
only two cases have been reported so far . though gitelman syndrome is an inherited disorder ,
given the paucity of reports , we believe that ss presenting as acquired gitelman syndrome may be relatively rare . acquired gitelman syndrome should be considered in differential diagnosis of renal involvement in patients with ss . | we report a case of a young female patient who presented with weakness of upper and lower limbs . on evaluation , she had hypokalemia , hypomagnesemia , metabolic alkalosis and hypocalciuria .
anti - ro ( ssa ) antibody was positive .
she had an acquired gitelman syndrome due to primary sjgren 's syndrome ( ss ) .
ss presenting with features of gitelman syndrome is very rare . |
inadequate organization has been identified as an important cause of inappropriate courses of treatment for patients in the health care system [ 1 , 2 ] .
a response to this challenge has been to focus on integrated health care and co - ordination between the actors involved in the process [ 37 ] .
an illustrative example of this can be found when looking at frail elderly patients on their discharge from hospital .
studies show that readmission can be diminished by increased collaboration between hospital , district nurses and general practitioners [ 8 , 9 ] .
follow - up home visits by the district nurse and general practitioner to frail elderly patients after discharge have shown promising results [ 10 , 11 ] .
the aim of follow - up home visits to elderly and frail persons is to enable them to remain in their own home , avoid readmission to hospital , admission to nursing homes or other forms of sheltered housing and to improve the functional ability and general wellbeing of the elderly .
a follow - up home visit comprises a home visit by a general practitioner ( gp ) and district nurse within about one week after discharge from hospital .
the visit is expected to last approximately one hour , a relatively long period compared to other types of doctor - patient contact .
this visit can be supplemented with two subsequent home visits or visits to the gp if this is considered necessary .
the latest study from denmark has shown that the readmission rate in the intervention group was 40% while it was 53% in the control group .
at the same time , studies have shown that there are indications that patients prefer primary care follow - up to specialist care .
these results are overall convincing and , as a result , on - going work is being done by health authorities to test and further develop follow - up home visits as an initiative , including a programme in copenhagen carried out from 20082011 .
the copenhagen programme was conducted as a randomized controlled trial which implies strict procedures for the implementation process .
a total of approximately 300 patients from bispebjerg hospital ( a major hospital in copenhagen ) were included in the intervention and control group .
approximately 100 follow - up home visits to patients in the intervention group have been made . in the remaining approximately 200 cases in the intervention group it has not been possible to carry out a follow - up home visit , first of all because gps could not meet the requirements . in this feasibility study gps
were not carefully selected for the programme as they were in an earlier study , and , as a result , more gps chose not to take part in the programme .
the effect of the copenhagen programme will be reported in 2012 by the danish institute of health services research .
this paper will focus on the implementation of follow - up home visits using a qualitative methodological approach .
we know some of the general dynamics in the area but we do not know more precisely what the practical limitations of the home visitor 's programmes are and how these limitations could make a difference to the outcomes assessed [ 14 , 15 ] . why do so few gps take part in the follow - up home visit programme ?
why is the documented effect of such visits not motivation enough for gps to encourage them to do so ?
addressing such questions could help us understand how to design and implement these visits in order to fully exploit the potential benefits of the intervention .
moreover , such results could generate useful hypotheses which could be addressed in future trials attempting to examine the effects of home visiting programmes .
the aim of this study is to explore one particular aspect of follow - up home visits : the motivation and rationale of local care providers to invest time and effort in follow - up home visits .
what is the main motivation of gps , district nurses and hospital staff to take part in a cross - sectoral activity like follow - up home visits ?
the case study is placed in a danish setting . in order to understand the organizational dynamics involved a short description of the setting
is needed : the danish health care system is mainly a public system based on general taxation , and characterized by universal access to health care services .
gps have a central position in the system since they have a kind of gatekeeper function , they are usually the first professional confronted with the patient 's problems and they see it as their responsibility to guide patients through the system .
gps are self - employed , general practices are small and typically privately owned ; only a minority of gps are organized in health centres .
home care organizations ( district nurses ) have occupied an ever more central role since a major administrative reform in 2007 gave the local level the main responsibility for preventive health care .
co - ordination between organizational units in primary and secondary health care is determined by agreements between regional and municipality authorities , which include procedures for such cases as when frail and elderly people are discharged from hospital .
in general , the danish system is considered quite integrated , in particular because the climate of trust in denmark makes co - operation possible [ 7 , 16 ] . as a result ,
follow - up home visits and other cross - sector interventions , such as follow - home arrangements and case managers have been quite regularly implemented on an experimental basis as a possible alternative to hospital - based measures .
the interventions have shown promising results but studies in the area also point to problems in connection with implementing the interventions [ 7 , 8 , 10 , 12 , 17 , 18 ] .
empirical data were gathered to develop hypotheses and theoretical concepts . at the same time , however , a rough theoretical framework was used as an underlying foundation in the study to ensure that a relevant focus and approach were applied .
resource dependency theory tells us that network formation can largely be explained by an organization 's need to reduce costs or gain resources and power .
organizations only work together when administrative and economic structures are designed which makes co - operation a profitable pursuit .
organizations thus typically follow their own agenda rather than involve themselves in cross - sectoral activities . in the health sector some support can be found for this basic claim .
studies have shown that network formation in general is limited and poor co - ordination predominant in the area [ 13 , 21 ] .
resource dependency theory also tells us , however , that organizations need to reduce uncertainty . to reduce uncertainty , organisations tend to co - operate with organisations they trust and depend on to achieve common goals [ 19 , 22 ] .
hence , in order to understand co - operation you also need to look at the question of trust and the quality of inter - organizational networks , as done in inter - organizational network theory .
this perspective has shown us that we need to have a comprehensive view of the factors which make health sector organisations work together .
cross - sectoral programs and inter - organizational activities are not based purely on partners ' own needs and resources [ 23 , 24 ] .
motivation plays a key role in inter - organizational theory [ 22 , 25 ] .
working together is a challenging task ; it is typically regarded as uncertain whether it will bring benefits equivalent to the resources invested .
motivation and commitment to the process are therefore needed among the actors involved in order to keep things moving and overcome the uncertainties inherent in the process . what motivates organizations to work together ?
the literature points to two aspects in particular : the object needs to be seen as highly relevant and benefits should be obvious for all involved [ 22 , 25 ] .
this paper is based on an evaluation report made by one of the authors for health and social care , city of copenhagen .
the interviews were structured around the following questions :
to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant?how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ? are the benefits obvious ?
to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant ? how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ?
two focus groups and seven individual interviews were conducted in march may 2010 , giving a total of 23 respondents .
the focus group approach was chosen because the interaction between some of the key respondents ( hospital staff , district nurses ) was considered to be of importance , since a key goal in the study was to understand the shared views on collaboration among these key actors in the programme .
one focus group interview was conducted with seven employees at bispebjerg hospital who were involved in the follow - up home visit programme .
recruitment criteria were designed in order to include a suitable spread of respondents , i.e. to ensure that members of all staff groups directly involved in the project were represented .
we ensured that the project co - ordinator at the hospital responsible for the selection of patients for the programme was represented in the focus group . at the same time we also ensured that a doctor and nurses from the most relevant wards were represented .
another focus group interview was conducted with nine district nurses who had been involved in the project .
nurses from different districts in copenhagen were selected for the focus group in order to ensure that different ways of organising follow - up home visits were represented .
gps were not as motivated to take part in the research project as district nurses and hospital staff , so a less time - consuming method than focus groups was considered appropriate . as it turned out , in - depth interviews proved to be highly relevant for gps because gps had divergent and less firm views about follow - up home visits than other groups in the study and the methodological design allowed these views to be fully expressed .
five individual interviews were held with gps involved in the project . both gps with a positive attitude towards follow - up home visits and gps with a less positive attitude were recruited to ensure that a wide range of attitudes was included in the study .
these recruitment criteria were based on data registered by the city of copenhagen showing which gps had refused to participate in follow - up home visits and which had agreed . at the same time we ensured that the gps represented covered different districts in copenhagen .
two individual interviews were held with employees from referrals in the city of copenhagen home nursing services .
there is a purchaser - provider split in the city of copenhagen , and the referrals purchase while the district nurses provides .
the two referrals came from different districts and had different positions in the organization , ensuring that the most important experiences from this organization were covered .
the focus group interviews took place in a neutral meeting room arranged by the project manager , and the in - depth interviews took place in the gps ' offices .
the focus group interviews lasted between 90 and 120 minutes , and the in - depth interviews between 25 and 60 minutes . both were recorded with a digital voice recorder and transcribed verbatim .
the interview transcripts were analyzed under the two headings which form the basic empirical questions in the study : relevance of collaboration and benefits of collaboration .
quotes from the interviews were grouped under each heading in order to find the quotes which were most illustrative of the views of gps , district nurses and hospital staff . to ensure reliability ,
results were presented and discussed in a steering group with representatives from the main professional groups involved in the programme ( gps , district nurses and hospital staff ) .
a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .
gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .
the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .
the gps generally see the follow - up home visit as a relevant type of contact .
a gp said : it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .
, it can give the patient extra resources when you visit the patient on the patient 's home ground .
the district nurses also generally see the follow - up home visits as a relevant type of contact .
the gps find that the medication review is an especially important element in follow - up home visits .
a gp said : we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes .
at the same time the gps also stress that follow - up home visits are not only about reviewing medication : the purpose of a follow - up home visit is first of all about making a patient review .
it is not only the medicine we should look at , but the patient in this sense follow - up home visits are very relevant .
the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient : the guide creates a good structure for the home visit .
the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are .
the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital
the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector : the guide is quite voluminous .
we have already made a lot of the tests at the hospital so there is no need to repeat these at home .
you should make sure that the gp has read the discharge summary before the follow - up home visit
the target group of the programme is elderly and frail patients just out of from hospital .
a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital
a nurse at the hospital expressed it this way : it is typically this type of patient that we see again and again : deteriorating health and a small social network .
the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .
other patients would have benefited from a follow - up home visit but they were not included in the programme gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .
this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients : it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .
a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc .
a gp said : the hospital has contact with the patient at the time of discharge .
in the present situation there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .
it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems : if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .
but it is often difficult to find out who is responsible and it is difficult to sort out the problem ( gp ) .
the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur : we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .
a fax from the hospital to the home nursing services is not enough ( doctor , hospital ) . in the city of copenhagen
the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .
it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having : follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility .
the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .
today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do .
the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .
these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .
such information is essential since follow - up home visits are new to many gps .
the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low :
i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases
i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit
the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .
a gp said : you get to talk that is the biggest gain , to get the dialogue .
we work in two parallel organizational systems . by not having the dialogue we as gps miss some observations .
the gps want , however , more information about the efficacy of follow - up home visits .
gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health : if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .
information could be better ( gp ) . at the hospital the staff involved in the programme
a nurse at the hospital said : follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .
it is really nice to know that there is this form of support when patients get home .
communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .
doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote : at meetings we have often asked for the
good story. it would make it much more meaningful for us if we were told that mrs .
that is the most annoying that we are not told what the results of follow - up home visits are
a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .
gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .
the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .
the gps generally see the follow - up home visit as a relevant type of contact .
a gp said : it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .
, it can give the patient extra resources when you visit the patient on the patient 's home ground .
the district nurses also generally see the follow - up home visits as a relevant type of contact .
the gps find that the medication review is an especially important element in follow - up home visits .
a gp said : we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes .
at the same time the gps also stress that follow - up home visits are not only about reviewing medication : the purpose of a follow - up home visit is first of all about making a patient review .
it is not only the medicine we should look at , but the patient in this sense follow - up home visits are very relevant .
the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient : the guide creates a good structure for the home visit .
the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are .
the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital
the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector : the guide is quite voluminous .
we have already made a lot of the tests at the hospital so there is no need to repeat these at home .
you should make sure that the gp has read the discharge summary before the follow - up home visit
the target group of the programme is elderly and frail patients just out of from hospital .
a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital
a nurse at the hospital expressed it this way : it is typically this type of patient that we see again and again : deteriorating health and a small social network .
the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .
other patients would have benefited from a follow - up home visit but they were not included in the programme
gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .
this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients : it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .
a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc .
a gp said : the hospital has contact with the patient at the time of discharge .
there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .
it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems : if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .
but it is often difficult to find out who is responsible and it is difficult to sort out the problem ( gp ) .
the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur : we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .
a fax from the hospital to the home nursing services is not enough ( doctor , hospital ) . in the city of copenhagen
the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .
it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having : follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility .
the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .
today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do .
the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .
these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .
such information is essential since follow - up home visits are new to many gps .
the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low :
i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases
i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit
the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .
a gp said : you get to talk that is the biggest gain , to get the dialogue .
the gps want , however , more information about the efficacy of follow - up home visits . gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health : if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .
the staff involved in the programme is very positive towards follow - up home visits .
a nurse at the hospital said : follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .
it is really nice to know that there is this form of support when patients get home .
communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .
doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote : at meetings we have often asked for the
good story. it would make it much more meaningful for us if we were told that mrs .
that is the most annoying that we are not told what the results of follow - up home visits are
the analysis illustrates that it is a big challenge to co - ordinate activities across organizational boundaries and motivate actors to participate in cross - sectoral health programs .
resources clearly matter ( fees for gps etc . ) but the paper also points to other key factors which need to be taken into consideration when trying to motivate partners to engage actively in a cross - sectoral programme like a follow - up home visit programme . in the following these key factors will be discussed under three sub - headings : the focus of collaboration , a partnership of equals , and the will to co - operate .
the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .
the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result , gps and
district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .
collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .
the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result ,
communication between the gp and the district nurse about a patient 's medicine is typically improved in the treatment following the visit .
this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .
the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .
studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .
thus , the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .
inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .
providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .
they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .
the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .
this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .
gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .
moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .
this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .
hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .
the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .
the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .
this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .
it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .
if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .
there needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits .
research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .
research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .
for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .
they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .
the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .
gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .
danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .
the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .
hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .
as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .
district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .
basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular
there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .
follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .
the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .
the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .
the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result
, gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .
collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .
the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result , communication between the gp and the district nurse about a patient 's medicine
this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .
the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .
studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .
thus , the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .
inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .
providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .
they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .
the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .
this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .
gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .
moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .
this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .
hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .
the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .
the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .
this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .
it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .
if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .
there needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits .
research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .
research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .
for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .
they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .
the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .
gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .
danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .
the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .
hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .
as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .
district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .
basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .
follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .
the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .
the findings support a basic claim in inter - organizational theory that other factors than resources matter if we want to understand what drives efforts at collaboration .
this analysis shows that the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials .
one lesson for current policy is that motivational factors need to be addressed in future collaborative programs in order to fully exploit the potential health benefits .
the follow - up home visit programme in copenhagen suffered from a lack of motivation among key actors ( especially gps ) to participate and , as a result , readmissions to hospital can be expected to be higher than anticipated prior to the programme .
susanna bihari axelsson , senior lecturer , associate professor nordic school of public health , box 12133 , nya varvet building 25 , se-402 42 gteborg sweden janne seemann , assistant professor , phd , department of sociology and social work , aalborg university , denmark j.d.h .
van wijngaarden , dr , assistant professor , institute of health policy and management , erasmus university rotterdam , p.o . | objectivesthe aim of follow - up visits by the general practitioner and district nurse ( within a week after discharge from hospital ) is to reduce hospital readmissions and improve the overall wellbeing of the patient .
there is strong evidence that these programmes are effective , but are difficult to implement because of a number of organizational obstacles , including co - ordination between the organizations involved in the process . in this paper
we look at the factors that affect motivation to participate in a cross - sectoral programme in copenhagen , denmark , implementing follow - up home visits to elderly persons.theory and methodsthe analysis is based on inter - organizational network theory in an attempt to explain the role of motivation in network formation between organizational systems .
the empirical findings are based on focus groups and in - depth interviews with hospital staff , general practitioners , and district nurses.resultscare providers are motivated to collaborate by a number of factors .
the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials.conclusionsthe study concludes that we need to focus on specific care fields and actors to reduce complexity in the area and more fully understand what motivates care providers to participate in cross - sectoral activities , such as a follow - up home visit programme .
one lesson for current policy is that motivational factors need to be addressed in future collaborative programmes in order to fully exploit the potential health benefits . |
alzheimer s disease ( ad ) is a progressive neurodegenerative disorder starting with mild memory loss and manifesting in severe cognitive decline .
neuropathologically , the disease is characterized by an extensive deposition of amyloid ( a ) peptides in extracellular amyloid plaques and by intracellular neurofibrillary tangles ( nfts ) of hyper - phosphorylated tau protein .
sporadic late - onset ad is the most common form of dementia in the elderly and is strongly associated with age . in rare cases
( < 5% ) , ad is inherited and results in an early disease onset .
one genetic risk factor for late - onset ad that has been reported throughout many studies is apolipoprotein e ( apoe ) which physiologically functions as a ligand in receptor - mediated endocytosis of lipoprotein particles [ 2 , 3 ] .
several single nucleotide polymorphisms in apoe lead to alterations in the coding sequence and result in three common isoforms called apoe2 , apoe3 and apoe4 with the e4 allele being an ad risk factor and e2 being protective [ 46 ] .
strong evidence suggests that apoe influences ad via its effect on a metabolism ; however , the details of this process have to be fully elucidated .
positional cloning studies of familial ad ( fad ) cases have identified mutations in three genes , amyloid precursor protein ( app ) , presenilin 1 and 2 ( ps1 , ps2 ) , which are tightly linked to the generation of a peptides .
proteolytic processing of amyloid precursor protein ( app ) by bace ( -site app cleaving enzyme ) is followed by cleavage by -secretase containing presenilins and results in the release of a peptides of different length . a40 and a42 ( 40 or 42 amino acids , respectively )
are the most prominent a peptides and since the c - terminus has important implication for toxicity , the longer a42 peptide exhibits stronger neurotoxic properties .
the fad - linked mutations cause an elevated production of amyloidogenic a , which is the basis for the amyloid hypothesis of ad , stating that a peptides are the key - players in neuronal dysfunction and subsequent neurodegeneration in ad .
recently , it has been suggested that the soluble oligomeric forms of a42 are the potentially harmful species and that these oligomers affect various important cellular mechanisms , resulting in decreased functionality and survival of neuronal cells .
however , a peptides are not exclusively generated under pathological conditions as they are part of the normal cellular metabolism and a significant load of amyloid plaques is also observed in brains of healthy aged individuals [ 10 , 11 ] .
this challenges the amyloid hypothesis of ad and up to date the nature of the disease - relevant alteration in aged neurons remains unclear .
although alois alzheimer already described protein deposits in the brain of demented patients , the question why distinct proteins that are potentially causative for the disease accumulate in discrete brain regions in the course of aging remains unanswered .
today , we know that the progressive deposition of misfolded and aggregation - prone proteins in defined regions of the nervous system is not unique to ad , but a well - described characteristic of several neurodegenerative disorders , including parkinson s disease ( pd ) , huntington s disease ( hd ) and amyotrophic lateral sclerosis ( als ) . recently , it has been proposed that the accumulation of disease - relevant proteins in aggregates might actually even be considered as a protective mechanism to dispose these proteins and remove them from the cellular metabolism [ 13 , 14 ] . generally , within the crowded cellular environment
, proteins are always at risk for denaturation . changing genetic and environmental factors , as during pathology or aging , challenge the integrity of the cellular protein homeostasis ( proteostasis ) . therefore , the stability and metabolism of the proteome needs to be controlled carefully and is carried out by a complex network of several hundred evolutionarily conserved proteins .
this network , including molecular chaperones , the ubiquitin proteasome system ( ups ) and the autophagy system is stringently regulated and indispensable for the maintenance of proteostasis .
all main players of this network are ubiquitously expressed and consequently also present in neuronal tissue .
molecular chaperones , most be prominently represented by the heat shock protein 70 ( hsp70 ) family and their regulators , are responsible for correct folding and refolding of proteins as well as for the transport of misfolded proteins to the protein - degradation systems .
the chaperone network is controlled by the activity of transcription factors , e.g. heat shock transcription factor 1 ( hsf1 ) , which rapidly adjusts the cellular chaperone capacity during stress conditions .
the ups is the major protein - degradation machinery of the cell and controls the metabolism of cytosolic proteins and the degradation of misfolded proteins .
proteins that are destined for proteasomal degradation become specifically tagged with the protein ubiquitin in a well - organized enzymatic cascade .
interestingly , also a ubiquitin - independent way of proteasomal degradation has been described recently that mostly involves small and misfolded proteins . as an additional mechanism for protein degradation
the major autophagic systems described so far are macroautophagy and chaperone - mediated autophagy ( cma ) .
macroautophagy is a complex process that involves the formation of a membrane structure , the autophagosome , which engulfs bulk cellular material ( proteins and organelles ) and subsequently fuses with the lysosome .
cma involves the constitutively expressed heat shock cognate 70 ( hsc70 ) that targets client proteins with a specific consensus motif directly to the lysosome .
in fact , hsp70 is also an important factor to assure selectivity of macroautophagy of degradation - prone proteins in a distinct pathway , mediated via the co - chaperone bcl2-associated athanogene 3 ( bag3 ) . aging and disease challenge the proteostasis network and the accumulation of instable proteins results in wide - spread protein aggregation [ 21 , 22 ] .
our discussion presented here attempts to integrate two major factors that are associated with neurodegeneration , protein homeostasis and aging .
most importantly , we focus on the role of specific pathways which are needed to restore and maintain proteostasis during aging and their interplay with a special focus on impaired proteostasis in ad , which may add to a novel view and understanding of ad pathogenesis .
since recently many excellent reviews on various aspects providing in depth discussions have been presented , we are concentrating here on the integration of these different aspects .
therefore , we frequently refer to review work with the recommendation of further reading rather than trying to fully cover the discussed issues by their original publications .
cells permanently encounter the problem to maintain the integrity and functionality of the proteome . within the crowded cellular environment ,
the correct conformation of proteins must be controlled and misfolded and irreversibly damaged proteins must be efficiently refolded or removed .
central players of the protein homeostasis system are molecular chaperones that sense misfolded proteins and , when refolding fails , direct them to the protein - degradation pathways .
molecular chaperones are specified as proteins that interact with and participate in folding or refolding of non - native proteins .
therefore , chaperones help unfolded proteins to achieve their functional conformation without being present in the final structure [ 15 , 2327 ] .
they exert a multitude of activities , including de novo folding , refolding of denatured proteins , transport to subcellular compartments , oligomeric assembly and disposal by proteolytic degradation [ 28 , 29 ] .
different classes of chaperones work together to form co - operative networks and are often termed heat shock proteins ( hsps ) , because they are sensitively up - regulated under stress conditions in which the amount of misfolded proteins is increased .
chaperones , which are involved in protein folding and refolding , such as hsp70 , hsp90 and chaperonin ( hsp60 ) , promote folding activity through atp / cofactor - regulated binding and release cycles [ 15 , 31 ] .
they recognize short hydrophobic amino acid stretches of misfolded proteins and can co - operate with atp - independent chaperones , e.g. small hsps ( shsps ) , to prevent protein aggregation .
hsp70 proteins are central players in proteostasis control and increasing hsp70 levels prevent protein aggregation in various disease models that are based on the expression of aggregation - prone proteins [ 32 , 33 ] .
the activity of hsp70 is atp dependent and is controlled by chaperones of the hsp40 family as well as nucleotide exchange factors ( nefs ) [ 34 , 35 ] .
after atp hydrolysis , a nef binds to the atpase domain of hsp70 and catalyzes the nucleotide exchange , which results in substrate release .
thus , chaperone binding to the hydrophobic region of misfolded proteins transiently blocks aggregation and the subsequent atp - dependent release allows controlled folding of the client protein .
hsp40/dnajs , hsc70-interacting protein ( hip ) , bcl2-associated athanogene 1 ( bag1 ) and bag3 are prominent co - chaperones that control the atp - dependent activity of hsp70 .
dnaj proteins generally enhance the atpase activity of hsp70 , but their action can lead to different fates of their bound substrates .
dnajb1 is an example for a dnaj protein that supports substrate folding , whereas dnajb2-bound clients are degraded by the proteasome .
the co - chaperone hip stabilizes the adp - bound state of hsp70 and co - operates with hsp70 in protein folding .
other hsp70-binding cofactors are the ubiquitin ligases carboxy terminus of hsc70-interacting protein ( chip ) and parkin , which provide a link between hsp70 , co - chaperones and the ups , resulting in client ubiquitination and degradation by the proteasome [ 34 , 38 , 39 ] .
recently , it has been shown that bag3 interacts with hsp70 and directs the chaperone and its substrates into the autophagic pathway , providing an association of hsp70 , co - chaperones and autophagy .
an extensive description of the hsp70 machinery , the exact structure - function relationship between hsp70 and its various cofactors , has been excellently reviewed elsewhere .
the functional variability of the chaperone system that is exerted by a multitude of single proteins highlights the complexity of this proteostasis network .
however , limited knowledge exists on the exact composition and capacity of the chaperone system within a cell or a certain tissue .
a general view assumes that total levels of cellular chaperones exceed the actual requirements and that a sufficient amount of chaperones is free of clients .
thus , the network has excess capacity to initially deal with sudden additional chaperone requirements , as exhibited during stress conditions .
an opposite view proposes that the capacity of the chaperone network is always closely titrated to the actual demand of chaperone activity and that the system is otherwise rapidly adapted to conditions of increased requirements .
the analysis of the chaperone capacity of neuronal and muscular tissue of caenorhabditis elegans ( c. elegans ) demonstrates that these tissues indeed display different folding activities and that neurons are particularly sensitive to protein denaturation during heat stress . however , the adaptation of the chaperone network during aging is critical as it is well acknowledged that protein aggregation and disruption of proteostasis are characteristic for aged cells .
the periodical application of mild heat stress decreases mortality in drosophila melanogaster and c. elegans , which is mediated by hsp70 activity and the overexpression of hsp70 in c. elegans increases the life span of the nematode [ 4345 ] .
several reports have analyzed chaperone protein or mrna levels in aged cells and found increased or basal amounts , whereas the stress - mediated induction of chaperone expression is impaired .
the transcription of chaperone genes in response to stress conditions is controlled by the transcription factor hsf1 , which shows an impaired dna - binding potential in aged cells .
a reduced activity of hsf1 in c. elegans results in a shortened life span and , conversely , the enhanced expression of the transcription factor increases the life span .
activity is also essential for the extended life span of the extremely long - lived daf-2/insulin / igf-1 receptor mutants of c. elegans [ 47 , 48 ] .
thus , hsf1 and chaperone activity can promote longevity , demonstrating a clear association of chaperones , proteostasis and aging .
some of the factors mentioned so far are involved in linking chaperone functions with cellular protein - degradation pathways , the ups and autophagy , for the removal of misfolded proteins . besides protein aggregation ,
one factor that induces ubiquitination is protein damage caused by free radical oxygen species ( ros ) and oxidative stress .
most likely , irreversible oxidation may activate chaperones and the ups to induce protein repair of misfolded proteins and lead to ubiquitination and protein degradation . during aging ,
in particular , the mitochondrial respiratory chain is strongly linked to the production of ros and as one consequence may cause protein dysfunction , apoptosis , necrosis , aging and disease [ 49 , 50 ] .
protein oxidation leads to a change in protein conformation and function and chaperones may sense such changes and in turn activate the ups as a quality control system .
depending on the degree of oxidation , irreversible oxidation and loss of protein function may lead to degradation and/or accumulation of damaged proteins and to the formation of so - called aggresomes , which display a high autophagic activity [ 51 , 52 ] .
the ups is a complex enzymatic pathway that starts with the ligation of ubiquitin , a 76-amino - acid - long and highly conserved protein , to other cellular proteins and thus labels them for degradation .
initially the c - terminal end of ubiquitin is activated by atp - dependent phosphorylation and formation of a thiol ester via an activating enzyme , e1 .
it is then transferred to a thiol group of an ubiquitin - carrier protein , e2 .
the e3 ligase directs ubiquitin from e2 to an -amino group of the target protein [ 53 , 54 ] .
the c - terminus of an additional ubiquitin protein can be ligated onto one of the seven lysine residues within the attached ubiquitin molecule . for degradation via the proteasome
ubiquitin linkages between either the c - terminus and lysine residues k48 or k63 are the major recognition signals for proteasomal degradation .
ubiquitin chains also occur among other lysine residues , whereas ubiquitin extension via k6 is associated with dna repair , k11 with endoplasmatic reticulum - associated protein degradation and cell cycle regulation , k27 with ubiquitin fusion degradation , k29 with lysosomal degradation and k33 with kinase modification .
monoubiquitination can modify the activity of the protein transport machinery and when attached to transmembrane proteins can serve as a sorting signal to direct their movement between different cellular compartments [ 5659 ] . the polyubiquitinated proteins destined for degradation
are processed by the multienzymatic proteasome complex : first they become deubiquitinated and then degraded by the 26s proteasome , a system that is composed of various proteasome subunits .
the eukaryotic 20s proteasome core complex consists of four heptameric rings comprising two classes of seven non - identical but homologous subunits .
the outer rings contain alpha - type subunits with gating function for substrate entrance and product release , while the beta - subunits exhibit peptide hydrolyzing activity .
the 19s regulatory complex binds to the 20s catalytic core in a flexible manner and consists of six atpases , forming a ring at the entrance of the core and exerting chaperone - like activity .
the ups is the major degradation system in the cell for the degradation of short - lived , misfolded and defective proteins .
an accumulation of polyubiquitinated proteins is reported for numerous neurological disorders , which suggests that ups dysfunction plays a prominent role in the pathogenesis of neurodegenerative diseases and moreover in aging . since proteasomal
activity decreases during aging over all , the degradation demand of the cell increases , which results in an age - associated induction of the autophagy pathway that delivers substrates for degradation ultimately to lysosomes [ 20 , 63 ] .
autophagy is negatively regulated via the evolutionarily conserved enzymatic mammalian target of rapamycin complex 1 ( mtorc1 ) . in turn , if mtor activity is inhibited by rapamycin , the autophagy pathway is switched on .
rapamycin exerts its stimulatory effect on autophagy by preventing mtor phosphorylation at ser-2448 and thereby subsequently blocks mtor signaling .
so far , many different autophagic systems have been described including macroautophagy and cma that form the main pathways .
macroautophagy is a multi - step process by which cytosolic material is sequestered into a double - layered membrane structure , the autophagosome , and is delivered to the lysosome for degradation .
it is a highly orchestrated process and involves autophagy - related ( atg ) proteins .
more than 30 genes have been identified in genetic analyses of yeast mutants which have defects in autophagic function .
after autophagy stimulation through mtor inhibition , the formation of phagophores ( autophagosome precursors / pre - autophagosomal structures ) is initiated in a not yet completely understood mechanism .
a subset of atg proteins is required for autophagosome formation : first , the atg9 system including atg9 , the atg1 kinase complex ( atg1 and atg13 ) , atg2 and atg18 ; second , the phosphatidylinositol 3-oh kinase ( pi(3)k ) complex consisting of vacuolar protein sorting ( vps)34 , vps15 , beclin 1/atg6 and atg14 and third , the ubiquitin - like protein ( ubl ) system composed of two ubl proteins ( lc3/atg8 and atg12 ) , an e1 enzyme ( atg7 ) , two analogues of e2 ubiquitin - conjugating enzymes ( atg10 and atg3 ) , an lc3/atg8-modifying protease ( atg4 ) , the protein target of atg12 attachment ( atg5 ) and atg16 .
beclin 1/atg6 regulates autophagic trafficking and membrane trafficking in a variety of physiological and pathological processes .
microtubule - associated protein 1 light chain 3 ( lc3/atg8 ) is commonly used as a marker for autophagic activity . with its e1-like activity , atg7 converts cytosolic lc3-i to the membrane - associated lc3-ii , which then is conjugated to phosphatidylethanolamine .
lc3-ii binds the inner and outer membrane of the forming autophagosome and provides a physical link between the autophagosome and lc3 interacting proteins , such as sequestosome 1 ( p62/sqstm1 ) or neighbor of brca1 gene 1 ( nbr1 ) .
these so - called autophagy receptors can simultaneously bind to ubiquitinated proteins and lc3 , employing their lc3-interacting motif and ubiquitin - associated domain .
the identification of autophagy receptors , such as p62/sqstm1 and nbr1 has provided a molecular link between ubiquitination and autophagy .
thus , p62/sqstm1 and nbr1 are possibly also important bridging factors between ups and autophagy . through self - oligomerization , which is stimulated by ubiquitin binding , p62/sqstm1 sequesters ubiquitinated substrates in form of inclusion bodies .
these inclusions are then specifically engulfed by the autophagosome membrane by recruiting lc3 [ 69 , 70 ] . while macroautophagy is considered as a rather unspecific robust degradation process , cma is a highly selective lysosomal pathway that removes a distinct subset of proteins containing a pentapeptide lysosome - targeting motif .
these substrates can on the one hand directly be translocated into the lysosome after docking to the lysosomal receptor lysosomal - associated membrane protein 2a or on the other hand unfolded by a chaperone complex containing hsc70 and the co - chaperones bag1 , hip , hsp70hsp90 organizing protein ( hop ) and hsp40/dnajb1 .
these co - chaperones provide a direct link to the ups and the folding and refolding activity of molecular chaperones .
in the course of aging , a variety of proteins tend to aggregate and impair the ups directly .
therefore , these proteins , most of which are ubiquitinated , can not be handled by the proteasome and have to be degraded by different protein clearance pathways .
recently , it has been shown that the cellular protein quality control ( pqc ) of polyubiquitinated proteins by proteasomal and autophagic systems is regulated by the hsp70 co - chaperones bag1 and bag3 , respectively .
proteasome activity decreases in an age - dependent manner in a cell model of replicative senescent human fibroblasts and is associated with an increased autophagic activity in aged cells .
this correlates with decreased levels of bag1 and increased levels of bag3 in aged cells , an age - related bag1/bag3 switch that serves as an induction control of the macroautophagic pathway ( bag3-mediated selective macroautophagy ) and may be considered as a backup of the ups in pqc ( [ 40 , 72 ] ) .
therefore , the expression shift from bag1 to bag3 during aging , but also upon acute stress ( e.g. proteasome inhibition , oxidative stress ) , can be considered as a physiologically important adaptive response [ 20 , 40 ] .
the bag3-mediated selective pathways have been shown to be involved in a variety of disease causing processes . in a model for hd ,
bag3 in concert with hspb8 facilitates the disposal of polyq43-huntingtin by stimulating macroautophagy and this complex is also involved in z - disc maintenance in flies , mice and men [ 73 , 74 ] . additionally , the transport of target proteins to the aggresome , a compartment with high autophagic activity , has been shown to be mediated by bag3 .
this aggresome - targeting pathway involving bag3 and hsp70 is distinct from other before - described mechanisms as it does not depend on substrate ubiquitination . taken together the molecular chaperone machinery , the ups and the autophagy system are involved in pqc and maintaining proteostasis within aging and disease to prevent protein misfolding and aggregation .
a summary of the main routes for protein degradation is shown in fig . 1 .
as aggregated proteins are found in ad patients and aging is a prevailing risk factor for ad it is important to further characterize how these pathways are regulated and how misregulation possibly contributes to the pathology of ad.fig .
1the ubiquitin proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria ) the ubiquitin proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria )
ad is characterized by the accumulation of intracellular a ( oligomers ) , extracellular high molecular weight deposits of a peptides ( fibrilliar forms ) , and the intracellular aggregation of hyper - phosphorylated tau . indeed ,
modification of the cellular proteostasis and the metabolism of a and tau have been proven to cause neuronal dysfunction and cell death .
the malfunctioning proteostasis control results in the accumulation of misfolded and aggregated proteins , which is a general hallmark of aging and neurodegeneration , as observed in pd , hd , als and ad . aging in particular is characterized by decreasing proteostasis capacity and increasing protein damage which are in combination a major challenge for the cell due to the inability to maintain metastable proteins in folded states . to counteract a cascade of protein destabilization caused by metastable proteins which escaped the pqc
the proteostasis network including molecular chaperones , the ups and autophagic pathways must manage the increasing burden of protein misfolding to maintain proteome stability .
since aging is the major risk factor of ad [ 7880 ] , the age - dependent loss of proteostasis might be an important contributor to the pathology of ad .
an overview of proteins that affect proteostasis in ad is shown in table 1.table 1proteins affecting proteostasis in alzheimer s diseaseproteinfunctionreferencea40/42decreases proteasome activity , modulates autophagy through mtor signaling[99102 , 104 , 143146]tauinhibitory effect of tau aggregates on proteasome activityhsf1induces app gene during stress[81 , 82]grp78 ( er isoform of hsp70)modulates app maturation and reduces a40/42 secretion[84 , 85]hsps ( hsp22 , 27 , 70 , 90)small hsps ( hsp22 , hsp27 ) bind to fibrillar amyloid plaques and inhibit their fibrillarisation ; hsp70 , hsp90 inhibit early stages of amyloid aggregation[88 , 89]chipe3 enzyme for phosphorylated tau[93 , 117]bag1regulates proteasomal degradation of tau together with hsp70ps1increases production of a42 , essential for lysosomal proteolysis and autophagy by enabling the acidification of lysosomes required for protease activation[163 , 165]ubb+1potently inhibits the degradation of polyubiquitinated substrates and therefore induces neuronal cell death[120 , 121]uch - l1dub needed for proteasomal degradation of client proteins , oxidized and down - regulated in ad[124 , 125]ubqln1ubqln1 activity is necessary to regulate the production of app and app fragments[127 , 128]mtorinhibitory and/or activating modulation of mtor through a42 but not a40[143146]beclin1beclin1 is down - regulated in ad brains and consequently increases app levels and its metabolites[161 , 162]a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum proteins affecting proteostasis in alzheimer s disease a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .
interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .
as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .
there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .
the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .
in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .
the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .
moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .
hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,
furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro .
these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .
besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .
post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .
these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .
hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .
hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .
protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .
vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models
, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .
tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .
collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . the ubiquitin
proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .
a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .
a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .
it still has to be elucidated which form of a is now affecting proteasome function .
there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell
free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .
there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .
consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .
so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons
, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain
, a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism
thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .
this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .
the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .
furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .
it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .
in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .
in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .
these data show that different aggregation states of tau modify its turnover via the proteasome .
it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,
the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .
in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .
moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .
taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .
ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .
overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .
oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .
it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .
uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .
interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .
several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .
for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .
recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .
in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .
taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .
this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .
ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .
interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .
in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .
these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .
these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .
a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .
neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .
furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .
genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients .
however , enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .
the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .
attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .
interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself . the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and
hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .
furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .
in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .
similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .
interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .
the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .
these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .
interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .
later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .
this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .
the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .
thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner .
consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .
the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .
the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .
this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .
interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .
as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .
hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .
another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .
beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .
in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .
moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .
however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .
recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .
ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,
the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .
this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .
-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .
in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .
the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease .
taken together , all these findings suggest an important role for autophagy in the pathogenesis of ad .
the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .
interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .
as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .
there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .
the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .
in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .
the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .
moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .
hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,
furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro .
these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .
besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .
post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .
these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .
hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .
hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .
protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .
vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models
, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .
tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .
collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation .
the ubiquitin proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .
a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .
a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .
it still has to be elucidated which form of a is now affecting proteasome function .
there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell
free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .
there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .
consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .
so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons
, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain ,
a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism
thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .
this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .
the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .
furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .
it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .
in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .
in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .
these data show that different aggregation states of tau modify its turnover via the proteasome . particularly intriguing
it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,
the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .
in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .
moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .
taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .
ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .
overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .
oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .
it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .
uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .
interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .
several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .
for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .
recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .
in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .
taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .
this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .
ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .
interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .
in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .
these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .
these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .
a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .
neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .
furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .
genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients . however ,
enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .
the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .
attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .
interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself .
the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and
hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .
furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .
in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .
similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .
interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .
the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .
these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .
interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .
later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .
this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .
the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .
thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .
the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .
the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .
this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .
interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .
as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .
hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .
another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .
beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .
in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .
moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .
however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .
recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .
ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,
the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .
this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .
-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .
in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .
the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . taken together
, all these findings suggest an important role for autophagy in the pathogenesis of ad .
there is quite some evidence that in ad , the proteostasis network is impaired . based on pathological analysis , ad mouse models , in vitro and cellular investigations a molecular link between chaperones , the ubiquitin proteasome system , autophagy pathways and pathogenetic mechanisms of ad can be suggested .
deregulation and changes of chaperones and proteasome activity might have serious implications for aging as well as for age - associated diseases .
autophagy pathways are key mechanisms and of vital importance for cellular function and , especially , for cell survival under adverse conditions .
consequently , an effect of proteostasis control on neurodegeneration or , vice versa , of neurodegeneration on proteostasis is reasonable . as with other pathomechanisms that are investigated in the search for the cause of ad , it is still open whether an impairment of proteostasis is an upstream or downstream event during ad onset and progression .
experimental approaches employing mouse models clearly demonstrate that stabilization or induction of proteostasis can be neuroprotective . whether this can be translated into the human condition and , most importantly ,
whether supporting proteome integrity can be a real target for pharmacological intervention for the prevention and treatment of ad is currently still open .
also , one has to consider that , e.g. the stimulation of general autophagy may in the long term lead to the stimulation of proliferation of non - neuronal cells , eventually resulting in tumor formation . indeed , a permanent autophagy induction is known as one cellular mechanism that promotes the escape of cells from proliferation control . as beneficial
it may be to support autophagy in post - mitotic neurons that are confronted with disturbed proteostasis and an impairment of proteasome function , this stimulation should target specific autophagy pathways , such as selective chaperone - mediated macroautophagy involved in the degradation of disease - associated protein aggregation .
although there are obviously still many questions to be answered to understand the role of proteostasis in ad ( and also in other age - associated neurodegenerative disorders ) , it is a big step forward in ad research to consider a possible role of pathogenetic pathways that are not directly linked to the usual suspects a and tau
. a better understanding of how the proteostasis network is regulated might help to identify targets which lead to prevention of the deleterious loss of neuronal cells and tissue in ad . | alzheimer s disease ( ad ) is one key medical challenge of the aging society and despite a great amount of effort and a huge collection of acquired data on molecular mechanisms that are associated with the onset and progression of this devastating disorder , no causal therapy is in sight .
the two main hypotheses of ad , the amyloid cascade hypothesis and the tau hypothesis , are still in the focus of ad research . with aging
as the accepted main risk factor of the most important non familial and late onset sporadic forms of ad , it is now mandatory to discuss more intensively aspects of cellular aging and aging biochemistry and its impact on neurodegeneration .
since aging is accompanied by changes in cellular protein homeostasis and an increasing demand for protein degradation , aspects of protein folding , misfolding , refolding and , importantly , protein degradation need to be linked to ad pathogenesis .
this is the purpose of this short review . |
the union rate and hardware - related complications remain a major concern in these patients , especially following multilevel spinal fusion surgeries .
developing an adjunct therapy that essentially supplements the mainstay surgical management is of great importance to these patients .
multitudinous research to gain insight into use of teriparatide ( 134 recombinant human parathyroid hormone ) for enhancing bone formation in osteoporosis is readily available in the literature .
its action as a direct anabolic agent in bone tissue , consistently increases bone mineral density and thus improves skeletal architecture . since its availability in market
, indications are unravelling and is being attempted in many conditions including fracture healing , dental stability , hypoparathyroidism , and hypocalcaemia .
another evolving field of interest is the role of teriparatide for enhancing bone graft union and osseo - integration of implants .
it is said to accelerate bone graft union and reduce the incidence of pedicle screw loosening after lumbar spinal fusion surgeries .
we intended to study this effect and analyze if teriparatide can be an effective adjunct therapy to achieve solid posterolateral fusion mass and reduce the incidence of subsequent screw loosening after multilevel instrumented lumbar fusion surgeries in elderly patients .
an open - labeled , nonplacebo controlled , retrospective , single center study is formulated by shortlisting 62 elderly patients ( male 9 ; female = 53 ) between the age group of 65 to 78 years ( mean age standard deviation [ sd ] = 70.1 3.7 ) , who underwent instrumented lumbar fusion for various degenerative pathologies over a period of time .
all of them had received multilevel lumbar fixation ( 3 levels or 4 vertebral segments ) with pedicle screw constructs and postero - lateral intertransverse fusion with a combination of local bone graft and allograft .
the prime indication for surgery was chronic unresolving low back pain , with or without neurological symptoms , associated with any one of the following conditions : a)multisegmental spinal instability ( spondylolisthesis grade 1),b)multilevel degenerative spondylosis with or without segmental instability , andc)degenerative scoliosis with or without segmental instability .
multisegmental spinal instability ( spondylolisthesis grade 1 ) , multilevel degenerative spondylosis with or without segmental instability , and degenerative scoliosis with or without segmental instability .
we excluded : a)those patients operated with oligo - level spinal fusions ( 2 levels or 3 vertebral segments).b)those with 1 or more osteoporotic vertebral compression fractures among the fused levels.c)those with previous surgery in the same level.d)those in whom infection or tumor was an indication for surgery.e)those with 1 or more potential data missing .
those patients operated with oligo - level spinal fusions ( 2 levels or 3 vertebral segments ) .
those with 1 or more osteoporotic vertebral compression fractures among the fused levels . those with previous surgery in the same level . those in whom infection or tumor was an indication for surgery . those with 1 or more potential data missing .
selection was irrespective of individual bone mineral density ( bmd ) status , as bmd scoring was not done as a part of routine preoperative evaluation in patients undergoing multilevel spinal fusions .
our sample included elderly patients 65 years who are expected to have detoriating osteogenesis potential .
associated medical comorbidities included diabetes mellitus ( dm ) and bronchial asthma ( ba ) in 25 and 8 patients , respectively .
a mixture of fresh frozen allograft and local bone chips were used as graft . local bone consisted of spinous process , lamina , and facets from the decompression site .
occasionally , additional interbody cages were used at insufficient discs especially at the caudal end of the construct to increase stability . following surgery
, patients were given an option of postoperative teriparatide use and opting to it was solely based on their own decision . in total , 30 patients who accepted for long - term daily use of teriparatide were in the experimental group and remaining 32 patients were in the control group .
teriparatide ( self - administered ; 20 mcg sc injection ) was started for those patients in the experimental group from the first postoperative day , once daily using delivery device .
the control group received similar standardized and accepted treatment protocols as those in the experimental group except for the use of postoperative teriparatide .
all patients were instructed to take calcium and vitamin d supplements every day following surgery .
outcome analysis was done using radiological data which included antero - posterior ( ap ) , conventional lateral view , and dynamic stress lateral view radiographs taken at 12 months follow - up ( figs . 1 and 2 ) .
( c ) mri images showing severe degeneration of disc levels and canal stenosis from l1l5 .
( d ) 12 months follow - up ap view x - ray which was considered as solid fusion ( lenke type 1 ) .
ap = antero - posterior , mri = magnetic resonance imaging . radiological outcome illustration of a random case from the control group . ( a )
( b ) preoperative dynamic stress ( extension and flexion ) lateral view radiographs showing degeneration from l3s1 segments .
( c ) mri images showing severe degeneration of disc levels and canal stenosis from l3s1 .
( d ) 12 months follow - up ap view x - ray showing bilateral signs of pseudo - arthrosis interpreted as nonunion ( lenke type 4 ) .
they divide postero lateral fusion mass into 4 grades based on the appearance in an ap radiograph .
grade a represents definitive fusion with bilateral thick bony masses , grade b represents probable fusion with unilateral thick bony mass and thin bony mass on other side , grade c represents no probable fusion with thin bony mass on the one side and pseudo - arthrosis on the other side , grade d represents no fusion with thin bony mass on both the sides showing obvious pseudo - arthrosis or resorption of graft bilaterally .
we interpreted grade 1 and grade 2 of lenke 's criteria as solid fusion which is explained as unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebra showing continuous trabeculation in an ap radiograph .
although most of the patients had long fixations , segmental cobb 's angle change involving the upper and lower ends of the construct as seen in dynamic ( flexion and extension ) stress lateral views will also be considered as nonunion .
screw loosening was determined when there was occurrence of radiolucency ( halo sign ) around the pedicle screws in either ap or lateral view radiographs .
change in the screw position noticed in dynamic stress lateral view radiographs will additionally be considered as screw loosening , which may occur at the cephalic and caudal ends of the construct .
results were tabulated and statistical analyses were carried out using graph pad prism 5 ( graphpad software inc . , san diego , ca ) .
analyses were done using unpaired t test for continuous variables and fisher 's exact test for categorical variables .
probability values of less than 0.05 were considered statistically significant . written informed consent was obtained from all patients including acceptance of daily usage of teriparatide delivery device from those in the experimental group .
this study was approved by the institutional review board of chang gung memorial hospital with irb registry no - 201600800b0 and was performed in accordance with the ethical standards stated in the most recent version of the 1964 declaration of helsinki .
sixty - two elderly patients ( age = 6578 years ; mean = 70.11 years ; male = 9 , female = 53 ) are divided into 2 groups based on teriparatide administration following surgery .
group 1 or the experimental group ( n = 30 ; mean age sd = 69.8 3.8 years ) was those in whom teriparatide was administered and group 2 ( n = 32 ; mean age sd = 70.4 3.6 years ) was the control group .
the presence of concomitant medical comorbidities in group 1 ( dm = 14 ; ba = 4 ) and group 2 ( dm = 11 ; ba = 4 ) was matched .
the demographic variation in characteristics of the groups is statistically insignificant ( table 1 ) .
surgical parameters of the experimental group ( fusion levels = 137 ; pedicle screws = 269 ) and the control group ( fusion levels = 144 ; pedicle screws = 283 ) had no statistically significant variance .
duration of teriparatide administration averaged 7.4 2.4 months in the experimental group .
follow - up data were available for a mean duration of 25.4 months for those in the experimental group and 27 months for those in the control group . even though follow - up was done for a longer duration , analyzing outcome further after 1 year may not be related to teriparatide use .
considering the duration of administration of teriparatide , radiological outcome analysis was decided to be done at 1 year follow - up .
none of our patients missed the 1st year follow - up and required x - rays were available in our database for all the patients .
radiological outcome analysis was done using ap , lateral , and dynamic stress lateral view radiographs taken at 12 months follow - up .
two or more observers concurred stating that there was unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation confirming solid fusion in 20 patients ( 66.7% ) of the teriparatide group and 16 patients ( 50% ) in the control group .
nonunion was noted in 10 patients ( 33.3% ) of teriparatide group and 16 patients ( 50% ) in the control group . the 16.7% advantage procured with teriparatide use was not statistically significant ( p = 0.20 ) ( table 2 ) .
screw loosening was calculated comparing the number of screws showing signs of loosening to total number of screws in each group .
the incidence of screw loosening was 13.4% in the teriparatide group and 24.4% in the control group .
this difference was statistically significant with p = 0.001 ( table 2 ) .
elderly patients with chronic unresolving symptoms due to degenerative spine disease are prone to have poor surgical outcomes .
they also predispose for more hardware - related complications , especially following long spinal fusions . considering the possibility of poor outcomes , treating these conditions
can not be ignored but rather a potential solution needs to be developed which effectively supplements the mainstay surgical management .
it is said to play a reasonable role in treatment of osteoporosis . apart from being used in osteoporotic individuals , insights into its action on many other conditions are unraveling .
animal studies yielded promising results for teriparatide usage to enhance postero - lateral fusion mass in rat models . in human studies relating to teriparatide
, most authors studied its effect only in osteoporotic individuals . in our sample , patients were between 65 and 78 years of age and are more susceptible to have poor bmd .
patients had comorbidities such as dm and bronchial asthma that have negative influence over bmd .
dm is said to reduce bmd at a higher rate in elderly women , even if the baseline bmd was high .
along with postero - lateral fusion , additional interbody cages were used especially at very insufficient discs often seen in the caudal end of the construct .
this step also allows the graft to access the pluripotent stem cells of the bone marrow .
since a large amount of graft is required for long fusions , there usually is a lack of host bone .
static and dynamic radiographs are used for radiological assessment of spinal fusion by many authors , but computerized tomography ( ct ) is considered more accurate .
most of the radiographic criteria developed to assess fusion mass consider movement in dynamic radiographs as a main component for assessment .
they lack reliability as the absence of motion does not confirm fusion , especially in the case of long instrumented levels . based on ap radiographs ,
we considered grade a ( definitive fusion ; bilateral thick bony mass ) and grade b ( probable fusion ; unilateral thick bony mass ) described by lenke et al as solid fusion .
fusion masses that did not favorably appear to have definitive or probable fusion were considered as nonunion .
we also considered change in segmental cobb 's angle , visualized in dynamic stress lateral view radiographs as an additional criterion to interpret as nonunion .
formation of a radiolucent zone ( halo sign ) around the pedicle screws seen in conventional ap / lateral view radiographs and change in screw position seen in dynamic stress lateral view radiographs were considered definitive indicators for screw loosening .
teriparatide is said to have potential to increase the quality of lumbar spine bone marrow and pedicle cortex and thus reduces the incidence of pedicle screw loosening .
it is even believed to increase the insertional torque of pedicle screws when administered from at least 1 month prior to surgery .
long - term teriparatide use can be associated with significant improvement in bone microarchitecture and trabeculation .
it is also said that ; more than 6 months of daily injection facilitates effective bone union following postero - lateral fusion . considering this ,
we administered teriparatide for a mean duration of 7.4 months and none of the patients had any drug induced complications .
most of the similar studies in literature included only short segment postero - lateral fusions .
our study is foremost to discuss the effect of teriparatide on multilevel posterolateral fusions in elderly patients .
according to our preliminary results , the percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group .
besides that , use of teriparatide effectively reduced the incidence of subsequent screw loosening which proved to be statistically significant .
our study evaluates the radiological outcome following postoperative teriparatide administration in patients undergoing multilevel instrumented lumbar fusion surgery .
even though percentage of solid fusion was more among patients who used teriparatide , the difference was not statistically significant . however , teriparatide was more significantly influential in reducing the incidence of subsequent pedicle screw loosening .
being a retrospective study , there are few methodological shortcomings that may have influenced our outcome .
the lack of preoperative bmd data , variation in surgical indications , and differential treatment durations of teriparatide administration may have biased the sample .
use of ct as an evaluation tool to assess bone graft union could have been more empirical .
considering the selected sample size , the study may be underpowered to draw potential conclusions . | abstractelderly patients with chronic nonresolving symptoms due to degenerative spine pathologies are prone to have poor surgical outcomes and hardware - related complications , especially following multilevel instrumented lumbar fusion surgeries .
with intention of analyzing if teriparatide can be an effective adjunct therapy to surgical management , radiological outcomes are studied .
sixty - two elderly patients were divided into 2 similar groups .
group 1 ( n = 30 ; mean age = 69.83 years ; fusion levels = 137 ; screws = 269 ) had taken teriparatide ( 20 mcg sc injection , once daily ) for a duration of 7.4 2.4 months following surgery and group 2 ( n = 32 ; mean age = 70.38 years ; fusion levels = 144 ; screws = 283 ) did not take teriparatide .
radiological evaluation was done to determine the state of postero lateral fusion mass and to investigate the incidence of pedicle screw loosening at 1-year follow - up .
unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation suggestive of solid fusion was obtained in 66.7% patients in the teriparatide group and 50% patients in the control group ( p = 0.20 ) .
13.4% of the total no . of screws showed signs of loosening in the teriparatide group , compared to 24.4% in the control group ( p = 0.001 ) .
percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group .
this difference may have clinical importance but was not statistically significant .
however , teriparatide use was more significantly influential in reducing the incidence of subsequent pedicle screw loosening . |
according to the world health organization 's recent update , diabetes , hypertension , and obesity are one of the top five continuing risk factors for cardiovascular deaths in the world .
obesity is increasing substantially and is one of the major contributors of disease prevalence due to its pathophysiological link to other cardiovascular risks such as hypertension and diabetes .
it is estimated that , in 2010 , 6.4% of adults would have diabetes mellitus affecting 285 million in the world and it will increase to 7.7% by 2030 , affecting 439 million adults .
of special note is that there will be a 67% increase in the prevalence of diabetes in developing countries from 2010 to 2030 .
metabolic syndrome ( ms ) is a constellation of overweight / obesity , hypertension , and disturbances of lipid and carbohydrate metabolism .
the definition of ms was debated for a long time to produce a standardized clinical criterion .
the world health organisation describes ms as the presence of type 2 diabetes or impaired glucose tolerance with any two of the following characteristics : obesity , high levels of triglycerides , low levels of high - density lipoprotein , and hypertension .
the international diabetes federation ( idf ) takes central obesity as a prerequisite for the diagnosis of ms with the association of any two of the other factors , that is , high blood pressure , abnormal blood glucose , high levels of triglycerides , and low levels of high - density lipoprotein .
also , the idf has derived specific reference values for central obesity for different ethnicities .
the national cholesterol education programme ( ncep ) expert panel on detection , evaluation , and treatment of high blood cholesterol in adults ( adult treatment panel , or atp , iii ) , the national heart , lung and blood institute , and the american heart association have released a report on the criteria for diagnosing and managing ms .
the panel describes ms as the presence of any three of the following : abdominal obesity , dislipidemia ( high levels of triglycerides , low hdl ) , increased blood pressure , and elevated fasting glucose .
for the purpose of this paper , the atp iii and idf 's definitions are used and compared .
each component of ms is a known risk factor for the development of type 2 diabetes , atherosclerosis , and coronary artery disease ( cad ) .
people with ms are 310 times more likely to develop cardiovascular disease commensurate with a high risk of morbidity and mortality [ 5 , 6 ] .
central obesity , one of the components of ms , predicts the occurrence of diabetes and overall cardiovascular risk .
the ncep atp iii states that ms is equal to cigarette smoking as a contributing factor for premature cardiovascular disease .
the prevalence of metabolic syndrome is increasing all over the world with different regions having individual clusters of epidemic risk factors [ 6 , 8 ] , and in particular there is evidence of a high prevalence of ms and diabetes in south asians .
substantial increase in the prevalence of type 2 diabetes in asia in recent years has raised serious concerns about cardiovascular consequences for these populations [ 5 , 10 ] . however , in developing countries , many of these subclinical conditions are not diagnosed until the onset of complications such as myocardial infarction or stroke .
it is essential to initiate early detection of these chronic diseases in underdeveloped countries in asia , such as nepal , so that preventative action can minimize the consequences .
this study aims to establish the prevalence of hypertension , diabetes , obesity , and metabolic syndrome in the participants of a major health screening programme in nepal .
this study also aims to establish the relationship between the components of ms and lifestyle of the participants .
nepal is one of the poorest countries of the world at the 136th position of human development index .
the subjects were the participants of the programme for detection and management of chronic kidney disease , hypertension , diabetes and cardiovascular disease , a community - based screening programme in eastern nepal . in this community - based programme
a series of community awareness programmes were conducted in a specific locality with the help of local leaders , medical students , and community volunteers .
various screening centres such as permanent centers ( in health clinics , community centers , etc . ) and temporary screening centers ( in schools , clubs , houses of worship , and private homes ) were used to screen the population .
each center used a group of five to seven people as community volunteers and consisted of a local leader ( priest , administrator , school teachers , and local political leaders ) , a laboratory technician , and nurse .
medical students ( approximately 100 in number ) and nursing students ( around 25 ) assisted the community volunteers . prior to screening ,
the community volunteers went from door to door to record the number of family members residing permanently and to inform the members of the family , about the need of the project .
all people of 20 years were invited to come to a predefined place in very close vicinity to their house .
pregnant or menstruating women at the time of analysis , people with a fever or acute illness , and those who had recently engaged in heavy exercise were excluded .
the research team also collected general information on the participants ' demographic data , diet , smoking , alcohol consumption , and physical activity . the data recorded included family and medical history for kidney disease , high blood pressure , diabetes , cardiovascular disease and any current medication or treatment .
blood pressure was measured by the auscultatory method with a random zero mercury sphygmomanometer and standard cuff ( 12 34 cm ) .
the blood pressure measurement was taken in the seated position , quietly in a chair with feet on the floor and an arm support at the heart level .
hypertension was defined according to the guidelines of the seventh report of the joint national committee on prevention , detection , evaluation and treatment of high blood pressure , that is , systolic blood pressure 140 mm hg or diastolic blood pressure 90 mm hg and/or concomitant use of antihypertensive medications .
body weight and height were assessed with all subjects standing without shoes and heavy outer garments to the nearest 0.1 kg and 1 cm , respectively .
waist circumference was measured over light clothing at a level midway between the lower rib margin and the iliac crest in centimetres rounded up to nearest 0.5 cm .
abdominal obesity is defined as an abdominal circumference > 102 cm ( 40 in ) in males and > 88 cm ( 35 in ) in females for ncep criteria and > 90 cm in males and > 80 cm in females for idf criteria for south asians .
plasma glucose concentration was determined by the glucose oxidase - peroxidase method ( vitalab selectra-2 , merck , germany ) .
the diagnosis of diabetes was defined by either casual plasma glucose 200 mgdl associated with symptoms of diabetes and on fasting samples plasma glucose 126 mgdl .
individuals with self reported , prior physician - diagnosis of diabetes were classified as having previously diagnosed diabetes .
serum lipids that include total cholesterol , high - density lipoprotein ( hdl ) , low - density lipoprotein ( ldl ) , and triglycerides ( tg ) were also measured ( vitalab selectra-2 , merck , germany ) .
the results from any person having a history of hypertension or found to have hypertension were verified by qualified doctors .
the biochemical tests were completed in semiautomatic analysers ( microlab 300 , vital scientific , the netherelands ) .
epidata refers to a group of applications used in combination for creating documented data structures and analysis of quantitative data . in this study ,
the idf and ncep atp iii 's criteria for metabolic syndrome were used to calculate and compare the frequency of metabolic syndrome .
the relationships between the prevalence of cardiovascular risk factors , demographic details , lifestyle , and physiological test results were analysed using the spearman correlation test .
further , the differences in the categorical variables were examined using chi - squared test .
odds ratios ( ors ) and their 95% confidence interval were calculated using binary logistic regression ( for gender and age ) and multinomial logistic regression ( for life style factors ) .
in total , 14,425 people , aged 20100 ( mean age 41.4 15.1 ) , were included in the study . among them ,
the percentage of education level is illustrated in accordance to the number of years in education ( 15 years
the participants were divided into four categories according to their work : labourer / farm , office , house , and none / unknown .
participants ' physical activities were defined according to the time spent every day on physical activity as > 60 min , 3060 min , < 30 min / day , and none .
abdominal obesity was observed in 11.5% ( n = 1607/14002 ) of the participants as per ncep criteria ( mean waist circumference : male107.38 6.19 cm , female94.84 5.84 cm ) and in 34.7% ( n = 5006/14418 ) of the participants as per idf criteria . according to the revised bmi , 10.6% ( n = 1534/14423 ) were underweight ( bmi < 18.5 ) , 28.2% ( n = 4065/14423 ) were overweight ( bmi = 2224.9 ) , and 32.5% ( n = 4689/14423 ) were obese ( bmi > 25 ) .
diabetic prevalence was 6.3% ( 889/14008 ) of which 4.8% ( n = 673/14008 ) were under treatment .
a figure of 12.3% ( n = 1718/14009 ) had a family history of diabetes .
hypertension was observed in 33.9% ( n = 4894/14422 ) of the participants ( mean systolic 138.72 18.03 mm hg and mean diastolic 93.09 8.45 mm hg ) .
only 12.9% ( 1812/14009 ) were previously diagnosed , and 8.5% were receiving treatment for hypertension .
a history of coronary artery disease was present in 1.6% ( n = 218/14007 ) , and 1% ( n = 142 ) were under treatment for ischemic heart disease or stroke .
table 2 shows the goodness of fit for the prevalence of obesity , hypertension , and diabetes .
prevalence of hypertension showed no difference from these data , and obesity showed only a small difference .
the percentages of the participants who had abnormal lipid profile that includes total serum cholesterol , serum ldl cholesterol , serum hdl cholesterol , serum triglycerides are listed in table 3 . there were 2191 sets of data eligible to meet the criteria for metabolic syndrome .
ms was observed in 22.5% ( n = 494/2191 ) of the participants according to the idf criteria and 20.7% ( 454/2191 ) according to the ncep criteria .
the percentages of individual ms risk factors among the total participants and the participants with ms are illustrated in figures 1 and 2 .
generally , among the total participants and the specific participants with ms , the presence of abnormal lipids was higher than the other factors defining ms . however , the presence of abdominal obesity was higher among ms participants using idf criteria ( figure 2 ) .
the females had a higher prevalence of ms than males . according to the ncep criteria , the age groups 4160 and 6180 had a higher prevalence of ms than the lower age group .
according to idf criteria , the age groups 4160 and 2040 had a higher prevalence of ms .
the participants who worked at home had a high incidence of ms according to both the criteria used .
the sedentary group had a higher incidence of ms than the participants who were physically active .
the univariate correlations between cardiac risk factors are shown in table 5 , and the chi - squared independence of them in the metabolic syndrome prevalence is listed in table 6 .
the prevalence of ms ( ncep scores ) had a significant positive relationship with education levels and physical activity .
there were significant positive correlations between physical activity and the three individual ms components : high glucose ( r = 0.03 , p < .01 ) , high bp ( r = 0.04 , p < .01 ) , and low hdl ( r = 0.23 , p < .01 ) .
there was no correlation between physical activity and the other two ms components : high triglyceride ( r = 0.003 , p > .05 ) and abdominal obesity ( r = 0.003 , p >
the ncep scores had a positive correlation between the family history of diabetes ( r = 0.83 , p < .01 ) and hypertension ( r = 0.115 , p < .01 ) .
although a number of these correlations show high levels of significance , the common variance is extremely low , suggesting that the sample size is having a major impact on the significance . as a result of this
table 7 lists the chi - squared independence , odds ratios , and confidence intervals in the association between age , gender , and specific lifestyle factors in metabolic syndrome prevalence .
gender , age , education level , and physical activity show a positive association with the prevalence of metabolic syndrome .
it is important to observe the prevalence of diabetes , hypertension , and obesity individually and also the combination of risk factors as metabolic syndrome to predict the risk of cardiovascular disease .
any association between lifestyle factors and these risk factors would provide the opportunity to encourage a change in lifestyle to promote lower levels of subsequent cvd .
the large number of poorly educated people and the large number of school dropouts could be linked to the disease prevalence .
the prevalence of hypertension and metabolic syndrome in poorly educated people was large when compared with the educated participants .
though the results are not generalized , the relationship between education levels and the prevalence of hypertension agrees with earlier studies [ 19 , 20 ] .
these found that education levels significantly influence the knowledge of hypertension and the awareness of cardiovascular risk .
this suggests that there is a need to improve the awareness of health and use education to prevent or reduce the risk of ms and cardiovascular risks in these groups .
the office workers had a lower prevalence of ms ( ncep scores ) than the other groups .
a considerable number of office workers ( 64% ) undertook regular physical activity of more than 30 min / day .
the home workers education levels and physical activities were comparatively lower than the other work groups .
these findings clearly show that education and physical activity have an influence on the prevalence of ms .
most of the females were home workers ( 75.5% ) , and their education was comparatively lower than the males .
the amount of physical activity involved in home workers is unknown , but the results suggest it is less than that undertaken by other workers .
asian populations continue to modernize , and levels of physical activity are declining as ( i ) home and work place jobs become more automated and sedentary and ( ii ) transportation is more readily available .
the prevalence of ms among the participants who had no physical activity was surprisingly no different than others .
this may be due to a higher than average number of missing values in these data ( 2191/14425 complete data to meet the criteria for ms ) or to other unknown socioeconomic factors .
controversially , there was a high prevalence of ms among people who regularly ate fruit and vegetables .
. found that a higher intake of macronutrients such as fruits and vegetables is associated with general obesity .
however , it is not clear how the vegetables and fruits were eaten , for example , overcooked , processed , and so forth .
the exact quantity of the dietary intake was not recorded as it was not the primary area of focus of the study . in these populations ,
these tend to report a low intake of mono - unsaturated fats ( mufas ) , n-3 polyunsaturated fats ( pufa ) , and transfatty acids ( mostly related to widespread use of vanaspati , a hydrogenated oil ) .
the healthy traditional plant - based diets are being replaced by cheaper calorie dense high - fat foods .
these changes are resulting in a rapid increase in the prevalence of obesity throughout asia and the subsequent development of ms .
ness and powles also found in their review that many studies were reporting the null or negative effects of fruit and vegetable intake on the prevalence of cardiovascular diseases .
however , the correlations found in those studies were generally low , as seen in our study .
further , they suggest that a food - based analysis would complement the nutrient - based analysis to clarify these issues . in nepal ,
the regular diet in addition to fruits and vegetables , that is , such as rice , which is high in carbohydrates , and the methods of cooking may be dietary causes of metabolic syndrome .
the age groups 4060 had a large prevalence of ms in this study . also , it is important to note that this middle - aged group had a high incidence of overweight or general obesity and abdominal obesity .
the other age groups had a lower prevalence of ms than the 4060 years old , yet it was still relatively high .
inadequate maternal nutrition in pregnancy , low birth weight , and childhood obesity may be important factors for the development of metabolic syndrome and diabetes . specifically in children and young individuals , a high intake of n-6 pufa is correlated with hyperinsulinaemia . in adults ,
unger described metabolic syndrome as a failure of the system of intracellular lipid homeostasis which prevents lipotoxicity in organs of overnourished individuals . in this study
in addition to low levels of hdl , the hdl particles are small , dense , and dysfunctional in south asians .
hypertriglyceridaemia is a direct reflection of an insulin resistance condition , and it is interrelated to the low hdl concentrations in developing endothelial dysfunction . in nepal ,
a high number of the participants had abdominal obesity and were overweight / obese , according to their bmi .
the bmi is a simple useful measure for overall abnormal weight , yet not a standard measure for obesity .
bmi can not differentiate between whether the condition was due to unusual muscular development or the accumulation or distribution of fat in the body [ 26 , 27 ] . despite
the low prevalence of general body obesity compared to western countries , metabolic syndrome is growing into a significant public health problem in asia .
this may be mainly due to the large number of people with central obesity , a feature which was also observed in this study
. the higher prevalence of ms in females is also more likely to be due to a higher incidence of abdominal obesity .
abdominal obesity is an important factor because metabolic syndrome and increased abdominal fat are related to a reduction of adiponectin , an adopicyte - derived hormone with antiatherogenic and anti - inflammatory properties .
the abdominal adipose tissue results in release of free fatty acids directly in the portal veins and altered lipid levels in the blood .
further abdominal adiposity increases insulin secretion , and it would be exaggerated by decreased hepatic clearance leading to hyperinsulinemia .
thus abdominal obesity is an important indicator of cardiovascular disease due to its link to dyslipidemia , hyperinsulinemia , hypertension , and impaired fibrinolytic capacity .
state that if the ncep 's criteria were applied to the asian population , it might underestimate the prevalence of metabolic syndrome and the risk of cardiovascular disease .
idf 's specific reference values for abdominal obesity make a substantial difference to the prevalence of ms between the two criteria . the idf 's cut - off points for south asians ' waist circumference
are lower than the ncep 's general cut - off points ( 90 cm versus 102 cm in men and 80 cm versus 88 cm in women ) .
another study on chinese population also found a large increase in the prevalence of metabolic syndrome using idf criteria compared with ncep criteria .
however , in our study both definitions demonstrated a higher prevalence of metabolic syndrome ( 20.722.5% ) in nepal when compared with the studies done in other southeast asian countries such as thailand ( 1218% using ncep definition ) and india ( 18.3% using idf definition ) .
these findings suggest the need for specific attention to control the disease prevalence in nepal .
although data were prospectively collected , they may not be generalizable outside of eastern nepal .
the results did not show substantiate relationship between smoking histories , diet , family history of cardiovascular , and metabolic syndrome .
abdominal obesity , with the revised reference values , is an important risk due to its physiological relationship to the other ms risk factors .
the ms prevalence may be due to lack of awareness and unhealthy lifestyles , so health education and more preventive measures should decrease the prevalence of ms and cardiac risks in nepal . | background . this study was carried out to establish the prevalence of cardiovascular risks such as hypertension , obesity , and diabetes in eastern nepal .
this study also establishes the prevalence of metabolic syndrome ( ms ) and its relationships to these cardiovascular risk factors and lifestyle . methods .
14,425 subjects aged 20100 ( mean 41.4 15.1 ) were screened with a physical examination and blood tests .
both the international diabetic federation ( idf ) and national cholesterol education programme 's ( ncep ) definitions for ms were used and compared .
results .
34% of the participants had hypertension , and 6.3% were diabetic .
28% were overweight , and 32% were obese .
22.5% of the participants had metabolic syndrome based on idf criteria and 20.7% according to the ncep definition .
prevalence was higher in the less educated , people working at home , and females .
there was no significant correlation between the participants ' lifestyle factors and the prevalence of ms .
conclusion
. the high incidence of dyslipidemia and abdominal obesity could be the major contributors to ms in nepal .
education also appears to be related to the prevalence of ms .
this study confirms the need to initiate appropriate treatment options for a condition which is highly prevalent in eastern nepal . |
balance is very important in daily activities of the elderly and greatly affects their
quality of life . due to age - related changes , balance ability deteriorates .
osteoarthritis is
a degenerative disease that causes severe pain in elderly patients with low pain thresholds .
research has shown that it results in a high disability score , low quality of life , and
limited range of motion , in addition to weak muscle strength , poor balance ability , and high
risk of falls1,2,3,4,5 .
as osteoarthritis causes
severe pain and functional disorders , in general , the clinical treatment is focused on pain
management and amelioration of related disorders .
the treatments include muscle strength
training , electrical stimulation , manual therapy , aquatic therapy , balance therapy , and
pharmacotherapy6 .
as balance ability and falls are closely related in the elderly , exercise therapies need to
focus on the maintenance of such ability7 .
horseback riding exercise is one such therapy . in research conducted by arajo et al .
8 where elderly individuals performed
horseback riding exercise for eight weeks , the researchers reported that the exercise
significantly improved the participants balance and muscle strength .
unlike other
interventions , horseback riding exercise can arouse the interest of patients and motivate
them while improving their balance , bone density , muscle strength , and mental state9,10,11 . according to some studies ,
horseback
riding exercise increased the static muscular contraction of the rider against the movement
of the horse and improved the muscle strength in the quadriceps femoris and knee flexors of
young riders12 , 13 .
most people do not indulge in horse riding because of where they live ( i.e. , urban rather
than rural areas ) or the costs and risks involved in horse riding .
hence , simulator
horseback riding exercise equipment using virtual reality has been developed for use at home
and in small spaces .
previous studies reported that horseback riding exercise significantly
increased oxygen intake , minute ventilation , met , calorie consumption , and muscle14 , 15 .
although various therapeutic approaches have made with horseback
riding exercise in the previous studies there is a lack of research on the effects of
horseback riding exercise equipment on the balance and gait abilities of elderly patients
with osteoarthritis .
experimental procedures were performed in accordance with the protocols established by the
institution of animal care and use committee ( iacuc ) at the daegu university . include criteria was aged 65 years or over ( table
1table 1.general characteristics of the subjectsvariablessubjects ( n=15)gender ( m / f)6/9age ( years)76.5 7.7height ( cm)160.6 9.7weight ( kg)54.7 9.8mean sd ) , and with a history of none falls in previous year , lower pain for perform
experiment , and visual or auditory damage , nervous system or of the vestibular organ
problems , who ca nt understand the contents of the experiment were excluded . after providing
information study all subjects received a participation agreement . in this study , the subjects , while providing conventional physical therapy , 8 weeks three
times a week the movement of the horse riding equipment , was carried out for 30 minutes .
before performing horse riding simulation exercise
, subjects were educated about exercise
program through a research assistant s demonstration . in this study , used the slimrider
( shinhwa , mx-0004se , korea ) , which is a horse riding simulator . while performing a horse
riding equipment exercise , subjects were effort to maintain the correct posture .
exercise
intensity was increased gradually according to the state of the subjects while performing
the exercise , the research assistant were always observed to prevent falling . in order to evaluate the balance of the elderly , it was using the frt ( functional reach
test ) and sbbsf ( short form berg balance scale ) .
the spss for windows ( version 18.0 ) was used for data analysis of the study . in order to
compare the differences between before and after , it was analyzed using the paired t - test .
in this study , sfbbs scores and range of frt increased significantly after horse riding
simulator exercise ( table 2table 2.comparison of measurement values at pre - test and post - testvariablesprepostsfbbs ( score)18.6 5.822.3 4.4**frt ( cm)19.7 9.526.3 7.5*mean sd .
osteoarthritis causes severe pain in elderly patients who have a low threshold for pain and
high disability scores , thereby resulting in a low quality of life , increased risk of falls ,
and poor balance ability1 , 2 , 6 . in the present
study
, the static , dynamic , and functional balance abilities of osteoarthritis patients who
undertook horseback riding exercises on exercise equipment were measured using a frt and
sfbbs .
the findings are in
accordance with those of study that stroke patients performed horseback riding exercise for
35 min five times a week for eight weeks and arajo8 in which elderly patients performed horseback riding exercise for
eight weeks .
they are also in accordance with the results of han jun - young16 in a study of stroke patients who
performed horseback riding exercise for 12 weeks that an eight - week - long horseback riding
exercise significantly improved the bbs scores of osteoarthritis patients . in general
elderly , the average bbs score is 20.60 4.0817 . in the present study ,
after eight weeks of the exercise , the average score
was 22.3 4.4 , higher than the average .
the results suggest that horseback riding exercise
equipment can improve dynamic and functional balance abilities .
furthermore , according to the frt results , the static balance ability of the participants
improved .
these results are in line with the research findings of yasuhiro et al.18 and kim sung - gil11 where the frt scores improved significantly after
12-week - long and 8-week - long horseback riding exercise , respectively .
an increase in the frt
score is considered an improvement in static balance ability .
based on the findings of the
present study , horseback riding exercise equipment seems to improve the static balance
ability of the elderly . | [ purpose ] the aim of this study was to examine the effects of horse - riding simulator
exercise on balance in elderly with knee osteoarthritis . [ subjects and methods ] fifty
elderly patients with knee osteoarthritis were recruited , a horse - riding simulator group
performed exercise three times a week for eight weeks . and each exercise was performed for
30 minutes .
[ results ] the horse - riding simulator group showed significant differences
after the intervention in short form berg balance scale , functional reaching test .
[ conclusion ] the results of this study indicate that horse - riding simulator exercise is
effective on knee osteoarthritis .
therefore , horse - riding simulator exercise can be used
balance training for knee osteoarthritis . |
, medulloblastomas can be classified in 4 different molecular subtypes : wnt , shh , group 3 and group 4 .
each of these molecular subtypes has specific underlying molecular features , gene expression , demographic characteristics and prognosis . sonic hedgehog ( shh ) medulloblastomas
are characterized by activation of the hedgehog ( hh ) signaling pathway and are often driven by mutations in hh pathway components . here
we focus on shh medulloblastoma and discuss how cell senescence shapes the natural history and molecular evolution of this childhood cancer .
for many years , it was thought that tp53 mutations were infrequent in medulloblastomas and that p53 signaling was dispensable for medulloblastoma tumor suppression .
however , recent studies reported that tp53 mutations are frequent in human wnt ( with a rate of 16% ) and shh primary medulloblastomas ( with a rate of 13% to 21% , depending on the study ) and are indicators of poor prognosis exclusively in shh medulloblastoma .
moreover , tp53 mutations have recently been identified as a key event in the pathogenesis of medulloblastoma recurrence .
although the specific roles of p53 in medulloblastoma are still largely unknown , the presence of recurrent tp53 mutations suggests that p53 signaling plays an important role in driving medulloblastoma tumorigenesis .
human genomic studies also established that tp53 mutations co - exist with mutations or amplifications of hh signaling components such as shh , smo , sufu , gli2 and mycn .
these studies , however , do not establish the sequential order of genetic events that lead to medulloblastoma formation and how these mutations correlate with presumptive histopathological stages of medulloblastoma .
nevertheless , patients with li - fraumeni syndrome , caused by germ - line mutations in tp53 , offer an opportunity to study how medulloblastomas arise at the genetic level .
li - fraumeni patients are cancer - prone and sometimes develop medulloblastoma ; therefore , at least for this subset of patients , tp53 mutations are the first genetic event leading to medulloblastoma formation ( fig .
notably , most li - fraumeni medulloblastomas belong to the shh subgroup , indicating that tp53 mutations specifically predispose to shh medulloblastoma . because cerebellum granule cell precursors ( gcps ) are the cells of origin of shh medulloblastoma
moreover , these shh medulloblastomas seem to be the result of chromothripsis , a massive genome rearrangement caused by chromosomal shattering likely occurring in a single event . these chromothriptic events
likely lead to mutations in components of hh signaling , such as gli2 , boc and mycn amplifications .
since hh signaling is the most important mitogenic pathway for gcps , it is therefore expected that acquisition of hh pathway mutations efficiently causes shh medulloblastoma in li - fraumeni patients .
( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .
chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .
( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .
spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated .
( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .
chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .
( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .
spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated .
although p53 knockout mice or mouse models of li - fraumeni syndrome do not develop medulloblastoma , elegant studies have demonstrated that the inactivation of p53 together with other dna repair factors such as as xrcc4 , ligase iv , xrcc2 and brca2 leads to shh medulloblastoma .
interestingly , those medulloblastomas also harbored spontaneous mutations in hh signaling components such as ptch1 , mycn and gli2 .
this result not only highlights how important it is to maintain genomic stability to prevent shh medulloblastoma formation , but it also indicates that hh signaling activation seems to be necessary for medulloblastoma formation even when p53 and dna repair mechanisms are absent . in summary ,
germ - line p53 mutations in both mouse and human cause genomic instability and lead to mutations in hh signaling components , conducing to medulloblastoma formation ( fig .
this is an interesting paradigm showing that hh signaling mutations happen subsequent to tp53 mutations in li - fraumeni syndrome .
half of the shh medulloblastomas with tp53 mutations have a germ - line ( li - fraumeni ) origin and are potentially explained by the mechanism described above .
however , it is not known how the other shh medulloblastoma cases ( including the ones with somatic tp53 mutations and the ones without tp53 mutations ) develop and the temporal order in which they acquire their mutations .
another unresolved , yet related , question is whether advanced medulloblastomas arise in a step - wise manner from subclinical precancerous lesions . for many epithelial cancers ,
the availability of preneoplastic lesions allowed the establishment of tumor progression models with sequential histopathological stages and the molecular changes that characterize them .
however , the problem for understanding brain tumor development lies in the inability to detect and obtain precancerous lesions ; therefore , genome sequencing of advanced brain tumors only offers a snapshot of the mutations present in advanced tumors but does not show the order in which mutations are acquired during tumor progression . to address this question , we used ptch1 heterozygous mice , a well - established model of shh medulloblastoma ( fig . 2 ) .
ptch1 mice develop preneoplastic lesions with high frequency , but only a fraction of those animals develop advanced medulloblastoma .
therefore , we hypothesized that an unidentified tumor suppressive mechanism might restrain the progression of medulloblastoma preneoplasia into advanced tumors .
we found that apoptosis levels are the same between preneoplastic lesions and advanced medulloblastoma , eliminating apoptosis as an essential tumor suppressor in this model of shh medulloblastoma .
surprisingly , when we looked at cell senescence , we found that while preneoplastic lesions display high numbers of p21 and p16 positive cells ( which are effectors and markers of cell senescence and cell cycle arrest ) , advanced medulloblastomas have very low levels of senescence . these high levels of senescence
are paralleled by lower levels of proliferation and correlate with loss of heterozygosity ( loh ) of the ptch1 wild - type allele in preneoplastic lesions ( fig .
1b ) , suggesting that high levels of hh signaling may contribute to cell senescence .
using laser capture microdissection , we found that one - third of all advanced medulloblastomas acquired p53 mutations that were not present in preneoplastic lesions , supporting the notion that p53 mutations allow senescence evasion and medulloblastoma progression ( fig .
moreover , we found that engineered p53 mutations prevent cellular senescence and accelerate medulloblastoma formation , showing that p53 mutations lead to senescence evasion . further supporting the idea that senescence evasion is necessary for medulloblastoma progression , advanced tumors without p53 mutations display low expression of p16 due to promoter methylation . in summary
, we found that , contrary to li - fraumeni syndrome ( where tp53 germ - line mutations lead to hh signaling mutations ) , hh signaling hyperactivity leads to cell senescence in preneoplastic lesions and creates selection pressure for the inactivation of p53 or p16 , which allows the progression from preneoplasia to advanced medulloblastoma ( fig .
figure 2.medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,
the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .
after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .
loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .
medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,
the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .
after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .
loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .
importantly , the finding of spontaneous p53 mutations is not limited to the ptch1 model , as we also found spontaneous p53 mutations in tumors from olig1-gnas mice , another model of shh medulloblastoma .
1b ) , this may highlight some possible interactions between p53 and hh signaling in the brain .
shh , a protein secreted by purkinje cells of the cerebellum , is the most potent mitogen for gcps .
in contrast to these proliferative effects of shh , p53 activity supresses proliferation and self - renewal of adult neural stem cells of the subventricular zone , and this effect might be mediated by p21 .
this may explain why p53 activity is downregulated during neurogenesis to allow cell proliferation and brain formation .
interestingly , gli activity has been shown to downregulate p53 protein levels in cell lines .
specifically , it has been proposed that high levels of hh signaling in cell lines caused by expression of constitutively active smo mutants or overexpression of gli1 and gli2 leads to an mdm2-dependent degradation of p53 .
consistently , low levels of mdm2 in vivo increase p53 levels , lead to cerebellar hypoplasia and reduce medulloblastoma development in ptch1 mice , showing that p53 signaling negatively controls cerebellum growth and implying that p53 signaling is important for medulloblastoma tumor suppression .
similarly , the proto - oncogene ppm1d , a negative regulator of p53 , is overexpressed in medulloblastomas and increases medulloblastoma formation in mice when overexpressed together with shh .
together , these findings provide strong evidence that hh signaling leads to a functional inactivation of p53 signaling that allows gcp proliferation and , in some instances , medulloblastoma formation .
however , the presence of somatic p53 mutations in ptch1 medulloblastoma demonstrates that the ability of shh signaling to functionally suppress p53 signaling is not always sufficient to inactivate p53 activity in a tumorigenic context . for many years
, it has been known that p53 deletion accelerates medulloblastoma formation and increases medulloblastoma incidence in ptch1 mice ; however , the mechanism responsible for this was never investigated .
the fact that p53 mutations , which are acquired spontaneously during medulloblastoma formation , lead to senescence evasion provides an explanation for the presence of somatic tp53 mutations in human shh medulloblastoma .
recently , we also found that the ligand shh induces dna damage in gcps , an effect that requires the presence of the hh receptor boc and cyclind1 .
these results support the hypothesis that gcps are sensitive to replicative stress and that hedgehog signaling likely causes replication stress .
it has been demonstrated that oncogene activation leads to oncogene - induced cell senescence ( ois ) , a tumor - suppressive mechanism that prevents transformation of premalignant lesions into tumors .
moreover , oncogene - induced dna damage seems to be required for ois . in light of this
, the fact that we found cell senescence and ptch1 loh in medulloblastoma preneoplastic lesions suggests that high levels of hedgehog signaling in preneoplasia likely cause high levels of oncogenic stress and this leads to cell senescence .
therefore , we propose that high levels of hedgehog signaling shape the molecular evolution of medulloblastoma by leading to ois and creating selection pressure to inactivate p53 in order to evade ois .
in addition to oncogenes , loss of tumor suppressor genes has been shown to cause ois .
for example , in the context of prostate cancer , pten inactivation causes senescence and leads to the acquisition of p53 mutations .
neurofibromin ( nf1 ) loss also leads to ras - mediated induction of cell senescence . since we observed a strong association between ptch1 loss and cell senescence in medulloblastoma preneoplasia , ptch1 seems to be a new tumor suppressor whose absence may be capable of causing ois .
however , the molecular mechanism leading to tp53 mutations has never been investigated in wnt medulloblastoma .
interesting work has shown that , at least in cell lines , overexpression of the downstream wnt effector - catenin leads to p53 stabilization and activation .
wnt activation in colorectal cancer has been associated with decreased levels of proliferation and accumulation of p53 protein in early tumor stages , suggesting that wnt signaling may lead to cell senescence in precancerous lesions and this could be the cause of the acquisition of tp53 mutations at late stages of colorectal cancer .
we thus speculate that wnt activation in the brainstem may also lead to changes compatible with cell senescence and this may be an explanation for the presence of tp53 mutations in advanced wnt medulloblastomas
. this is supported by the fact that expression of an active - catenin mutant in the lower rhombic lip of mice leads to the formation of hyperplastic lesions that only progress to advanced medulloblastomas when p53 is also deleted .
it has been shown that low - grade astrocytoma lesions display a dna damage response and that loss of components of the atm - chk2-p53 pathway accelerates astrocytoma development .
although cell senescence was not interrogated in those reports , another study showed that pilocytic astrocytomas ( pa ) , the most benign type of astrocytomas , display mapk activation and ois ; interestingly , pa are indolent tumors that display long periods of growth arrest , rarely become high - grade astrocytomas , and do not display tp53 mutations .
this is in agreement with the idea that pa are long - term senescent astrocytic lesions that do not progress .
eglexternal granule - cell layergcpsgranule cell precursorshhhedgehogiglinternal granule - cell layeroisoncogene - induced senescenceshhsonic hedgehog external granule - cell layer granule cell precursors internal granule - cell layer oncogene - induced senescence
work done in the charron lab was supported by grants from the canadian institutes of health research ( cihr ) , the fonds de recherche du qubec - sant ( frqs ) , and the canada foundation for innovation ( cfi ) .
sms is recipient of the mcgill james o. and maria meadows fellowship and ircm challenge - gupards et gazelles gourmands scholarship . | abstractthe mechanisms leading to brain tumor formation are poorly understood . using ptch1+/ mice as a medulloblastoma model
, sequential mutations were found to shape tumor evolution .
initially , medulloblastoma preneoplastic lesions display loss of heterozygosity of the ptch1 wild - type allele , an event associated with cell senescence in preneoplasia .
subsequently , p53 mutations lead to senescence evasion and progression from preneoplasia to medulloblastoma .
these findings are consistent with a model where high levels of hedgehog signaling caused by the loss of the tumor suppressor ptch1 lead to oncogene - induced senescence and drive p53 mutations .
thus , cell senescence is an important characteristic of a subset of shh medulloblastoma and might explain the acquisition of somatic tp53 mutations in human medulloblastoma .
this mode of medulloblastoma formation contrasts with the one characterizing li - fraumeni patients with medulloblastoma , where tp53 germ - line mutations cause chromothriptic genomic instability and lead to mutations in hedgehog signaling genes , which drive medulloblastoma growth .
here we discuss in detail these 2 alternative mechanisms leading to medulloblastoma tumorigenesis . |
a 33-year - old woman , ( gravida 3 , para 2 ) , at 34 weeks of gestation , presented with episodes of lightheadedness , and dyspnea .
she was asymptomatic until 18 weeks of gestation , when she started having episodes of palpitations associated with presyncope .
initially , the episodes were infrequent ( less than once per day ) but a gradual increase in frequency and severity was noted by the patient .
one month prior to presentation , she noted episodes of paroxysmal nocturnal dyspnea , but no dyspnea on exertion , chest pain , diaphoresis , pedal edema , orthopnea , or neurological symptoms .
she had no significant past medical history and had not had complications relating to this pregnancy .
all prior pregnancies had progressed to full term without similar symptoms or complications , and resulted in spontaneous vaginal deliveries of healthy newborns .
physical examination revealed an afebrile patient with a regular pulse of 80 beats per minute , blood pressure 110/70 mmhg , and an oxygen saturation of 100% on room air .
the examination was normal except for mild non - pitting edema and fine bibasilar crackles .
laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers and tsh .
cardiac monitoring showed multiple episodes of monomorphic , non - sustained ventricular tachycardia ( vt ) with left bundle branch morphology , consistent with vt originating from the right ventricular outflow tract ( fig .
urgent two - dimensional echocardiogram showed normal left ventricular size and wall thickness with no segmental wall motion abnormalities and normal systolic and diastolic function with a left ventricular ejection fraction of 57% .
a 12-lead electrocardiogram showed sinus rhythm with average rate of 80 beats / min with a normal axis , pr - interval , and qrs duration with no evidence of chamber enlargement or infarction .
the clinical working diagnosis was pregnancy - related vt of right ventricular outflow tract origin .
a 24 year woman ( gravida 4 , para 2 ) presented to her local emergency department at 24 weeks gestation because of shortness of breath , cough , and chest tightness developing over the previous day .
she had a history of mild asthma but had not had asthma - related symptoms for several years and had never had an asthma exacerbation requiring inpatient or emergency department care . past medical history and family history
there had been no similar symptoms or medical complications in her previous three pregnancies , one of which had ended with spontaneous abortion and the other two with term delivery of healthy children .
she smoked one - half to one pack of cigarettes per day but did not drink alcohol or use recreational drugs .
physical examination revealed an afebrile patient with a regular pulse of 78 beats per minute , blood pressure 100/60 mmhg , and an oxygen saturation of 96% on room air . on respiratory exam
the rest of the examination was normal except for mild non - pitting peripheral edema .
laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers , and tsh .
the patient was diagnosed with an asthma exacerbation and treated with nebulized salbutamol 2.5 mg , which relieved her symptoms . shortly thereafter , she developed runs of asymptomatic nonsustained vt with a left bundle branch block pattern ( fig .
nebulized salbutamol 5 mg was once again administered , whereupon the patient developed sustained runs of vt of similar morphology .
intravenous lidocaine and magnesium sulfate were administered , leading to resolution of the ventricular arrhythmia .
no further salbutamol was administered , and two further days of electrocardiographic monitoring showed only occasional pvcs .
twelve lead electrocardiogram showed sinus tachycardia at 102 beats per minute with occasional premature ventricular beats , normal corrected qt interval and no evidence of ischemia or chamber enlargement .
echocardiography showed normal left ventricular size with normal systolic function with an ejection fraction of 57% , and no valvular abnormalities .
given the absence of structural heart disease and risk factors for coronary disease or arrhythmia , the clinical diagnosis was catecholamine - sensitive pregnancy - related vt of right ventricular outflow tract origin .
ventricular tachycardias ( vt ) may be seen in pregnancy and can manifest as a new onset arrhythmia or be exacerbated by pregnancy and can cause concern for the well - being of both the mother and the fetus .
a study of 11 pregnant women who experienced a new onset of vt during pregnancy showed that onset was distributed equally over the three trimesters and disappeared completely during the postpartum period.1 the characteristics and underlying mechanisms of new - onset vt during pregnancy have not been adequately investigated .
although the majority of vts that occur during the pregnancy are benign , in women with structural heart disease , arrhythmias ( especially those of ventricular origin ) is one of the five independent predictors of having an adverse outcome during pregnancy.2,3 peripartum cardiomyopathy ( ppcm ) needs to be considered in women presenting with vt in the last month of pregnancy.4,5 the criteria for the diagnosis of ppcm include : development of congestive heart failure secondary to left ventricular systolic dysfunction in the last month of pregnancy or within 5 months after delivery , absence of preexisting cardiac dysfunction , and absence of identifiable cause of heart failure and lv systolic dysfunction demonstrated by classic echocardiographic criteria.6 hence in women presenting with vt in the last month of pregnancy , an urgent echocardiographic study can assist in establishing a diagnosis of ppcm .
women with certain types of congenital heart disease ( chd ) either repaired or not , are particularly vulnerable to vt . for example
, patients with previous surgeries for correction of tetralogy of fallot may experience vt arising from the right ventricular outflow tract as a result of right ventricular volume overload due to decades of severe pulmonary regurgitation as a consequence of patch augmentation of the right ventricular outflow tract at the time of original corrective surgery.7 a study of 28 female patients with repaired chd demonstrated women with chd had a significantly higher incidence of tachyarrhythmia during pregnancy and in the postpartum period compared to controls.8 congenital long qt syndrome ( lqts ) is a hereditary disorder of cardiac ion channels causing abnormal electrical activation of the heart.9 women with congenital lqts may experience life - threatening polymorphic vt ; however recent reports indicates that their incidence appears to be significantly increased during the postpartum period.10,11 patients with congenital lqts are particularly vulnerable to adverse arrhythmic effects of electrolyte depletion ( further prolongation of qt interval ) a possible complication in some pregnant women with hyperemesis .
cautious use of mediations that may further prolong the qt interval is necessary in these individuals to avoid precipitation of polymorphic vt .
in addition to cardiac diseases , several systemic non - cardiac disorders , including severe electrolyte derangements , thyroid function abnormalities , pulmonary embolism , anemia , and drug overdose may present with ventricular arrhythmias .
there are few reports of new onset vt during pregnancy in absence of structural heart disease.12,13 increased sympathetic activity , as well as physiological changes associated with normal pregnancy such as increased heart rate , decreased peripheral resistance , increased stroke volume and psychological stresses are thought to be the most common precipitants of vt in pregnant women with a structurally normal hearts.14,15 the majority of outflow tract vt in structurally normal hearts originate from the right ventricular outflow tract ( rvot ) just below the pulmonary valve .
this is also the most common focus of initiation of pregnancy - related idiopathic vt and usually does not deteriorate to an unstable rhythm .
it will often manifest as ectopic beats , bigeminal rhythms or short runs of non - sustained vt .
norepinephrine concentration in the myocardial synaptic cleft has been suggested as a potential mechanism for this arrhythmia.16 only 10% of vt originates from the left ventricular outflow tract ( lvot).17 to make a diagnosis , a 12-lead ecg should be recorded ideally during the arrhythmia , although this can be difficult .
a typical resting ecg of a pregnant woman may show an increased heart rate with decreased pr , qrs and qt intervals , but there is usually no significant change in the amplitudes of p and t - waves and qrs - complex.15 premature atrial and ventricular beats as well as q wave and inverted t waves in the inferior leads can also be seen during pregnancy.18 on ecg , rvot vt appears as wide qrs complex tachycardia with left bundle brunch block ( lbbb ) morphology and an inferior axis.17,19 two phenotypic forms of rvot vt can occur , non - sustained monomorphic vt and paroxysmal sustained vt.20 in contrast , lvot vt morphology depends on the site of origin with either lbbb and early precordial transition in leads v12 , or rbbb pattern in v1 with broad monophasic r in the precordial leads .
in addition , the ecg in patients with vt originating in the lvot will show r : s amplitude ratio of 30% or more or an r : qrs duration ratio of 50% in leads v12.21 a routine 24 to 48 hour - holter monitor is helpful in capturing frequently occurring paroxysmal arrhythmias . to elicit symptoms ,
an exercise ecg can be reasonably carried out during pregnancy in absence of obstetrical contraindications for exercise .
no fetal adverse events are reported for adenosine , except for one case of transient fetal bradycardia.22 the first step in acute management of vt in pregnancy to determine the hemodynamic stability of the pregnant woman .
if the woman is unstable or there is evidence of significant fetal compromise which is thought to be related to the vt , dc cardioversion with 50100 j should be given immediately .
dc shocks can be repeated at higher levels of energy ( 100360 j ) if indicated . the risk for the fetus is minimal for all stages of pregnancy , because the amount of current reaching the fetus is small.23 in stable vt , an accurate diagnosis of the type of arrhythmia should be made with a twelve - lead ecg prior to any intervention .
invasive electrophysiological studies are rarely required during pregnancy , as the arrhythmias can be effectively managed pharmacologically .
conservative medical treatment of vt arising during pregnancy is indicated in any patient with sustained vt as long as the patient is hemodynamically stable.14 if possible drug therapy should be avoided during the first trimester and drugs with the longest records of safety should be used as the first - line therapies.24 the literature safety data has examined digoxin , adenosine , flecainide , procainamide , propranolol , propafenone , quinidine , sotalol and verapamil , however ; the experience is limited to single or small case series .
in stable patients with sustained vt acute therapy can be initiated with either intravenous procainamide , which is safe , well - tolerated and is not associated with teratogenicity .
alternatively lidocaine can also be used to treat stable vt.14 idiopathic vt originating from the rvot and with a lbbb morphology responds well to beta - blockers , while idiopathic lvot vt with rbbb morphology generally responds well to verapamil .
given the sensitivity of majority of vts in pregnancy to catecholamines , a cardioselective beta - blocker , in particular metoprolol , in the absence of contraindications is considered first line therapy.12
in our cases , further investigations did not reveal a secondary cause of the patients arrhythmias .
based on the ecgs ( qrs morphology of a left bundle branch block pattern with an inferior axis , and clinical findings ) both patients were diagnosed with idiopathic vt originating from the rvot .
the lower dose for the second patient was chosen because of suspected asthma exacerbation ; however the dose was slowly increased to 50 mg po every 12 hours .
both patients tolerated treatment well and had no further documented episodes of vt on long - term cardiac monitoring .
patient 1 was scheduled for elective c - section at a high risk obstetric center and was discharged home . on the follow - up
patient 2 had a spontaneous vaginal delivery at 36 weeks and continued to be on metoprolol after discharge .
pregnant patients may present with ventricular tachycardia during any trimester careful and timely clinical , electrocardiographic , as well as echocardiographic assessment help identify those individuals with structural cardiac abnormalities such as congenital heart disease or peripartum cardiomyopathy , as well as those with long qt syndrome who will need specific management beyond therapy for the arrhythmia . in those stable patients with structurally normal hearts ,
identification of the location of origin of tachycardia will help in choice of appropriate medical therapy . | ventricular tachycardia although not common , can occasionally complicate pregnancy .
its presence may indicate an underlying cardiac structural abnormality , or undiagnosed congenital arrhythmic disease .
however , some pregnant patients with ventricular tachycardia have structurally normal hearts .
two cases of ventricular tachycardia in pregnant patients with structurally normal hearts are presented and an approach to diagnosis and management of such patients are discussed . |
common sources include backyard burning , incineration of plastics , and chlorine bleaching of pulp in paper mills .
documented health effects include acute and chronic effects , including chloracne , various types of cancers , reproductive diseases , circulatory and respiratory diseases , and diabetes .
the traditional approach to environmental remediation includes a host of physical and chemical methods , depending on the characteristics of the polluted site and the extent of contamination present .
bioremediation , which is the use of biogenic materials and organisms for environmental cleanup , has also been proposed , including phytoremediation using plants and microbial degradation using primarily bacteria and fungi .
bioremediation is an attractive strategy , as it can destroy the pollutant rather than transferring it from one environmental compartment to another .
common bioremediation strategies include the addition of nutrients , degradative microorganisms , or both .
sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy .
one of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds .
monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels .
traditionally , the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate , the rate of substrate removal , and the resulting metabolites [ 69 ] . to optimize the bacterial degradation of pollutants , it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities .
this includes analyzing not only the degradative pathways [ 1012 ] , but also the peripheral processes and mechanisms that are involved in taxis ( i.e. , directed motion in a chemical gradient ) , uptake , and transport during exposures to specific substrates .
analysis of dna and rna can shed light on an organism 's metabolic potential ; however , these measurements poorly correlate to actual protein expression profiles .
therefore , global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation .
in addition to identifying important degradative enzymes in a variety of important microbes [ 1517 ] , proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates .
the imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome , certain elements in the pathway are located on a plasmid , and there may be alternative pathways at work .
the present study builds on previous work and utilized difference gel eletrophoresis ( dige ) coupled with mass spectrometry ( ms ) to exploit recently gathered rw1 genome data . when used together , these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium 's degradative activity toward this recalcitrant compound .
the aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran , a nontoxic surrogate for dibenzo - p - dioxin , as compared to nonselective growth media .
acetate was selected as the nonselective alternate substrate , as growth on this compound was observed to influence expression of select proteins , including the dioxin dioxygenase .
thus , any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects .
cultures of s. wittichii strain rw1 ( 100 ml to 1.0 l ) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium ( ph 7.05 ) supplemented with either dibenzofuran crystals or 50 mm acetate as growth substrates .
cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units ( 560 nm ) . following biomass processing ,
protein levels in the samples were on the order of 75200 g / ml .
protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer .
culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar .
harvested biomass was washed , spun again , and the resultant pellet suspended in a small volume of 100 mm ammonium bicarbonate ( ph ~7.0 ) . this microbial suspension
was then sonicated under cooling with ice , using a microtip sonicator ( fisher scientific , pittsburgh , pa , usa ) in a sequence of three 10-second bursts delivered in thirty - second intervals .
the sonicated cells were then immediately centrifuged at 10,000 xg for 10 minutes at 4c .
briefly , 8 parts of 10% tca in acetone ( 20c ) were added per volume of supernatant and , following mixing on a vortex , the resultant dilution was incubated at 20c overnight .
following centrifugation ( 10,000 xg , 10 minutes , 4c ) , harvested biomass was washed in cold acetone for 10 minutes at 20c .
following a subsequent centrifugation , the pellet was resuspended in sample preparation buffer ( 7 m urea , 2 m thiourea , 2% chaps , 0.2% dtt , 0.02% bromophenol blue ) and stored at 20c until analyzed .
protein concentrations were measured using the bicinchoninic acid assay ( pierce , rockford , il , usa ) following dilution to reduce the concentration of interfering agents .
twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran ( n = 3 ) and acetate ( n = 3 ) were labeled using cy dyes ( ge healthcare ) as described elsewhere .
briefly , samples were adjusted to 1 g/l using sample preparation buffer and the ph checked .
subsequently , 0.25 l of 1 pmol/l cy dyes were added to samples for 30 minutes in the dark on ice . to stop the labeling reaction ,
0.5 l of 10 mm lysine was added to the samples , which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis .
unless stated otherwise , all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes .
samples were randomized to reduce the effect of dye bias and in - gel variations .
a global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard .
one cy-3- and one cy-5-labeled sample were added to each gel , as defined by the experimental randomizing procedures . to each sample
, an additional 175 g of unlabeled sample were added ; the volume was increased to a total of 450 l using sample preparation buffer .
the reducing agent dtt ( dithiothreitol ; 1.3 mg per tube ) and ipg ( immobilized ph gradient ) buffer ( 0.5% ) were added , and the samples incubated and mixed in the dark at room temperature for approximately 1 h. samples were then applied to 24 cm ph 47 ipg strips ( ge healthcare ) and focused for 60 kvh using the following protocol : 12 h rehydration at 30 v ; 1 h step and hold at 500 v ; 7 hour gradient to 1,000 v ; step and hold at 1,000 v for 1 hour ; gradient to 8,000 v for 3 h ; step and hold at 8,000 v until 60 kvh .
strips were then reduced and equilibrated using 10 mg / ml dtt ( 15 min ) followed by 25 mg / ml iodoacetamide ( 15 min ) .
the ipg strips were overlaid on 24 26 cm 816% gradient tris - hcl ph 8.8 precast gels ( nextgen sciences , ann arbor ,
approximately 1 ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip .
the gels were then run 1 - 2 w per gel overnight ( ~2224 hours ) at 20c until the marker dye ran off the gel .
gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 ( ge healthcare ) bva batch processor tool .
images were uploaded to decyder ( version 6.5 ) and spurious image objects ( water spots , streaks , and mismatches ) were identified and excluded from further analysis .
following allocation of changed proteins , individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height , area , and slope .
pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions ( p < 0.05 ) following digital image analysis using decyder , and the corresponding spots were automatically picked using an ettan spot picker ( ge healthcare ) with ettan spot pick software v.1.1
. spots were delivered in 100 mm ammonium bicarbonate to a 96-well plate and digested using established protocols .
briefly , gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac ( savant ) drying .
gel pieces were rehydrated in 40 l of 10 ng/l trypsin in 100 mm nh4hco3 on ice for 45 minutes .
the supernatant was removed and replaced with 100 mm nh4hco3 and digested at 37c overnight .
peptides were then extracted using 50% acetonitrile/0.1% tfa ( trifluoroacetic acid ) for 30 minutes at 37c .
the peptides were microextracted using omix c18 tips ( varian , palo alto , ca , usa ) following the manufacturer 's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10 mg / ml 2,5-dihydroxybenzoic acid
. mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms ( applied biosystems , foster city , ca , usa ) in positive reflector mode with delayed extraction using the following parameters : laser energy , 1400 arbitrary units ; mass range , 5005,000 da ; 120 nsec delay , 100 laser shots per spectrum .
external calibration was conducted using a standard peptide mixture ( bradykinin , insulin b chain , p14r , and acth ) , and internal calibration was carried out using trypsin autolysis peaks .
data were processed in data explorer v1.1 ( applied biosystems , foster city , ca , usa ) using noise reduction ( 2 standard deviations ) and peak deisotoping .
peak masses were searched using the mascot online search engine ( http://www.matrixscience.com/ ) with the following settings : database , ncbi entire database ( 5.6 million entries ) ; no missed cleavages ; monoisotopic peaks ; no fixed modifications ; variable modification of methionine oxidation ; error tolerance of 150 ppm .
database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . where ambiguous names were encountered , blastp searches were used to identify homologous proteins from orthologous species .
image analysis of 24 cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots .
differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates ( figure 1 ) . of the 937 identified spots ,
595 were matched between all the gels used to statistically compare the quantitative abundance of proteins .
statistical analysis compared triplicate biological observations for each condition , normalized to the internal pooled standard ( figure 2 ) .
crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that , of all proteins observed , 22 proteins were modulated in response to changes in culture conditions .
these candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows : 16 showed an apparent increase and 6 an apparent decrease ( figure 2 ) .
these proteins , along with 22 proteins selected due to their high abundance in both growth conditions , were further analyzed and identified using mass spectrometry ( figure 3 ) .
a mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins ( 52% ) .
all protein identifications corresponded to the genome of s. wittichii strain rw1 . among the 16 proteins upregulated during growth on dibenzofuran ,
7 were successfully identified ( table 1 ) . among the 6 proteins downregulated during growth on dibenzofuran ,
an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions ( table 3 ) .
of the 7 identified proteins increased during growth on dibenzofuran , 2 were directly related to the dibenzofuran degradation pathway ( figure 4 ) ; the others were involved in downstream metabolic processes ( catechol 1,2-dioxygenase , adenosylhomocysteinase ) , cell growth ( elongation factor ts ) , and cell protection ( cold shock dna - binding domain protein , alkyl hydroperoxide reductase ) .
the three identified proteins whose expression was decreased ( fumarylacetoacetate hydrolase , tonb - dependent receptor , and acyl - coa dehydrogenase ) are involved in biosynthesis , catabolism , and transport .
the unchanged proteins represented basic cell functions , although biosynthesis , catabolism , and transport proteins dominated the identities .
the alpha subunit of the dioxin dioxygenase , the first step in the dioxin degradation pathway , was also identified as unchanged .
dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [ 2830 ] .
the complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . using simple extraction and purification techniques followed by dige
, over 500 protein spots were resolved on a large ( 24 cm ) 2-dimensional gel and matched between the three biological replicates , representing approximately 10% of the entire protein content .
of these 500 proteins , 22 were found to be regulated in response to growth condition changes .
of the genes they identified as being changed in their experiments , only two overlapped with the proteins identified in our study : swit_3144 ( tonb - dependent receptor , downregulated ) and swit_3376 ( chaperonin groel , upregulated ) .
both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . in our study
, we also identified a number of proteins that are related to dioxin / dibenzofuran degradation ( e.g. , dioxin dioxygenase , meta - cleavage product hydrolase , and 2,3-dihydroxybiphenyl 1,2-dioxygenase ) .
other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . the increase in the presence of antioxidants such as alkyl
hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran , perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation .
increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress .
however , proteins of the cold shock family and related ones are also known to have transport and protein processing roles . among the proteins in the dioxin degradation pathway , the most prominent on the gel was the meta - cleavage product hydrolase .
this identification was produced from two adjacent spots , likely representing an artifact due to the protein 's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme .
the one identified in the present study is the product of the swit_3055 locus , a gene also known as dxnb2 .
the glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus .
again , there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids .
the kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism .
the increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo .
the high degree of redundancy in the dioxin and dibenzofuran degradation pathways , that is , the presence of multiple ring - hydroxylating alpha and beta subunits , glyoxalase / bleomycin resistance protein / dioxygenases , and meta - cleavage product hydrolases , remains to be explained .
one possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans .
further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways .
although not directly implicated in dioxin degradation , the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein ( swit_5089 ) flanks the ferredoxin fdx1 ( swit_5088 ) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . of the multiple isoforms of this enzyme ,
the electron supply chain also contains two isofunctional reductases . neither the ferredoxin itself nor the reductases could be identified . in previous studies ,
the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase , so gel - based methods may not be sensitive enough to detect this protein .
if transcription of the ferredoxin is linked to the other genes at that locus , as is predicted , the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo .
the detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation .
when utilizing s. wittichii rw1 as a bioremediation agent , it may be possible to induce the expression of the dioxin degradation pathway using acetate .
induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium , and growth in a complex environmental medium ( landfill leachate ) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit .
previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity or viable cells [ 38 , 39 ] , respectively .
the addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 .
proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons .
the relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms .
furthermore , microbes are easily controlled and manipulated in the laboratory , both during growth and gene expression .
these factors will continue to drive biomarker discovery in microbial proteomes , including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments .
furthermore , the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation .
this study highlights a number of proteins that were changed in response to dibenzofuran exposure , opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins , and suggests ways to enhance the biodegradation of dioxins . | sphingomonas wittichii rw1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins , which represent priority pollutants in the usa and worldwide . although its genome has been fully sequenced , many questions exist regarding changes in protein expression of s. wittichii rw1 in response to dioxin metabolism .
we used difference gel electrophoresis ( dige ) and matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ) to identify proteomic changes induced by growth on dibenzofuran , a surrogate for dioxin , as compared to acetate .
approximately 10% of the entire putative proteome of rw1 could be observed .
several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated , thereby highlighting the utility of using proteomic analyses for studying bioremediation agents .
this is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy . |
among dermatological disorders , erythematous dermatitis , or eczema , and psoriasis are the most common.1 dermatitis is defined as a superficial skin inflammation , characterized by poorly delimited erythema , and usually itchy crusts and scales.2 conventional measures to prevent eczema include avoidance of irritants and potential allergens , as well as continued hydration of the skin with thick emollients .
other therapies including phototherapy , antimicrobials , antihistamines , and systemic immunosuppressives are also therapeutic options.1 psoriasis is an autoimmune , chronic , noncontagious , recurrent , inflammatory skin disorder . in western populations ,
its prevalence is estimated to be as high as 2.8%.3 the typical skin lesions of psoriatic patients are sharply demarcated erythematous plaques covered with silvery or opalescent scales.4 the inflammatory response is supported by the infiltration of polymorphic neutrophils in the skin .
neutrophils are triggered by the chemokines and lymphokines released by keratinocytes and t - lymphocytes , respectively , and , in turn , trigger the activity of lymphocytes and keratinocytes , thus generating a vicious cycle limited to acute lesions but linked in any case to the extended inflammation.5 treatments for psoriasis include immunosuppressive drugs such as methotrexate , cyclosporine , and fumaric acid esters.6 however , advances in mechanistic understanding of signaling pathways involved in the pathogenesis of psoriasis have led to the testing of biological therapies .
these include immune suppressive drugs ( eg , alefacept ) and anticytokine therapies ( antitumor necrosis factor [ tnf ] therapies [ adalimumab , etanercept , infliximab , ustekinumab]).7 topical application of anti - inflammatory molecules is a feasible alternative to systemic approaches to treat psoriatic and eczematous symptoms by directly acting on inflammatory processes and generating a marked soothing response in skin.8 a recent study on costs of psoriasis medications has demonstrated that biological treatments represent 67% of total medication costs , and only 5% of patients purchase them .
in contrast , topical medications are the most commonly purchased treatments and account for the 18% of total costs.9 furthermore , since almost 10% of patients require at least three topical therapy switches over a 1-year period to gain health benefit , the identification and formulation of novel anti - inflammatory compounds that could be used in topical application are a medical need for psoriasis treatment and a possible approach to reduce costs.9 boswellia serrata gum resin extracts ( bses ) ( or olibanum ) are used in traditional ayurvedic medicine to treat inflammatory diseases .
the prevalent component of lipophilic fraction of olibanum is represented by the pentacyclic triterpenes boswellic acids ( bas ) , several forms of which have been characterized by analytical techniques , including - and -configured bas ( figure 1 ) .
another structural variety is given by the presence of a carbonyl group and an acetyl group in 11-keto bas ( kba ) and 3-o - acetyl - bas ( akba ) , respectively.10 animal studies and pilot clinical trials support the efficacy of bses for the treatment of a variety of inflammatory diseases such as inflammatory bowel disease , rheumatoid arthritis , osteoarthritis , and asthma.1114 in 2002 , the european medicines agency classified bse as an orphan drug for the treatment of peritumoral brain edema.15 the main anti - inflammatory properties of bas are attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase ( 5-lo ) by kba and akba.15 the mechanistic model has to be fully elucidated , but there is some evidence of an indirect action of bas leading to an irreversible inhibition of 5-lo by a ca2 + -mediated pathway.16 bas also inhibit the human leukocyte elastase , released in inflammation- and hypersensitivity - based human disorders , thus further supporting ba s antiphlogistic properties.17 in activated human monocytes , akba conveys inhibition of nuclear factor ( nf)-kb and subsequent downregulation of tnf- expression , via its direct inhibition of i kappa b kinase ( ikk).18 noteworthy is that when the ikk inhibition by bas has been investigated in the cd18 hypomorphic ( cd18[hypo ] ) mouse model of psoriasis , the suppressive effects on nf - kb signaling have been confirmed , thus suggesting that targeting nf - kb with bas may be an effective strategy for the treatment of psoriasis.19 since both psoriasis and erythematous dermatitis present a prevalent inflammatory component and bas have a documented inhibitory activity in some pathways with a major role in inflammation , especially in nf - kb pathway , we developed a formulation containing bas with potential efficacy in patients with psoriasis and erythematous dermatitis . in this double - blind study ,
we compare , for the first time , a novel ba formulation ( containing bosexil , inci : lecithin , bse ) with a placebo formulation in the treatment of psoriasis or eczema .
a third arm of the trial received a formulation of vaccinium myrtillus seed oil , which was previously demonstrated as an efficient local treatment for psoriatic lesions.20
this is a randomized , placebo - controlled , double - blind study performed at the department of dermatology at the university of varese ( italy ) in collaboration with velleja research and the local health agency 1 ( asl1 ) in the piacenza ( italy ) area .
we separately considered in the study patients who were affected either by psoriasis or by erythematous dermatitis . the diagnosis of erythematous atopic eczema or psoriasis was made by the physician in charge of the department of dermatology involved in the trial .
patients had to be free from any topical or systemic anti - inflammatory treatment for at least 3 months .
patients were randomly assigned , in a 1:1:1 ratio and according to a computer - generated numerical table , to one of the following groups : bosexil , v. myrtillus seed oil , or placebo .
bosexil was formulated according to the phytosome delivery system that is aimed at improving the bioavailability and skin affinity of bioactive molecules.21 all preparations could not be distinguished without a chemical analysis .
patients were asked to apply the cream twice a day to the affected areas in a sufficient quantity to cover the whole skin area involved , for a total period of 30 days .
other concomitant pharmacological treatments were not interrupted or modified , except for those , dermatological or oral , finalized to treat psoriasis .
clinical assessments were carried out at baseline ( t0 ) and at the end of the trial ( t30 ) , and included the evaluation of scales and erythema in the case of psoriasis and the evaluation of itch and erythema in the case of eczema .
lesions and clinical manifestations were classified as absent ( score 0 ) , moderate ( score 1 ) , severe ( score 2 ) , or very severe ( score 3 ) .
efficacy was statistically assessed through a procedure based on the individual s permanence or migration state ( primary end point ) depending on the entity of the score assigned to a symptom at t30 in comparison with the corresponding baseline score ( t0 ) .
thus , the change of condition generated the following categories of patients : in remission : when individuals with scores > 0 at t0 have score = 0 at t30 ; improved : when individuals have lower scores at t30 than at t0 ; unchanged : when individuals have the same scores at t0 and t30 ; worsened : when individuals have higher scores at t30 than at t0 .
we validated this statistical procedure in a previous study on the efficacy of v. myrtillus seed oil.20 as an internal control , we compared the effects of bosexil and v. myrtillus with the same placebo . the psoriasis area severity index ( pasi ) and the eczema area and severity index ( easi )
the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure .
the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure .
each group consisted of ten patients affected from psoriasis and ten patients with erythematous dermatitis ( except the bosexil group , in which patients with dermatitis numbered nine ) . at t0 ,
all groups were similar in terms of severity of the disease . however , three patients in the placebo group , one in the v. myrtillus group , and two in the bosexil group suspended the treatment before t30 and were included with a score of 3 in the statistical analysis . no significant difference in terms of age or sex
the results from the change of condition ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,
signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .
in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .
treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,
p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively .
the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .
figure 2 shows pictures of some psoriasis patients one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo .
the results from the change of condition ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,
signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .
in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .
treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,
treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) ,
the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and
in patients with psoriasis ( table 5 ) , signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .
in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .
treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,
p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) .
in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening .
v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively .
the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .
figure 2 shows pictures of some psoriasis patients one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo .
this study aimed to compare , for the first time to our knowledge , the efficacy of topical preparation of bosexil and v. myrtillus seed oil vs a placebo in individuals with a diagnosis of psoriasis or erythematous dermatitis .
indeed , these formulations are particularly rich in polyunsatured fatty acids ( v. myrtillus seed oil ) or bas ( bosexil ) that exert anti - inflammatory effects , thus may provide benefits in skin inflammatory diseases .
bas in bosexil formulation were more active in the treatment of psoriatic scales than erythema , and were similarly efficient to treat itch and erythema in patients affected by eczema .
this formulation was designed according to the phytosome delivery system21 in order to improve the bioavailability and skin affinity of bas .
consistently with previous results,20 this study confirmed the efficacy of v. myrtillus seed oil preparation in the treatment of major symptoms in both psoriasis and eczema , such as erythema and scales and erythema and itch , respectively .
noteworthy is that v. myrtillus seed oil was associated with three remissions in the treatment of erythema in psoriatic patients .
bilberry fruits contain small seeds rich in alpha - linolenic acid and linoleic acid , which are considered essential nutrients ( the human body , in fact , lacks the necessary enzymes to introduce an unsaturation beyond position omega-9 ) .
linoleic acid and alpha - linolenic acid are involved in the skin barrier function.22 one issue in the management of chronic diseases such as psoriasis is the adherence to treatment .
few data are available in literature , but the most frequently mentioned reasons for nonadherence to topical treatment are low efficacy , time consumption , and poor cosmetic characteristics of topical agents.23 this study has tested a twice - daily administration which determined an effective improvement of disease . however , the study lasted a short period of time and included an overall limited number of patients ; therefore , long - term , larger studies should be recommended to assess the adherence to this protocol in real life .
moreover , earlier assessments ( eg , at day 15 ) could have been performed to collect information on the early efficacy of both bosexil and v. myrtillus seed oil .
last , other end points ( eg , patient - related severity score or the dermatology life quality index ) have not been evaluated in this study .
in addition to the anti - inflammatory / barrier - protecting effects of polyunsatured fatty acids or bas , a cosmeceutical formulation of these creams may provide the adjunctive benefits that can restore and protect the skin barrier function , increase the remission times between flare - ups , and enhance the pharmaceutical effects of active compounds.24
in conclusion , bas contained in bosexil and formulated according to the phytosome delivery system21 may be eligible components for the formulation of preparation with anti - inflammatory properties for the treatment of psoriatic and eczematous symptoms .
we speculate that this formulation could also be applied to other inflammatory skin disorders , including contact , atopic , seborrheic , nummular , chronic palmoplantar , and generalized exfoliative or chronic lichen simplex dermatitis . | backgroundboswellic acids ( bas ) show anti - inflammatory properties in a variety of inflammatory diseases , including rheumatoid arthritis , osteoarthritis , and asthma .
a topical administration route is currently used to deliver active compounds in psoriatic and eczematous patients . in this double - blind study
we compare a novel ba formulation ( containing bosexil , inci [ international nomenclature of cosmetic ingredients ] : lecithin , boswellia serrata resin extract ) with a placebo formulation .
a third arm of the trial received a formulation of vaccinium myrtillus seed oil , previously demonstrated as an effective local treatment for psoriatic lesions.methodspatients with psoriasis or erythematous eczema were randomly assigned , in a 1:1:1 ratio , to bosexil , v. myrtillus seed oil , or placebo . in order to evaluate the effects of treatment , the changes of scales and erythema from diagnosis to the end of treatment
were scored in psoriatic patients , while changes in itch and erythema were analyzed for erythematous eczema patients .
psoriasis area severity index and eczema area and severity index scores were also calculated.resultsin patients with psoriasis , scales and erythema improved both with bosexil and the v. myrtillus seed oil treatment in comparison with placebo . in particular , the treatment with bosexil formulation improved scales ( 70% of cases ) and erythema ( 50% of cases ) without any case of worsening . in patients with eczema , the administration of placebo did not result in any improvement in 90% of cases , and in the remaining 10% worsened both itch and erythema .
bosexil formulation improved both itch ( 60% of cases ) and erythema ( 60% of cases ) without any case of worsening .
v. myrtillus seed oil improved itch and erythema in 66.7% and 77.8% of patients , respectively.conclusiona topical formulation of bosexil may be promising for the treatment of psoriasis and erythematous eczema .
long - term studies are recommended to evaluate the adherence to this topical treatment and its clinical benefits in real life . |
cell lines , cultures and transfections : a madin - darby bovine kidney cell
line ( mdbk ) ( hsrrb , osaka , japan ) was cultured in dulbecco s modified eagle s medium ( dmem )
with 10% fetal calf serum ( fcs ) . a human cervical carcinoma cell line ( hela )
( riken cell
bank , tsukuba , japan ) was cultured as described in previous studies [ 10 , 14 ] .
a cattle xlf gene
( nm_001075393.1 ) with an artificial ecori site at the 5 end and bamhi site at the 3 end
was synthesized .
the fragment was confirmed by sequencing and ligated to the ecori and bamhi
sites of the peyfp - c1 vector to give the in - frame fusion gene .
peyfp - cattle xlf ,
peyfp - cattle xlf ( 162299 ) , peyfp - cattle xlf ( 162287 ) or peyfp - c1 was transient transfected
in cells using fugene hd ( promega , madison , wi , u.s.a . )
the cells were cultured for 2 days and then monitored under an fv300 confocal
laser scanning microscope ( olympus , tokyo , japan ) as previously described [ 9 , 11 , 12 ] .
immunoblotting : the extraction of total lysates and western blot analysis
were conducted based on the previous methods [ 11 ,
13 ] .
the membranes
were blocked in blocking one ( nacalai tesque , kyoto , japan ) for 30 min .
the following
antibodies were used : a rabbit anti - xlf polyclonal antibody ( a300 - 730a ) ( bethyl
laboratories , montgomery , tx , u.s.a . ) , a rabbit anti - gfp polyclonal antibody ( fl ) ( santa
cruz biotechnology , santa cruz , ca , u.s.a . ) , a mouse anti - ku70 monoclonal antibody ( n3h10 )
( neomarkers , fremont , ca , u.s.a . ) or a mouse anti--actin monoclonal antibody ( sigma , st .
three antibodies ( i.e. , anti - xlf antibody , anti - ku70 antibody and
anti - gfp antibody ) were diluted in signal enhancer hikari ( nacalai tesque ) , respectively .
the binding to each protein was visualized using a select western blotting detection system
( ge healthcare bio - sci .
piscataway , nj , u.s.a . ) , in accordance with the manufacturer s
instructions .
immunofluorescence staining : immunofluorescence staining was conducted as
previously described [ 9 , 11 ] .
briefly , the fixed cells were blocked for 10 min using a blocking
solution and then incubated for 30 min at room temperature with a mouse anti-h2ax
monoclonal antibody ( jbw301 ) ( upstate biotechnology inc . , charlottesville , va , u.s.a . ) or a
rabbit anti - xlf polyclonal antibody ( x4754 ) ( sigma ) . after washing with pbs , detection of
each protein
was performed using alexa fluor 568-conjugated secondary antibodies ( molecular
probes , eugene , or , u.s.a . ) .
local dna damage induction using laser and cell imaging : local dna damage
induction using laser and cell imaging was conducted as described previously [ 9 , 11,12,13 ] .
briefly , a
530% power scan ( for 1 sec ) from a 405 nm laser was used to induce local dsbs .
images of
living cells or fixed cells expressing eyfp - tagged cattle proteins or eyfp alone were
obtained using an fv300 confocal scanning laser microscopy system ( olympus ) .
expression and localization of cattle xlf in cattle cells : we examined the
expression and subcellular localization of xlf in cattle cells .
first , we examined the
expression of xlf and ku70 in the cattle cell line mdbk and the human cell line hela by
western blot analysis using the anti - xlf antibody and anti - ku70 antibody .
( a ) total cell lysates from each
cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed
by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an
anti--actin antibody .
( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells
during the cell cycle .
the images shown
are a representative example for interphase cells or mitotic phase cells ( c ) .
these results demonstrate that the core nhej factors , xlf
and ku70 , are expressed in cattle cells .
total cell lysates from each
cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed
by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an
anti--actin antibody .
( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells
during the cell cycle .
the images shown
are a representative example for interphase cells or mitotic phase cells ( c ) . to elucidate the localization of xlf in cattle cells
, we studied the distribution of xlf by
confocal laser microscopy ( fig . 1b and 1c ) .
indirect immunofluorescence staining using the anti - xlf antibody showed that fluorescence
was detected in the nucleoplasm of mdbk cells during the interphase . on the other hand ,
the
fluorescence was detected throughout the cytoplasm of mdbk cells during the mitotic phase ,
but not in the condensed chromosomes of the mitotic cells .
these observations indicate that
the localization of cattle xlf changes dynamically during the cell cycle .
to clarify the
localization of xlf in living cattle cells during the interphase , we examined the expression
and localization of eyfp - cattle xlf in mdbk cells .
the expression vector peyfp - c1 containing cattle xlf
( peyfp - cattle xlf ) was transfected into mdbk cells ( fig .
( a ) schematics of eyfp - cattle
xlf chimeric protein and control protein ( eyfp ) .
( b ) extracts from cattle ( mdbk ) cells
transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western
blotting using the anti - xlf , anti - gfp or anti--actin antibody .
living mdbk cells transiently expressing
eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .
eyfp images for
the same cells are shown alone ( left panel ) or merged ( right panel ) with differential
interference contrast images ( dic ) ( center panel ) . ) .
2b , a signal of
eyfp - cattle xlf was detected in the transfectants by western blot analysis using the
anti - xlf antibody and anti - gfp antibody . by confocal laser microscopy
, we clarified that
eyfp - cattle xlf was localized in the nuclei of living interphase cells in eyfp - cattle xlf
transfectants ( fig . 2c ) .
expectedly , in eyfp
transfectants , we confirmed that eyfp was distributed throughout the cell excluding the
nucleolus ( fig . 2c ) .
( a ) schematics of eyfp - cattle
xlf chimeric protein and control protein ( eyfp ) .
( b ) extracts from cattle ( mdbk ) cells
transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western
blotting using the anti - xlf , anti - gfp or anti--actin antibody .
living mdbk cells transiently expressing
eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .
eyfp images for
the same cells are shown alone ( left panel ) or merged ( right panel ) with differential
interference contrast images ( dic ) ( center panel ) .
eyfp - cattle xlf accumulates quickly at dsbs induced by laser
microirradiation : we examined whether eyfp - cattle xlf accumulates quickly at the
405 nm laser - induced dsb sites ( fig . 3afig .
( a )
the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser
irradiation were examined .
( b ) imaging of living eyfp - cattle xlf - transfected mdbk
cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left
panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) .
( c ) immunostaining of microirradiated
eyfp - cattle xlf - transfected cells with anti-h2ax antibody .
the cells were fixed and
stained with the anti- h2ax antibody at 5 min postirradiation . left panel ,
eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .
( d )
time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after
irradiation .
upper panel , eyfp - cattle xlf ; lower panel , differential interference
contrast images ( dic ) . ) . as shown in fig .
3b , we observed that
eyfp - cattle xlf accumulated at the microirradiated sites in the living cattle cells .
next ,
we investigated whether cattle xlf accumulated at 405 nm laser - induced dsb sites by
immunostaining with an antibody that detects h2ax . as shown in fig .
3c , eyfp - cattle xlf colocalized with the dsb marker h2ax at
microirradiated sites in mdbk cells .
next , we carried out time - lapse imaging of eyfp - cattle
xlf - transfected mdbk cells .
3d ,
we observed eyfp - cattle xlf accumulation at the microirradiated sites 5 sec after
irradiation . in eyfp - cattle xlf - transfected cells ,
these results reveal that after irradiation ,
eyfp - cattle xlf quickly accumulates and formes foci at laser - induced dsbs in living
cells .
( a )
the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser
irradiation were examined .
( b ) imaging of living eyfp - cattle xlf - transfected mdbk
cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left
panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) .
( c ) immunostaining of microirradiated
eyfp - cattle xlf - transfected cells with anti-h2ax antibody .
the cells were fixed and
stained with the anti- h2ax antibody at 5 min postirradiation . left panel ,
eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .
( d )
time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after
irradiation .
upper panel , eyfp - cattle xlf ; lower panel , differential interference
contrast images ( dic ) .
the c - terminal region ( ctr ) of cattle xlf is essential for the nuclear localization
and recruitment of xlf to dsbs in cattle cells : to determine the region essential
for nuclear localization of cattle xlf , we investigated the localization of cattle xlf and
its mutant .
firstly , the peyfp - cattle xlf and its mutants were transfected into mdbk cells .
as shown in fig .
4.the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of
cattle xlf to dsbs in vivo .
( a ) extracts from cattle ( mdbk ) cells
transiently expressing the indicated cattle xlf deletions were prepared and subjected
to western blotting using the anti - gfp or anti--actin antibody .
( b , c ) identification
of essential domain of cattle xlf for nuclear localization and for accumulation at
dsbs .
the localization
and accumulation of the chimeric proteins at laser - induced dsbs were investigated via
live cell imaging .
the results are summarized on the right : cellular localization ( n ,
nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated
sites ; - , not accumulated at microirradiated sites ) .
( d ) immunostaining of microirradiated eyfp - cattle xlf
( 162287)-transfected cells with anti-h2ax antibody .
the cells were fixed and stained
with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf
( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .
( e ) identity between
the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino
acids 288299 ) .
( f ) the alignment of the primary sequence among homologous xlf
proteins . for comparison , the basic ( red ) or non - basic residues ( black )
, a signal of each eyfp - cattle xlf mutant was detected in the extracts of each
transfectant by western blot analysis using the anti - gfp antibody . by confocal laser
microscopy
, we confirmed that eyfp - cattle xlf was localized in the nuclei of living
interphase cells .
we observed that n - terminal deletion mutant eyfp - cattle xlf ( 162299 )
localized predominantly in the nuclei , whereas eyfp - cattle xlf ( 162287 ) as well as eyfp was
distributed throughout the cell excluding the nucleolus in mdbk cells ( figs . 2c , 4b and 4c ) .
these results indicate that 12 c - terminal amino
acids ( amino acids 288299 ) of cattle xlf are vital for the nuclear localization of xlf in
cattle cells ( fig .
which region
of cattle xlf is essential for its accumulation at dsbs in vivo , we tested
whether the xlf mutant proteins could be recruited to dsbs induced by microirradiation .
we
observed that a n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) as well as the wild
type eyfp - cattle xlf , accumulated at the dsbs sites in the living mdbk cells ( fig . 3c and
data not shown ) . on the other hand , the mutant protein eyfp - cattle xlf ( 162287 ) failed to
accumulate at the dsbs sites , which was detected by the dsb marker h2ax ( fig . 4c and 4d ) , indicating that deletion of the
c - terminal end 12 amino acids abolished the recruitment of cattle xlf to dsbs .
the cattle
and human xlf proteins are 81.9% identical at the amino acid level , whereas the c - terminal
domain ( amino acids 220299 ) of cattle xlf retains only 56.4% identity to human ( fig .
interestingly , we confirmed that the basic
amino acids in the ctr of xlf are evolutionarily highly conserved among humans and domestic
animal species , e.g. , cattle , goats , horses and avian , but not in yeast ( fig .
4f and data not shown ) , which strongly suggests
the biological significance of the xlf ctr in domestic animals .
the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of
cattle xlf to dsbs in vivo . ( a )
extracts from cattle ( mdbk ) cells
transiently expressing the indicated cattle xlf deletions were prepared and subjected
to western blotting using the anti - gfp or anti--actin antibody .
( b , c ) identification
of essential domain of cattle xlf for nuclear localization and for accumulation at
dsbs .
the localization
and accumulation of the chimeric proteins at laser - induced dsbs were investigated via
live cell imaging .
the results are summarized on the right : cellular localization ( n ,
nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated
sites ; - , not accumulated at microirradiated sites ) .
( d ) immunostaining of microirradiated eyfp - cattle xlf
( 162287)-transfected cells with anti-h2ax antibody .
the cells were fixed and stained
with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf
( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .
( e ) identity between
the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino
acids 288299 ) .
( f ) the alignment of the primary sequence among homologous xlf
proteins . for comparison , the basic ( red ) or non - basic residues ( black )
to develop next - generation chemotherapeutics for cancer and other disease is important to
clarify the molecular mechanisms of c - nhej .
human xlf is the most recently identified core
nhej factor , and it appears to play essential roles in c - nhej .
expectedly , xlf - deficient cells derived from human patients and from
knockout mice show ionizing radiation sensitivity [ 3 ,
15 ] . in addition
, sirna - mediated downregulation of
xlf in human cell lines leads to radiosensitivity and impaired nhej . .
homologues of the xlf gene were predicted in several eukaryotic
organisms . on the other hand , the expression ,
function and regulation mechanism of xlf
have not been elucidated in animal species other
than mice and humans [ 1 , 3 , 11 , 16 , 21 ] .
domestic cattle are
important domestic animals as livestock and draft animals in not only japan , but also many
countries .
recently , cattle have been an ideal animal model for assessing chronic radiation
exposure [ 5 , 20 ] . however , the molecular mechanism of c - nhej is still unknown in cattle cells . in
this study
, we examined the expression and subcellular localization of cattle xlf and its
mutants in cattle cell line mdbk .
we found that xlf as well as other core nhej protein ku70
is expressed in cattle cells , and the localization of cattle xlf changes dynamically during
the cell cycle .
in addition , xlf might play a vital role in the repair of dsb immediately
after microirradiation of cattle cells .
moreover , our data showed that the ctr of cattle xlf
is vital for the nuclear localization of xlf and for the accumulation of xlf at dsbs
in vivo .
these findings suggest that the mechanisms regulating of the
localization and recruitment to dsbs play a key role in the function of cattle xlf .
cattle xlf ( nm_001075393.1 ) as well as goat xlf ( xp_005676612.1 ) and sheep xlf
( xp_004004987.1 ) is a 299-amino acid protein .
the cattle and goat xlf genes are 94.6%
identical at the amino acid level .
in addition , the cattle and sheep xlf genes are 94.3%
identical at the amino acid level . on the other hand ,
human xlf is a 299-amino acid protein , which contains an n - terminal head
domain ( amino acids 1141 ) , a coiled - coil central domain ( amino acids 142230 ) and a non
structured c - terminal domain ( amino acids 231299 ) [ 1 ,
2 , 17 ] .
comparison with other eukaryotic homologues shows a high degree of sequence similarity
within the 220 n - terminal amino acids , while the c - terminal domain ( amino acids 225299 ) is
less conserved [ 1 , 2 , 17 ] . on the basis of experimental
findings
, there are some reports concerning the role of the c - terminal domain of xlf in
humans , but not in animals including cattle .
yano et al . reported that a
10-amino - acid deletion at the c - terminal end completely abolishes the ku - xlf interaction and
the accumulation of xlf at dsbs . on the other
hand , malivert et al .
have reported that the c - terminal end ( amino acids
231299 ) of human xlf is dispensable for dna repair in vivo . in this study
, our data showed that a 12-amino - acid
deletion at the c - terminal end abolishes the accumulation of cattle xlf at dsbs . altogether
,
we conclude that the xlf ctr is important for the accumulation of xlf at dsbs in both human
and cattle cells , although the role of the c - terminal region of xlf remains controversial in
human cells .
it was demonstrated that there is a general absence of conservation in the 75 c - terminal
amino acids among humans and other species , although the extreme c terminus of xlf contains
a small conserved basic cluster , which was proposed as a putative nls ( krkk ) [ 1 , 2 ] .
our data
revealed experimentally that the ctr of cattle xlf is critical for the nuclear localization
of xlf and recruitment to dsbs , whereas the n - terminal domain is not essential .
in addition ,
basic amino acids in the ctr of cattle xlf are evolutionarily conserved among ctr of
domestic animals , which suggests the common biological significance of the xlf ctr in
domestic animals .
we consider that there is only one nls ( kvkrkklr ) in cattle xlf , and the
nls is a classical monopartite nls having a single cluster of basic amino acid residues .
we
speculate that the c - terminus of 75 amino acids is important for a specific function in each
species , whereas the xlf ctr is critical for the regulation of common functions in domestic
animals .
, we showed that xlf is expressed in cattle cells and the localization of
cattle xlf changes dynamically during the cell cycle .
in addition , our data showed that the
localization and recruitment of cattle xlf to dsb sites at an early stage following
irradiation are dependent on the ctr .
these basic informations might be useful to develop
the molecular - targeting therapeutic drug taking xlf as a target molecule for human and
domestic animals .
further studies to elucidate the mechanisms regulating cattle xlf at dsbs
will lead to a better understanding of the physiological function of xlf not only in cattle
cells , but also in cells of human and other domestic animals .
inherited mutations of core
c - nhej factors ( e.g. , dna - pkcs , dna ligase iv and xlf ) , have been discovered in humans
. on the other hand , inherited
mutations of the
dna - pkcs , which cause scid , have been identified in not only humans , but also domestic
animals , i.e. , mice , horses or dogs .
therefore ,
further comparative studies might provide available information for the development of new
clinical medicines and new chemoradiotherapies for humans and domestic animals including
cattle . | clinically , many chemotherapeutics and ionizing radiation ( ir ) have been
applied for the treatment of various types of human and animal malignancies .
these
treatments kill tumor cells by causing dna double - strand breaks ( dsbs ) .
core factors of
classical nonhomologous dna - end joining ( c - nhej ) play a vital role in dsb repair .
thus , it
is indispensable to clarify the mechanisms of c - nhej in order to develop next - generation
chemotherapeutics for cancer . the xrcc4-like factor ( xlf ; also called cernunnos or nhej1 )
is the lastly identified core nhej factor .
the localization of core nhej factors might
play a critical role in regulating nhej activity .
the localization and function of xlf
have not been elucidated in animal species other than mice and humans .
domestic cattle
( bos taurus ) are the most common and vital domestic animals in many
countries . here
, we show that the localization of cattle xlf changes dynamically during
the cell cycle .
furthermore , eyfp - cattle xlf accumulates quickly at microirradiated sites
and colocalizes with the dsb marker h2ax .
moreover , nuclear localization and accumulation
of cattle xlf at dsb sites are dependent on 12 amino acids ( 288299 ) of the c - terminal
region of xlf ( xlf ctr ) .
furthermore , basic amino acids on the xlf ctr are highly
conserved among domestic animals including cattle , goat and horses , suggesting that the
ctr is essential for the function of xlf in domestic animals .
these findings might be
useful to develop the molecular - targeting therapeutic drug taking xlf as a target molecule
for human and domestic animals . |
folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .
our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .
this modification can be used for successful targeting of cancer cells expressing the folate receptor .
many conventional cancer chemotherapies are ineffective because of an inability to reach the tumor site in effective concentrations.1 there is little doubt that nanoparticles offer new opportunities in many fields.2 nanotechnology is expected to revolutionize medicine .
nanostructures can play a major role in medicine , especially in cancer diagnosis and therapy.3 magnetic nanoparticles have been investigated for various biomedical applications , nanoparticles , and prospected in diagnostic research for magnetic resonance eg , fe3o4 imaging and application of nanotechnologies in medicine.4 magnetic nanoparticles could enhance therapeutic effects and reduce side effects of drugs when used in combination with conventional cancer treatment.5 the combination of fe3o4 magnetic nanoparticles with different chemotherapeutics may provide new strategies in the treatment of specific cancer cells.6 moreover , fe3o4 nanoparticles are the only magnetic nanomaterials approved for clinical use by the us food and drug administration , and the preparation method is relatively simple.7 we aimed to determine whether the anticancer effects of methacrylamido - folic acid ( ma - fol ) would have improved anticancer activity if incorporated into magnetic nanoparticles .
we demonstrated that magnetic fe3o4 nanoparticles coupled with folic acid can inhibit tumor proliferation and induce apoptosis of cancer cells in a dose- and time - dependent manner .
the folate receptor is significantly overexpressed on the surface of human cancer cells.8,9 folate receptor - mediated drug delivery is based on conjugation with folic acid , which is internalized by folate receptor - mediated endocytosis .
folic acid has been immobilized on superparamagnetic particles,10 polymer nanoparticles,11 and incorporated into dendrimer - based therapeutic nanodevices12 for selective targeting of tumor cells .
folate receptors exhibit limited expression on healthy cells , but are often present in large numbers on cancer cells.13 folic acid receptors are overexpressed by epithelial cancers in the ovary , mammary gland , colon , lung , prostate , nose , throat , and brain,14 so represent an important target for tumor - specific delivery of anticancer drugs .
apoptosis , or programmed cell death , is an active process characterized by cytoplasmic shrinkage , chromatin condensation , nuclear fragmentation , and activation of caspases.13 in addition , phosphatidylserine is exposed on the external surface of the cell in the early phase of apoptosis , and this exposure precedes membrane damage and dna fragmentation.15 on the other hand , necrosis is passive , and is characterized by cell swelling , rupture of the plasma membrane , and cell lysis , with leakage of cytoplasmic components , such as lactate dehydrogenase.13 in the present study , folic acid was coupled on the surface of fe3o4 for selective binding to cancer cells and immobilized on the surfaces of magnetic nanoparticles , to disperse particles and improve their cell internalization and target cancer cells , respectively .
further , the apoptotic effects of ma - fol - modified fe3o4 nanoparticles were determined in a 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and in a nih/3t3 control cell line ( normal mouse embryonic fibroblasts ) by flow cytometry and transmission electron microscopy ( tem ) .
nanoparticles are generally internalized into cells via fluid - phase endocytosis,16,17 receptor - mediated endocytosis , or phagocytosis .
one strategy to realize efficient and specific cellular uptake of nanoparticles is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis.18 the objective of this research was to assess the potential effects of fe3o4 magnetic nanoparticles modified with ma - fol on 5rp7 cancer cells and nih/3t3 cells .
ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .
a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .
completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .
reaction mixture was extracted with ethyl acetate ( 3 20 ml ) to remove 1h - benzotriazole .
water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .
h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 ) was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .
c nmr ( 125 mhz , dmso - d6 ) : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .
superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .
the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .
these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .
5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .
cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .
, the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .
the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .
cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem ,
ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .
the cells were cultured and then reseeded with the nano - particle - dispersed culture media . after 24 and 48 hours of incubation at 37c
, the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,
the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .
annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .
it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells . a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .
cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml , after which 100 l of solution ( 1 10 cells ) was transferred to a 5 ml culture tube .
annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .
uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .
cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,
the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .
they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .
ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .
a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .
completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .
reaction mixture was extracted with ethyl acetate ( 3 20 ml ) to remove 1h - benzotriazole .
water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .
h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 ) was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .
c nmr ( 125 mhz , dmso - d6 ) : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .
superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .
the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .
these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .
5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .
cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .
, the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .
the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .
cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem
, ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .
the cells were cultured and then reseeded with the nano - particle - dispersed culture media .
after 24 and 48 hours of incubation at 37c , the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,
the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .
annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .
it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells
. a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .
cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml , after which 100 l of solution ( 1 10 cells ) was transferred to a 5 ml culture tube .
annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .
uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .
cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,
the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .
they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .
a logical method to promote internalization of nanoparticles is to modify their surface with a ligand such as folic acid which can be efficiently taken up by cells through receptor - mediated endocytosis.23 folic acid binds to the folate receptors at cell surfaces with very high affinity and is internalized by receptor - mediated endocytosis.24,26 more importantly , folic acid is stable , poorly immunogenic , and has the ability to target cancer cells preferentially because the folate receptor is frequently overexpressed on the surface of cancer cells .
flow cytometry is a process whereby physical or biochemical parameters of single biological cells or particles are measured as the cells move through a fluidic channel.27 the annexin v binding assay provides a very specific , rapid , and reliable technique to detect apoptosis by flow cytometry or by fluorescence microscopy.28,29 fitc is a very useful fluorescent moiety that can be used to label essentially any protein .
fitc - conjugated annexin v is used to detect apoptotic cells in a diverse range of cell types and in response to many different proapoptotic stimuli.28,29 iron nanoparticles were successfully modified with ma - fol as described in the methods section .
ma - fol was used to target preferentially cancer cells with folate receptors expressed on their surfaces , and to facilitate the nanoparticles to transit across the cell membrane .
annexin v - fitc allows detection of cell surface changes that occur early during apoptosis , because annexin v binds to phosphatidylserine which becomes exposed on the outer surface of the plasma membranes by flipping from the inner side of the membrane.22,30,31 exposure of phosphatidylserine during apoptosis precedes nuclear changes .
this change in membrane surface is important for macrophages to recognize cells undergoing apoptosis.30,32 annexin v , an anticoagulant protein , binds to phosphatidylserine with high affinity .
thus , by staining cells with annexin v - fitc and additionally with dna - specific fluorochrome , eg , propidium iodide , it is possible to identify live cells , early apoptotic cells , as well as late apoptotic and necrotic cells , by flow cytometry.22,33 subsequently , annexin v - fitc binds to cells expressing phosphatidylserine , an early marker of apoptosis on the cell surface .
target cells are gated upon as propidium iodide - negative and quantified with respect to their annexin v positivity .
the shift from annexin v to annexin v is a discrete event , such that all target cells fall within discernible populations with respect to annexin v. dead or dying cells are then stained with annexin v - fitc.34 annexin v has been used to detect apoptotic cells in a wide variety of cell types .
externalization of phosphatidylserine has been demonstrated in plasma membrane permeability changes , as measured by propidium iodide uptake and annexin v binding , and precedes the morphological features of apoptosis as assessed by flow cytometric analysis of cell shrinkage.29,34 the sensitivity and early kinetics of annexin v binding make it an ideal marker for cell death in a flow cytometric assay.34 analysis of annexin v / propidium iodide - stained cells by flow cytometry allows quantitation of the fraction of cells that are annexin v - negative and propidium iodide - negative ( double negative ) , annexin v - positive and propidium iodide - negative ( single positive ) , or annexin v - positive and propidium iodide - positive ( double positive).28 a number of detectors are aimed at the point where the stream passes through the light beam , ie , one in line with the light beam ( forward scatter ) and several perpendicular to it ( side scatter ) and one or more fluorescent detectors . forward
scatter correlates with the cell volume , and side scatter depends on the inner complexity of the particle ( ie , shape of the nucleus , amount and type of cytoplasmic granules , or membrane roughness).35 data analysis was performed with diva software ( bd facs diva software 6.0 ; bd corporation , bedford , ma ) .
apoptosis was quantitatively confirmed by analyzing the percentage of early apoptotic cells using annexin v - fitc / propidium iodide double staining .
a marker of early apoptosis , measured by annexin - v , is phosphatidylserine , which is released as a result of redistribution of the plasma membrane of the cells.22 three main populations of cells were distributed in dot - plots for viable cells q3 ( annexin v - negative / propidium iodide - negative ) , early apoptotic cells q4 ( annexin v - positive / propidium iodide - negative ) , and late apoptotic and necrotic cells q2
the cytotoxicity of folic acid - modified nanoparticles was determined by exposing normal cells and cancer cells to various concentrations in dulbecco s modified eagle s medium for 24 and 48 hours .
the results and respective percentages of cells in apoptotic regions for both nih/3t3 and 5rp7 cells are given in figures 1 and 2 .
the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol - modified nanoparticles was 4.5 g / ml or below in cancer cells .
as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .
these results confirm that the cells are saturated with ma - fol - modified nanoparticles at a concentration of 4.5 g / ml . up to this concentration
figure 2 shows cell death due to the ma - fol - modified magnetic nanoparticles at 4.5 g / ml for 24 hours in cancer cells .
when cells were exposed to a nanoparticle concentration of 4.5 g / ml for 24 hours , cell survival decreased to 83% . when incubation time was extended to 48 hours
as shown in figure 2 , folic acid - modified nanoparticles caused worse damage to cancer cells than to normal cells .
the population of annexin v - positive live cells increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours ( figure 2 ) .
when cancer cells were incubated with modified magnetic ma - fol nanoparticles for 48 hours ( 9 g / ml ) , the population of annexin v - positive live cells was found to be 7.3% ( figures 2a and 2b ) .
cells in the late stages of apoptosis are located in the bottom right quadrant of the dot - plot as double positive annexin v - fitc- and propidium iodide - binding cells because , at this stage , the cell membranes are damaged and propidium iodide has penetrated into the cell . in the later stages of apoptosis , propidium iodide enters the cell , leading to a double positive population.28 cells undergoing necrotic cell death also stain as double positive for annexin v and propidium iodide , as discussed earlier .
flow cytometry by this double staining method enables clear detection of three populations of cells ( viable , apoptotic , and necrotic ) .
thus , the early apoptotic cells bind only to annexin - v fitc , and late apoptotic cells to both annexin v - fitc and propidium iodide , and viable cells do not take up any of the dye . the annexin v - fitc
/ propidium iodide population was considered to reflect normal healthy cells , whereas annexin v - fitc - positive / propidium iodide - negative cells were taken to show early apoptosis .
annexin v - fitc -positive / propidium iodide - negative cells were in late apoptosis or necrosis.35 our flow cytometric analysis has shown that48 hours of treatment with ma - fol - modified magnetic nanoparticles caused apoptosis in a 5rp7 cell line in a concentration - dependent manner .
ma - fol - modified magnetic nanoparticles at a dose of 4.5 g / ml showed 12.9% cells in the apoptotic zone in the case of nih/3t3 cells , and the same concentration produced apoptosis in 4.7% of cells in dot - plot analysis .
late apoptosis and necrosis increased steadily following the increase at a concentration of 4.5 g / ml . flow cytometric determination of cell cycle phase distribution has further confirmed that folic acid - modified magnetic nanoparticles cause cell cycle deformation in a cancer cell line .
the results of this study confirm that cancer cells have more folate receptors , and that a 4.5 g / ml concentration of folic acid - modified magnetic nanoparticles causes apoptosis in 5rp7 cells .
the uptake of folic acid - modified nanoparticles by cancer cells was also much higher than that of normal cells .
this indicates that folic acid modification not only facilitates the nanoparticles to target specific cells , but also increases the yield of cell internalization .
the interaction between folic acid and folate receptors expressed on the surface of cancer cells might have contributed to the improvement of nanoparticle uptake , based on receptor - mediated endocytosis.37 the schemes for the modification of nanoparticles display tem images ( figure 3 ) of ma - fol - modified with fe3o4 nanoparticles for this purpose , firstly , magnetic nanoparticles were imaged .
figure 4 shows the tem images of nih/3t3 cells not treated with ma - fol - modified magnetic nanoparticles .
figure 5 shows the uptake of folic acid - modified nanoparticles into normal cells at 24 hours and 48 hours .
the viability of normal cells after each incubation time with the nanoparticles was close to that of control cells , and was in the 95%96% range in flow cytometric analysis .
the uptake of magnetic nanoparticles by cancerous rat 5rp7 fibroblasts is shown in figure 7 .
after 48 hours of culture in media containing folic acid - modified nanoparticles , the morphology and viability of normal fibroblast cells containing the modified nanoparticles were close to that of the control cells , suggesting biocompatibility of the nanoparticles .
the tem and flow cytometry results showed that folic acid - modified nanoparticles were internalized into both normal cells and cancer cells .
the immobilization of folic acid on the nanoparticles was demonstrated by increasing the amount of nanoparticle uptake into cancer cells in comparison with normal cells .
this suggests that the modification of magnetic nanoparticles with ma - fol could be used to resist nonspecific uptake and thus avoid their recognition by macrophage cells , and simultaneously facilitate nanoparticle uptake to specific cancer cells for cancer therapy . in flow cytometry results , the population of annexin v - positive live cells in the cancer cell line increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours at a concentration of 4.5 g / ml .
the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol modified nanoparticles is 4.5 g / ml or below in cancer cells .
as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .
these results confirm that cells saturated with folic acid - modified nanoparticles are detected in the 4.5 g / ml concentration .
up to this concentration , magnetic nanoparticles were aggregated and had difficulty penetrating the cells . in tem analysis ,
the concentration of 4.5 g / ml was used to compare the cancer cells and normal cells at 24 and 48 hours . in normal cells , organelles and cell structures
were not affected by the presence of ma - fol - modified nanoparticles inside the cells ( figure 5 ) . however , cancer cells treated with the 4.5 g / ml concentration of ma - fol - modified nanoparticles underwent apoptosis .
a representative picture of intracellular nanoconjugate uptake by nih/3t3 cells during a 24-hour and 48-hour treatment is shown in figures 5 and 6 .
the amount of intracellular uptake of the nanoparticles can be seen in normal cells , whereas in the 5rp7 cell line the amount of the nanoconjugate was increased and cell modifications were damaged , figure 7 clearly illustrates the internalization of ma - fol - modified nanoparticles and their localization near the nucleus and a great number of apoptotic particles .
this picture shows clearly that nanoconjugate uptake was maximum , with 5rp7 cells having maximum receptor expression , and hence the most intense color of the pellet , whereas the uptake gradually decreased with reduction in folate receptor expression and , accordingly , the intensity of the color of the pellet in the case of nih/3t3 .
the picture confirms our hypothesis about the targeting efficacy of nanoconjugates containing folic acid , ie , that a positive correlation exists between maximum folate receptor expression and maximum uptake of nanoconjugates .
the 5rp7 cells with 90% receptor expression showed maximum uptake , and the nih/3t3 cells showed the least .
this is further substantiated by tem and flow cytometry analysis , confirming that folic acid - modified magnetic nano - particles can be used as a targeting agent .
ma - fol - modified magnetic nanoparticles represent a potential novel delivery system for compounds with anticancer activity because the folate receptor is frequently overexpressed in cancer cells .
moreover , it may increase our ability to target drugs to tumor cells , protect the drug from in vivo degradation , and reduce drug toxicity .
this modification containing folic acid can be used for successful targeting of tumor cells expressing the folate receptor .
future studies will focus on determining the stability and targeting efficacy of this modification in vivo .
this study has important implications in cancer cell imaging , tumor ablation , and drug delivery because of its targeting efficacy .
furthermore , it needs to be investigated whether nanoparticles could cause long - term changes in systemic activity .
however , the effect and usage of such combinations need to be investigated further in in vivo experiments . | background : magnetic nanoparticles show great promise for use as tools in a wide variety of biomedical applications .
the purpose of this study was to investigate the potential effects of methacrylamido - folic acid ( ma - fol)-modified magnetic nanoparticles on 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( normal mouse embryonic fibroblasts).methods : the cytotoxicity and viability of 5rp7 and nih/3t3 cells were detected .
the percentage of cells undergoing apoptosis was analyzed by flow cytometry using annexin v - fluorescein isothiocyanate staining .
nanoparticle internalization into 5rp7 and nih/3t3 cells was visualized by transmission electron microscopy.conclusion:in this study , folic acid coupled to the surface of iron oxide for selective binding to cancer cells and immobilized the surfaces of magnetic nanoparticles
. this complex improves cell internalization and targeting of cancer cells .
we detected increased apoptosis using flow cytometry and transmission electron microscopy.results:folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .
our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .
this modification can be used for successful targeting of cancer cells expressing the folate receptor . |
this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .
data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .
only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .
body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study
we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .
smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .
bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .
on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .
lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .
the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .
furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .
students who were injured more than one time were taken into account as subjects with lbp only for their first injury .
the scientific council of the university of sfax in tunisia gave written permission for the study protocol .
statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,
chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance
this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .
data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .
only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .
body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study
we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .
smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .
bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .
on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .
lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .
the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .
furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .
students who were injured more than one time were taken into account as subjects with lbp only for their first injury .
the scientific council of the university of sfax in tunisia gave written permission for the study protocol .
statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,
chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance
table 2 shows that the prevalence of lbp was significantly higher ( p<0.001 ) among females ( 17.6% ) than among males ( 12.5% ) .
lbp occurred in 17.1% of those classified by bmi as lean , 14.8% of those in the normal range , 14.3% among those who were overweight , and 12.7% of those who were obese .
in addition , 13.9% of students smoking < 20 cigarettes and 13.3% of students smoking 20 cigarettes had lbp .
prevalence of lower back pain ( lbp ) by gender , obesity and smoking lbp : low back pain ; standard body mass index ( bmi ) categories : underweight ( bmi of<18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obese ( bmi of 30 ) ; smoking : cigarettes per day ; no smoking : not currently smoking . in the analysis of risk factors for lbp in sports training , we found that in both sexes combined , lbp was positively associated with fatigue ( caused by the long time spent in training ) , gymnastics , judo , handball , and volleyball .
the increased prevalence of lbp is less in basketball and athletics and even lower in soccer , weightlifting , and swimming ( fig .
percent prevalence of lower back pain related to fatigue and type of sports in males and females combined .
figure 2 shows that there is no significant difference ( p>0.05 ) in the prevalence of lbp among males and females separately if they are compared in each practiced sport at the institute .
however , if we analyze the prevalence of lbp in terms of sex in each sport , we find that the prevalence of lbp was significantly higher among males than females in handball ( p<0.05 ) and significantly lower in athletics ( p<0.05 )
. percent prevalence of lower back pain related to fatigue and type of sports in males and females .
its incidence and prevalence are so high that it should be studied as an epidemic and social disorder ( 35 ) .
the first salient feature that emerges from this study is that lbp is widespread among students in high schools for athletes ( 14.8% ) .
the students were injured mainly in gymnastics , judo , handball , and volleyball , in addition to lbp caused by fatigue due to the long time spent in training .
these findings are generally in accordance with the literature . in a study focused on identifying the types of physical activity associated with increased occurrence of lbp in schoolchildren , skoffer and foldspang ( 6 ) noted that lbp was increased by the number of hours jogging , playing handball , and doing gymnastics .
in addition , sato et al . ( 33 ) found that the odds ratio for the risk of experiencing lbp was significantly higher in most sports items compared with the no - sports group .
the most frequently implicated sports were volleyball , athletics , judo , gymnastics , golf , and rugby , where the odds ratio exceeded 2 .
kirialanis et al . reported that injuries among gymnasts are anatomically widely distributed ( 36 ) .
as in our study , much research ( 6 , 33 , 37 ) noted that gymnastics was associated with an increased prevalence of lbp .
judo is a grappling style martial art with emphasis on dynamic throws and submissions , which can lead to substantial injuries due to the high velocity of the maneuvers ( 38 ) .
we registered a high prevalence of lbp in judo , as noted in the study of sato et al .
handball , which also includes repeated sudden and often rather violent movements , has been shown to be associated with lbp in our population and in other studies ( 6 , 33 ) .
we observed a high prevalence of lbp associated with the fatigue caused by the long time spent in physical activity ( 16.5 h per week ) .
. however , this point of view is attractive in terms of prevention as physical activity is amenable to behavioral intervention , and physical training programs at school and leisure activities may be adjusted to aim at strengthening the back ( 6 ) .
moreover , both physical inactivity and intensive sports activity have been associated with lbp in some studies ( 28 , 32 , 3941 ) .
our results show that a greater amount of physical activity increased the risk of lbp mainly in gymnastics , judo , handball , and volleyball .
the time spent in athletics and basketball was associated with lbp to a lesser extent .
moreover , it appears that soccer , weightlifting , and swimming are correlated with relatively low prevalence of lbp .
skoffer and foldspang ( 6 ) reported that of several sports activities , only swimming was associated with a decreased prevalence of lbp .
swimming activates the muscles of the trunk , for example , the erector muscles of the spine , so swimming might be a means to prevent lbp . however , our results on soccer are at odds with those of bejia et al . ( 42 ) , who reported that soccer was positively associated with chronic lbp in tunisian schoolchildren and adolescents . similarly , our findings on weightlifting contrast with those of calhoon and fry ( 43 ) , who reported that lbp was one of the most frequent injuries in this sport .
the relatively low prevalence of lbp in our study in soccer and weightlifting may be due to the system of this sports and physical education institute which is focused mainly on knowing how to teach these sports ( gestural technique and pedagogy ) and not on researching performance .
our study shows that the percentage of females ( 17.6% ) with lbp was significantly higher than that of males ( 12.5% ) , as frequently reported in the literature ( 1217 ) . however , the prevalence rates of lbp do not differ significantly between males and females ( p>0.05 ) if they are compared in each practiced sport separately .
there was no significant difference in lbp by bmi ( p>0.05 ) , in males and in females combined as reported in other studies ( 44 , 45 ) .
in addition , many other studies have reported that active smoking is a risk factor for back pain ( 47 , 48 ) .
however , we did not find a significant relationship between smoking and lbp ( p>0.05 ) .
( 49 ) was that parental smoking seemed to increase the risk of spinal pain for both genders .
we believe that students should perform exercises to strengthen the muscles of the arms and back , and increase coordination of the upper extremities and the lumbar region with regular activities that have been proven to be effective for the prevention of lbp .
swimming or gymnastics are the nearest sports to reach this aim as they seem to reduce lbp significantly ( 50 ) .
this study was a survey based on data from the institute health service files of 8 years and therefore had some limitations . to closely evaluate the relationship between lbp and sports activities ,
more information on the students is needed , including basic information such as their physical activity outside the institute . in this study , the diagnostic of lbp was performed by the doctor of the institute health service and mostly without medical imaging , which could have shown the severity of lbp .
imaging is used to show the severity of the injury rather than the severity of the pain associated with it .
lbp was strongly associated with the amount of time reserved to practice the different sports planned in this sports and physical education institute .
the sports identified as posing the greatest risk for lbp were gymnastics , judo , handball , and volleyball , followed by basketball and athletics .
imed ajili ) and the administration of the sports and physical education institute of sfax ( tunisia ) for their assistance .
| introductionfor obvious reasons , athletes are at greater risk of sustaining a lumber ( lower ) spine injury due to physical activity . to our knowledge , no previous studies have examined the prevalence of low back pain ( lbp ) in a tunisian sports and physical education institute.aimto assess the prevalence of lbp in different sports among students studying in a sports and physical education institute in tunisia , to determine the causes of the injuries , and to propose solutions.methodsa total of 3,379 boys and 2,579 girls were studied .
a retrospective cross - sectional survey was conducted on a group of students aged 18.524.5 years at the higher institute of sport and physical education of sfax to estimate the prevalence of lbp and its relation to the type of sports .
data on age , weight , height , smoking , and the sport in which the student was injured in the low back were collected from the institute health service records from 2005 until 2013.resultslbp was reported by 879 of the 5,958 study participants ( 14.8% ) .
the prevalence of lbp was significantly higher ( p<0.001 ) in females ( 17.6% ) than in males ( 12.5% ) .
lbp prevalence did not differ by body mass index or smoking habit ( p>0.05 ) .
the sports associated with the higher rates of lbp were gymnastics , judo , handball , and volleyball , followed by basketball and athletics.conclusionlbp is frequent among undergraduate students of a sports and physical education institute in tunisia .
it is strongly associated with fatigue after the long periods of training in different sports .
gymnastics , judo , handball , and volleyball were identified as high - risk sports for causing lbp . |
in modern society , health is a n important component , which requires sustained development ( 1 ) . in recent decades
, a person s health has become one of the most important human rights among social groups throughout the world ( 2 ) .
the meaning of health has been extended beyond borders , and different definitions have emerged , owing to diverse geographic regions and cultural views in societies ( 3 ) .
the definition of health may vary by cultural background , and may influence health practices and compliance to medical treatment ( 4 ) .
individuals perceive health on the basis of their experiences of ill health ( 5 ) .
so , understanding health as a subjective experience can enable health care providers to manage the care giving procedure based on their clients needs .
furthermore , one of the most important aspects with regard to health perception , manifests when patients suffer from a chronic disease , through which their perception of health is affected ( 5 , 6 ) .
diabetes is incurable , and has the third highest mortality rate on the planet ( 8) . the probability of the incidence and prevalence of this disease has increased recently ( 9 ) . in 2004 , it was estimated that by 2030 , this number will have grown to 366 million ( 10 ) and in 2010 , the number of people afflicted by diabetes was estimated to increase up to 439 million by 2030 ( 8) .
current estimates indicate that the number of diabetic patients in iran is around 3.5 million out of a total population of just over 68 million ( 11 ) .
previous studies have demonstrated that chronic illnesses such as diabetes can influence a person s perception of health ( 12 ) .
in addition , the attitudes and beliefs regarding health undoubtedly play an important role in promoting healthy behaviour ( 13 ) .
therefore , the understanding of diabetic patients perceptions can help the development of precautionary programmes for diabetics ( 14 ) .
although the concept of health is gaining more importance and it has constantly been improved and changed by the passage of time ( 15 , 16 ) , few studies are reported to have focused on health and illness , particularly with regard to diabetic patients belonging to different ethnic groups ( 17 ) .
health is a relative concept ; nonetheless , several criteria have been proposed for exploring it through various ethnic and cultural groups ( 2 , 18 ) .
prior studies have shown lack of awareness about the concept of health among iranian kurdish diabetic patients .
kurds are an ethnic group living in the middle east , particularly in iran , who are estimated to be the third largest ethnic group , constituting 9 percent of the total population ( 19 ) .
consequently , investigating the perception of health - related meaning among this ethnic group can increase the knowledge about the nature of health and its effects .
therefore , this study explored the meaning of health as perceived by iranian kurdish diabetic patients in order to develop an appropriate multidisciplinary outlook toward this ethnicity of patients .
considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .
content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .
the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .
a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .
the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .
the sample size was determined only when the interviewing stage reached saturation ( 22 ) .
the sample was restricted to 20 because no new data emerged after the twentieth interview .
therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . in - depth , face - to - face , semi - structured interviews were used for data collection .
the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,
an interview guideline was defined to direct the interviews toward the aims of the study .
the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .
examples of questions asked during the interview were as follows : when do you feel healthy ?
further , the patients were asked to describe all the factors that could affect their health .
twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .
each interview spanned 4090 minutes ( average duration : 60 minutes ) . each interview was transcribed and then used as a guide for the other interviews .
two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .
the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .
the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers . through these inquiries ,
the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .
this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .
this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .
all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .
the participants were assured that they could leave the study at any time even after they had signed the consent form .
they were assured that their care would not be affected if they chose not to participate in the study .
they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .
all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions . in this study ,
the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .
this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .
the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .
this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .
researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,
one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .
considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .
content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .
the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .
a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .
the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .
the sample size was determined only when the interviewing stage reached saturation ( 22 ) .
the sample was restricted to 20 because no new data emerged after the twentieth interview .
therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon .
in - depth , face - to - face , semi - structured interviews were used for data collection .
the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,
an interview guideline was defined to direct the interviews toward the aims of the study .
the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .
examples of questions asked during the interview were as follows : when do you feel healthy ?
further , the patients were asked to describe all the factors that could affect their health .
twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .
two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .
the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .
the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers .
through these inquiries , the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .
this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .
this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .
all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .
the participants were assured that they could leave the study at any time even after they had signed the consent form .
they were assured that their care would not be affected if they chose not to participate in the study .
they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .
all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions .
in this study , the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .
this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .
the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .
this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .
researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,
one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .
twenty diabetic patients participated in this study ; the participants included seven men with an age range from 53 to 80 years ( average age : 68 years ) and thirteen women with an age range from 32 to 55 ( average age : 40 years ) .
meanwhile , the average number of years which had lapsed since diagnosis of diabetes was seven years with a range of 517 years . based on the thoughts and feelings of the participants , the following three main themes emerged : the syndrome of the healthy body and the happy heart life without compulsory limitations the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .
this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .
it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .
the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .
similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .
one of the participants in the study said , a person with a healthy and contented heart is well - behaved and kind to others ( p1 ) .
the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .
another participant said , health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy
the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises .
health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .
health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety ( p 11 ) .
another participant said , health means that we do not have any problems or complaints of illness
( p6 ) , and , health brings calmness , and it is like praying to god without having to think about stressful matters (p2 ) , and , when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .
so , the complaints gradually go away ( p 18 ) all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .
one of the women ( 43 years old ) said , when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak (
sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me
when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines , this freedom is very fantastic
, spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .
participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .
most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old ) said , some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and
, a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace
( p 18 ) , and , healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet (p 11 ) another participant said , when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy (p 6 ) .
the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .
this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .
it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .
the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .
similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .
one of the participants in the study said , a person with a healthy and contented heart is well - behaved and kind to others ( p1 ) .
the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .
another participant said , health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy
the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises .
health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .
health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety ( p 11 ) .
another participant said , health means that we do not have any problems or complaints of illness
( p6 ) , and , health brings calmness , and it is like praying to god without having to think about stressful matters (p2 ) , and , when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .
all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .
one of the women ( 43 years old ) said , when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak ( p 13 ) .
sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me
for some participants , the limitations had led to the feeling that they were different from others
when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines , this freedom is very fantastic
in general , spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .
participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .
most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old )
said , some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and , a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace
( p 18 ) , and , healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet (p 11 ) another participant said , when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy (p 6 ) .
as the results for this group of diabetic patients indicated , health is composed of the syndrome of the healthy body and the happy heart , life without compulsory limitations , and exalted spirituality .
many studies have shown that health perception is a reflection on , not only health but also health as a culture .
for all patients , health perception and points of view are the important factors that affect health behaviour and determine any related behaviour ( 25 , 26 ) . an individual may be ill and yet may feel healthy ; therefore , many people can compare their health status during suffering from illness with that in the past ( 22 , 27 ) .
the themes that emerged in our study showed these instances briefly . in this study , participants talked about the effects of diabetes on physical and psychological health , which indicated that our results were similar to those of other studies ( 28 , 29 ) .
the most important theme of this study was the syndrome of the healthy body and the happy heart .
the patients always used local terminology for health description , and these terminologies have extended meanings . in this context , health is considered as a feeling that can affect all components of the human body , including their physical , psychological , social , emotional , and spiritual facets .
this view about health is mainly accepted about diabetes because diabetes affects all aspects of a patient s life ( 11 , 28 , 29 ) . in this study , participants admitted that , not only physical health but also vivacity , satisfaction , and calmness of mind , good relationship with family and good economic status are all considered as aspects of health . hence , health care professionals should strongly consider the issue that health implies lack of having any problems , complaints , illnesses , etc .
furthermore , physical illness and its adverse effects are important topics in the health experiences of diabetic patients ; however , when these patients were healthy , they said that without peace of mind one could not feel healthy .
moreover , in a study about health in spanish immigrants , ailinger and causey found that some concepts of health are related to the patients psychological point of view and peace of mind , calmness in family , absence of complaints , and so on ( 30 ) .
assessed the effectiveness of self - efficacy and family inter - relationships among young turkish patients afflicted with diabetes type i ( 29 ) .
their study showed that calmness of mind and some other related factors such as relationship among family members , conflicts or compatibility in the family , and emotional situations can affect self - efficacy , and accordingly , these factors affect their overall health .
hence , considering the important effect of calmness of mind on diabetic patients health perception and points of view , we , as health care professionals , ought to be obliged to associate a disturbed mind with bad health .
overall , by using these implications as the bases of patients culture , we can improve health programmes for all patients .
one of the most plausible themes in our interviews was health as life without compulsory limitations .
miklaucich studied the limitations of life as meaningful health experiences in patients with coronary angina ( 31 ) .
abazari et al . also demonstrated that diabetic patients have dietary restrictions as well as limitations with regards to bearing children ( 18 ) .
thomson & gifford indicated that trying to keep a balance was the preferred meaning of health for diabetic participants ( 32 ) .
it is more important for the professional health care teams to help diabetic patients feel that they are not different from others and in this regard , consultation with dieticians can help to obviate some part of this limitation of life .
studies also show that having a regular activity program and regular follow- up consultation with other health care professionals can help diabetic patients to expel limitations ( 33 , 34 ) . in this article
, all participants talked about the importance of the spiritual dimension of health and achieving exalted spirituality .
some other studies showed that spirituality is one of the significant factors that can affect health ; hence , spirituality plays a very important role in gaining adaptation to a lifestyle , with regards to chronic diseases , and helps patients to manage their health - related problems ( 35 ) .
this theme was plausible in traditional societies ; hence , nursing professionals must pay more attention to this influential factor .
patients can adopt a spiritual approach by becoming a member of a religious group , praying , and meditating .
exalted spirituality , in fact , helps patients to cope with the disease , and feel better about their lives ( 36 , 37 ) .
he showed that praying helps patients to not only connect with the higher power , but also to relate to each other .
their act of praying is a response to their anxieties about themselves or their families .
walton showed that women with chronic diseases , used prayer to calm themselves and connect with others to support each other and cope with diseases ( 39 ) .
therefore , health care professionals should be aware of the effects of spirituality on patients having a chronic disease .
the results of our study primarily indicated that the factors that affect health perception include complaining about the illness , side effects and outcomes of the disease , and the extent of the complaints that make the patient feel sick .
patients with chronic diseases encountered some social and emotional problems due to changes in their lifestyle ( 36 ) .
diabetes affects patients quality of life , and especially their perception and attitude toward health ( 12 ) .
it fully depends on how patients and health professionals define health ( 40 , 41 ) . due to chronic and acute complaints of diabetic patients ,
we need to create a programme aiming to reduce these adverse effects and promote self - care for diabetic patients . especially , a program must be developed based on paying attention to body and mind simultaneously , trying to remove the limitation in life of patients by considering the extent of the limitations and limitations that exist for the patients which compel them to believe that the disease has been brought about just for them .
this program should not affect or limit the beliefs and spiritual activity of patients because a person s previous experiences influence the perception of present time , which also impact on their future .
besides , having been brought up in a different society and within a different ethnic group , influences a person s health - related behavior .
so , health care providers must manage care situations by enhancing health experience to enable patients lives with the least limitations , and giving them an opportunity to preserve exalted spirituality .
studying health perception among patients with chronic diseases is an important factor that facilitates the development of appropriate programmes , to improve health levels among such patients . in this regard ,
the professional health - care provider team ought to consider health perception among patients , and then they apply this knowledge for setting up suitable health improvement programs .
nurses need to pay more attention to diabetic patients limitations and facilitate a programme to improve their health in conjunction with other health care teams .
although nursing and other health care professionals have the responsibility to help those patients achieve peace of mind , the patients families should also play an active role in the health care programme and treatment process . | introductionhealth is an exclusive and subjective phenomenon , and one of the most important situations with regard to perception of health , arises when patients suffer from a chronic disease.this study was conducted within the qualitative research framework and aimed to explore the meanings of health as perceived by a group of iranian diabetic patients.methodsa descriptive qualitative analysis design was used .
data were collected through semi - structured interviews with 20 participants among diabetic patients , who were admitted to the diabetes care centre of tohid hospital of the kurdistan university of medical sciences , sanandaj , iran during a ten - month period in 2014 .
interviews were transcribed and analysed through conventional content analysis.resultsbased on the findings of the study , three major health - related themes emerged : 1 ) the syndrome of the healthy body and the happy heart ( physical well - being vivacity , satisfaction , and calmness of the mind ) , 2 ) life without compulsory limitations ( lack of dietary limitations , no activity limitations , lack of social limitations ) , and 3 ) exalted spirituality ( satisfying self and others , trusting god , remembering god).conclusionhealth care providers should consider the meaning of health in special groups , chiefly in patients with chronic diseases .
it facilitates the development of appropriate programmes to improve desirable health levels among diabetic patients . |
however , proper amount of daily water intake in a healthy individual is controversial . there is a prevalent thought that we all have to drink eight glasses of water daily
, we try to clarify this topic from the perspective of avicenna and traditional persian medicine ( tpm ) .
the most important traditional persian medical encyclopedia , al - qanun fil - tibb ( canon of medicine ) , was reviewed .
furthermore , medline , embase , scopus , iranmedex , and science iranian database ( sid ) focusing on the keywords traditional medicine , water intake ,
fluid , and complementary and alternative medicine were reviewed to find relevant information .
avicenna believed that the demand for daily water is not the same in different individuals .
the determinant variables mentioned in his book , the canon of medicine , comprises mizaj ( temperament ) , health status , age , sex , season , place , habits , occupation , etc .
he believed that water in extra amount quenches the hararat - e - ghariezi , which is the basal internal heat to convey normal homeostasis and metabolism in the body .
several factors determine the actual need of any person to drink water . consequently , recommending a specific amount of daily water intake for all is illogical .
moreover , important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders . | background : fluid intake is an essential factor in having a normal homeostasis .
however , proper amount of daily water intake in a healthy individual is controversial .
there is a prevalent thought that we all have to drink eight glasses of water daily , however , it lacks enough supportive evidence at least for healthy individuals . here , we try to clarify this topic from the perspective of avicenna and traditional persian medicine ( tpm).methods : the most important traditional persian medical encyclopedia , al - qanun fil - tibb ( canon of medicine ) , was reviewed . furthermore , medline , embase , scopus , iranmedex , and science iranian database ( sid ) focusing on the keywords traditional medicine , water intake ,
fluid , and complementary and alternative medicine were reviewed to find relevant information.results:avicenna believed that the demand for daily water is not the same in different individuals .
the determinant variables mentioned in his book , the canon of medicine , comprises mizaj ( temperament ) , health status , age , sex , season , place , habits , occupation , etc .
he believed that water in extra amount quenches the hararat - e - ghariezi , which is the basal internal heat to convey normal homeostasis and metabolism in the body.conclusion:several factors determine the actual need of any person to drink water .
consequently , recommending a specific amount of daily water intake for all is illogical . moreover , important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders . |
a 34year - old male presented for general examination on day 49 of a 50-day self - imposed fast , seeking approval to continue his fasting behavior . during this period ,
the patient reported only consuming water , tea , coffee ( with no milk or sugar ) , and a daily multivitamin tablet ( centrum performance ; pfizer ) .
patient history prior to fasting behavior revealed a family history of obesity and the presence of pathologies related to body mass .
personal history involved an oscillating mass between 85 and 134 kg over a 10-year period . at the start of fasting ,
the patient reported body mass as 96.8 kg , and 29.2% body fat , as recorded by a personal bioelectrical impedance device .
history following the onset of fasting behavior was unremarkable , but included severe abdominal cramps between days 7 and 21 of fasting , development and frequent occurrence of vasovagal syncope on transition from sitting to standing , and the near - cessation of fecal movement .
self - reported physical activity and mobility was reduced , but not prevented ; this participant was not bedridden .
substantial water and coffee intake was noted ( 812 cups of coffee daily and 23 bottles of water daily ) . as expected , body mass loss during fasting was severe and noticeable ( fig .
resting metabolic rate ( 30 minutes by indirect calorimetry ) was 1626 kcal day . during the 30-minute rest period
resting ecg appeared morphologically normal , patient was bradycardic ( 52 bpm ) and notably hypertensive ( 150/78 mmhg ) .
body composition was measured by whole - body air displacement plethysmography ( bod pod ) as 19.8% body fat , while height was 180.9 cm and mass was 75.4 kg , with a bmi of 23.5 kg m , all recorded on calibrated laboratory devices .
sum of four skinfolds1 was 30.0 mm , approximately half of that of a reference population of similarly aged males ( 62 25 mm , aged 3039 ) .
it was visually noted that subcutaneous fat in the abdominal region was still present , despite severe losses in the peripheral limbs , protruding ribs , and lack of prominent chest and shoulder musculature .
2 ) . during a six - minute walk test , this individual covered 342 m , which was below expected performance for a healthy male adult.2 a mild scoliosis was observed while walking ; however , it is unclear if this is related to fasting .
venous blood was taken for serum markers of metabolism , liver , and renal function .
noteworthy findings include plasma glucose of 3.1 mmol l and alkaline phosphatase of 36 u l. total bilirubin was 19 mol l , direct bilirubin was 7 mol l. uric acid was 620 mol l. urine was straw colored , lacked sediment , and was noticeably sweet in aroma
. rapid dipstick analysis ( multistix 2161 ; siemens ) was negative for blood glucose , contained trace proteins , and indicated urine ph of below 5 .
one month following initial presentation , the patient returned for follow - up examination during which body composition , resting metabolic rate , urine , and venous blood were recorded .
resumption of feeding initially involved liquids such as vege table soups , with the resumption of solid foods three days following breaking of fast .
days 35 involved one solid meal per day , with 23 solid meals during days 57 , and the resumption of three solid food meals per day for 7 days following breaking of fast . in the following 23 days
, diet was reported to be of caffeine and alcohol free , low carbohydrate ( a piece of bread every 13 days ) , and high servings of fruit and vegetables .
self - estimation of diet by volume was 20% animal protein ( chicken and fish ) and 80% fruit or vegetables ; validated , quantifiable measures of intake and caloric content are not available .
heart rate remained bradycardic at rest ( 48 bpm ) , and blood pressure was notably reduced ( 132/78 ) .
metabolic rate was reduced ( 1413 kcal day ) , and body composition was unchanged ( 75.0 kg ; bmi , 23.4 kg m ; 16.1% fat mass ) .
blood biochemistry returned to within normal range , with the exception of direct bilirubin ( table 1 ) .
it is noteworthy , therefore , in this case study that moderate physical function appears maintained at day 49 , as assessed by general observation and results of the six - minute walk test .
frommel et al.3 suggested that fasting in healthy lean individuals was well tolerated until 18% of body mass was lost ; the patient reported here lost 20.7% of body mass without substantial loss of function .
one prior report of long - term fasting in obese individuals for 3040 days demonstrated body mass losses between 10.6% and 20.5% , but does not report on function of individuals.4 it seems self - apparent and supported by this limited evidence , such that the starting mass is a key variable in survival of extreme fasting , and not the amount of loss .
this patient started fasting while obese and ended his fast when anthropometric variables were within the targeted ranges for the general population .
indeed , among the few ( nonpublished ) examples of complete fasting and mortality,5 patients were frail between 30 and 50 days , and death was noted to occur between days 43 and 70 , which this case study has reached without obvious or significant frailty occurring .
current world medical association guidelines prioritize autonomy , recommending nonintervention in fasting to the point of harm and/or death , if the patient makes a written informed statement of intent.5 lack of research into this field precludes evidence - based decision - making or advice . in the limited light of this case study and historic data of fasting in obesity,4
it is tempting to suggest that guidelines to physicians for advice to the fasting individual reflect body mass , and specifically mass of adiposity at start of fasting behavior .
survival in starvation is ultimately governed by physics , the calories available to maintain metabolic function .
availability of adipose tissue for metabolic usage may thus delay the catastrophic degradation of muscular protein , increasing survival time . that said , small animal models reveal organ - specific effects of starvation , with loss of liver and gut size and function noted after only four to six days.6 functional damage to digestive organs can not be ruled out , and indeed , the elevated direct bilirubin is suggestive of reduced liver filtration . of concern ,
direct bilirubin remained elevated 30 days post breaking of fast . elevated uric acid is likely due to reduced kidney filtration , as has been reported in caloric restriction previously.7 further , hypertension was an unexpected finding .
this finding is counter to one of the few published examinations of cardiology in severe starvation , where bradycardia and hypotension ( ~20 mmhg below fed state ) were noted in juvenile pigs following 29 days of complete starvation.8 the porcine heart contains several structural differences relative to the human , most related to bipedal vs. quadrupedal stance.9 as frequent syncope was also noted in that our case study , it is plausible that dysregulated autonomic regulation of blood pressure underlies the observed hypertension .
blood pressure was measured in a supine position , increasing venous return in the human relative to standing position .
simple frank starling mechanics combined with a failure of autonomic regulation may thus explain elevated blood pressure .
thus , it is tempting to suggest that differences observed may be species dependent ; however , this is highly speculative on a single case study .
further , long - lasting cardiovascular deficits were noted in the above porcine model on refeeding , as well as chronically following experimental conclusion.8 as death resulting from starvation often occurs via cardiovascular complications , this elevated blood pressure during fast is key to note ; this should be carefully monitered in fasting individuals
. caloric restriction and weight loss in overweight or obese individuals is often associated with subsequent regain of weight.10,11 having access to this case study 30 days following resumption of feeding was of interest due to a chance to examine any occurrence of such a rebound effect .
it is of interest to note that this was not seen here ; however , future gains in weight can not be ruled out . here , we report the maintenance of physical function of an individual after 49 days of complete fast .
this report does not indicate support for such behavioral choices , and the projections of metabolic energy provision by starting adiposity assume no other perturbations to homeostasis , such as impaired immune response , liver and renal function , or cardiovascular events . | a 34-year - old obese male ( 96.8 kg ; bmi , 30.2 kg m1 ) volitionally undertook a 50-day fast with the stated goal of losing body mass . during this time , only tea , coffee , water , and a daily multivitamin were consumed .
severe and linear loss of body mass is recorded during these 50 days ( final 75.4 kg ; bmi , 23.5 kg m1 ) . a surprising resilience to effects of fasting on activity levels and physical function is noted .
plasma samples are suggestive of early impairment of liver function , and perturbations to cardiovascular dynamics are also noted .
one month following resumption of feeding behavior , body weight was maintained ( 75.0 kg ; bmi , 23.4 kg m1 ) .
evidence - based decision - making with the fasting or hunger striking patient is limited by a lack of evidence .
this case report suggests that total body mass , not mass lost , may be a key observation in clinical decision - making during fasting and starvation . |
tangential tractions on the vitreomacular interface are usually responsible for the development of idiopathic macular holes .
the prevalence is 3.3/1000 and it shows a strong female predominance.1 in the past , it was known as a rare , untreatable disorder leading to central loss of vision . in 1991 ,
kelly and wendel2 reported that idiopathic macular holes could be closed by vitrectomy and gas tamponade . with a better understanding of the pathogenesis of macular hole development and innovations in macular surgery ,
one of the important developments in the surgical approach is peeling of the internal limiting membrane ( ilm ) .
some authors have argued that ilm peeling has beneficial effects on the surgical outcome,3,4 whereas others have stated that this approach does not affect surgical success,5,6 and may lead to complications by increasing the surgery duration.7,8 the ilm is formed by mller cell extensions and functions as a kind of basal membrane between retina and vitreus , acting as a surface for glial cell proliferation .
vitreoretinal interface changes due to cellular proliferation lead to ilm distortion and eventually to the development of epiretinal membrane ( erm ) , macular hole , and recurring macular edema.9 with the removal of the ilm , successful results may be obtained in surgical treatment of such vitreoretinal diseases .
the most difficult aspect of surgery is due to the tight attachment of the ilm to the retina and its extreme thinness and transparent nature . to increase the visibility of the ilm
indocyanine green ( icg ) and trypan blue have been used by many researchers in peeling of the erm and ilm .
there are different opinions on the amount and concentration of dye required , and exposure duration in the vitreous space . in many studies , because of intraretinal accumulation of icg and its toxic effects on retinal pigment epithelium , visual field defects after surgery and possible optical nerve atrophy have been considered.8,10,11 recently , brilliant blue ( bb ) has been used in preclinical studies as it has minimal toxic effect and provides effective membrane staining.9,12 in this study , the anatomical and visual outcomes of pars plana vitrectomy ( ppv ) and ilm peeling with bb in the treatment of idiopathic macular holes has been examined .
fifty eyes of 48 patients who had 23 g ppv and ilm peeling as a treatment for macular holes between july 2007 and december 2009 were retrospectively evaluated in the authors clinic .
all patients underwent measurement of best - corrected visual acuity and intraocular pressure , anterior segment and fundus examinations before surgery .
the possible merits and risks of the treatment were explained to the patients , and informed consent was obtained from all patients in accordance with the helsinki declaration .
inclusion criteria were : diagnosis using noncontact - lens biomicroscopy and optical coherence tomography ( oct ) of stages 2 , 3 , and 4 idiopathic macular hole according to the gass classification .
exclusion criteria were : trauma history ; previous macular surgery , rhegmatogenous retinal detachment together with macular hole ; myopia higher than 10d ; macular hole for more than 2 years ; and previous retinal vessel disease .
macular examination was performed before surgery and 1 , 3 , and 6 months after surgery using spectral oct ( optovue inc , fremont , ca ) .
all patients had a 23 g transconjunctival three port pars plana vitrectomy . in four eyes having both macular hole and erm
, both ilm and erm peeling were performed . to visualize the ilm , 0.3 ml
, 16% c3f8 gas was injected into 28 eyes ( 56% ) and 18% c2f6 gas into 22 eyes ( 44% ) .
follow - up time points included 1-day , 1-week , 1-month , 3-month , and 6-month postsurgical evaluations and thereafter visits at 3-month intervals .
best - corrected visual acuity , intraocular pressure , and complications were recorded for each visit .
anatomical and functional outcomes of macular hole patients who underwent 23 g ppv and ilm peeling with bb were evaluated .
cases in different macular hole stages were compared in terms of anatomical and functional results .
all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,
imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .
brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .
after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .
after liquid air exchange , air gas exchange was performed ( 16% c3f8 or 18% c2f6 ) . none of the patients required sutures .
all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,
imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .
brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .
after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .
after liquid air exchange , air gas exchange was performed ( 16% c3f8 or 18% c2f6 ) .
the patient group consisted of 33 ( 66% ) female and 17 ( 34% ) male subjects ( mean age sd ; 63.34 9.6 ) . of these cases ,
preoperative oct examination revealed that , according to gass classification , 17 eyes ( 34% ) had stage 2 macular holes , 24 eyes ( 48% ) had stage 3 , and 9 eyes ( 18% ) had stage 4 .
anatomic success was assessed by the closure of the hole on oct imaging and the withdrawal of subretinal fluid .
the hole was closed in 46 ( 92% ) of the 50 eyes after surgery but not closed in 4 ( 8% ) eyes .
when postoperative oct controls revealed that subretinal fluid had disappeared , there was no need for a second surgery .
hole persistence was observed in only one patient 2 months after the surgery and it was closed by reoperation .
figure 3 shows pre- and postoperative third - month spectral oct imaging of a patient with a stage 4 macular hole .
the mean best - corrected visual acuity of all patients before surgery was 0.71 0.25 logmar .
postoperative mean best - corrected visual acuity ( and comparison with preoperative values ) was obtained as : 0.48 0.26 at 3 months ( p < 0.05 ) , 0.52 0.27 at 6 months ( p < 0.05 ) , 0.42 0.30 at 9 months ( p < 0.05 ) , and 0.41 0.31 at 12 months ( p < 0.05 ) . when assessed together , the final visits ( at 12 months ) demonstrated that visual acuity had increased in 41 eyes ( 82% ) , whereas , in 6 eyes ( 12% ) , visual acuity remained unchanged . because of recurrent macular holes , three cases showed a decrease in visual acuity .
two of these three cases had stage 4 macular holes , and one had a stage 3 macular hole .
best - corrected visual acuity results of patients before and after surgery are given in figure 1 and table 2 . when evaluated according to the stage of macular hole , in stage 2 patients , all measurements of visual acuity after surgery showed that it was significantly higher when compared with preoperative measurements ( p < 0.05 ) .
increase in visual acuity between 6 and 12 months after surgery was statistically significant due to cataract surgery ( p < 0.05 ) . in stage 3
patients , preoperative visual acuity compared with postoperative 3-month scores showed an increase , but this was not statistically significant ( p = 0.056 ) . during the postoperative period , visual acuity reduced between months 3 and 6 due to cataract development . in patients who underwent phacoemulsification after 6 months ,
therefore , comparisons of postoperative 912-month values showed significant increases ( p < 0.05 ) .
in contrast , in stage 4 patients , visual acuity scores of all time points after surgery failed to reach statistically significant levels with respect to preoperative values .
therefore , the first month visual acuity measurements were excluded from analysis . in 22 phakic patients ,
the mean preoperative visual acuity of stage 2 macular hole patients was higher than that of stage 3 or 4 patients .
consequently , the most prominent increase in visual acuity was observed in the stage 2 group after surgery .
there was no significant difference between stage 2 and 3 macular hole patients in terms of anatomical and visual outcomes , but the functional successes of these two groups were significantly higher than those of stage 4 patients .
iatrogenic retinal tears developed in four patients during surgery and were successfully treated intraoperatively . in one patient , a nasal retinal tear developed near the optic nerve while removing the posterior hyaloid .
one of the phakic cases had preoperative lens opacity and vitrectomy and phacoemulsification was performed together with intracapsular lens implantation .
since the first report of kelly and wendel2 on the treatment of macular holes by pars plana vitrectomy and gas tamponade , anatomical and functional success rates have increased with developments in vitreoretinal surgery .
previous studies reported success rates over 90% after one session of surgery13,14 and anatomical success rates as high as 100% in stage 2 or the initial phase of stage 3 macular holes.15 in histopathological studies,16,17 various surgical approaches have been proposed based on the idea that collagen - containing myofibroblasts and actin - containing cells in the structure of ilm and erm may cause contraction and lead to hole formation or widening of an existing hole .
many studies1821 have reported that ilm peeling increases anatomical and functional success in the treatment of macular hole , and during the late phases it reduces reformation of the hole .
kwok et al18 compared anatomical closure of stage 3 or 4 holes in 40 patients with or without ilm peeling .
success rate was 89% in the ilm peeling group whereas it was 59% in the group without ilm peeling .
the differences in anatomical closure success may be due to stage differences of macular holes , inclusion of traumatic cases , high myopic eyes , and recurrent cases .
although there is no consensus on the effects of ilm peeling on anatomical and functional success , publications stating that ilm peeling increases hole closure are in the majority.22,23 there are several opinions regarding which patients require ilm peeling .
previous studies have stated that ilm peeling should be considered especially in patients with a hole wider than 400 microns and that is chronic ( 6 months or longer ) , traumatic , or recurring.2125
ilm peeling was reported to increase anatomical success and prevent reopening of the hole by decreasing erm development.25,26 besides reports that suggest that ilm peeling increases anatomical success but not functional success,27 there are also reports that suggest functional outcomes are better in ilm peeling groups.18 in this study , ilm peeling was applied to all patients who underwent macular hole surgery .
anatomic closure was obtained in 46 ( 92% ) of the 50 eyes , but the hole was not closed in four patients .
visual outcomes were significantly better in stage 2 or 3 patients than in stage 4 patients ( p < 0.05 ) .
the authors consider that ilm peeling improved anatomical and functional success rates in stages 2 and 3 patients whereas it had no effect on visual outcomes in stage 4 patients due to photoreceptor loss .
cataract development is the most prevalent complication of macular hole surgery in phakic eyes . in this study group , cataract development
was observed in 23 ( 74% ) of 31 patients due to an endotamponade application . besides publications that report
early cataract surgery following macular hole surgery may increase the risk of re - opening of the hole,28 there are also studies that report no such risk.29,30 therefore , in patients developing cataract , phacoemulsification surgery was performed at least 6 months after macular hole surgery to prevent a recurrence . as the thin and semitransparent structure of the ilm complicates visualization during surgery , in patients with ilm peeling , there may be small asymptomatic paracentral scotomas , irregularity in nerve fiber layers , and retinal microhemorrhages due to iatrogenic retinal trauma.3136 chromovitrectomy has been developed as a method to improve ilm visibility , shorten the duration of surgery , and reduce iatrogenic retinal trauma.36 many dyes , such as indocyanine green ( icg ) , infracyanine green ( ifcg ) , trypan blue ( tb ) , bb , and triamcinolone acetonide ( ta ) , are used to dye the ilm .
since icg was first introduced for ilm peeling in macular hole surgery , several potential side effects have been noted.37,38 visual field defects , retinal pigment epithelium , and ganglion cell defects are among the most reported side effects.26,32,33 dose - dependent biochemical damage to the retinal pigment epithelium and ganglion cells , photo - oxidative cell damage due to the phototoxic properties of icg , and retinal pigment epithelium damage due to hypoosmotic solution are considered responsible for icg - mediated ocular toxicity.3941 because of the potential toxic side effects of icg in chromovitrectomy , other vital dyes with minimal toxicity have been tried.32,33,38,42 ifcg , unlike icg , does not contain iodine , and it is widely thought that its toxicity to the retinal pigment epithelium is less than that of icg.31 triamcinolone acetonide was reported as both toxic4345 and nontoxic46 to retinal pigment epithelium in preclinical studies . in the authors opinion , there is an insufficient number of randomized clinical trials to make a conclusion .
however , in some studies of ta - assisted ilm peeling , it was found similar to icg.43,47 another vital dye , tb , is recommended for erm staining because of its high affinity for proliferation - dense intraocular tissues.48 several preclinical studies with high - concentration tb reported toxic effects on tissue culture of retinal pigment epithelium.49,50 however , many other researchers claim that if it is used in lower concentrations , there will be no toxic effects.51,52 in this study , bb , a relatively new type of vital dye , was used to stain the ilm .
enaida et al9 studied icg on rats and found that low - dose icg administration resulted in no retinal cell damage but high dose icg led to morphological damage of retinal cells . in a rat study by hisatomi et al and enaida et al,53,54 low dose intravitreal bb administration caused no effects on retinal cells , but in higher doses electron microscopy revealed cyst formation in the inner layers of the retina .
the authors of this present study think that biological adaptation to bb is better than to icg . the use of bb is easier than icg and tb because of its granular structure and easy dissolution in intraocular irrigation .
when compared with bb , higher concentrations of icg are required to dye the ilm.12 in addition , bb is not a fluorescent dye , so its phototoxicity is lower than icg .
the authors of this study have observed intraretinal accumulation in postoperative fundus fluorescein angiography of patients for whom icg was used .
ilm peeling in macular hole surgery provides beneficial effects on anatomical outcome , independent of disease stage . in terms of visual outcome | purpose : the evaluation of anatomic and visual outcomes in macular hole cases treated with internal limiting membrane ( ilm ) peeling , brilliant blue ( bb ) , and 23-gauge pars plana vitrectomy ( ppv).materials and methods : fifty eyes of 48 patients who presented between july 2007 and december 2009 with the diagnosis of stage 2 , 3 , or 4 macular holes according to gass classification who had undergone ppv and ilm peeling were included in this study .
pre- and postoperative macular examinations were assessed with spectral - domain optical coherence tomography .
23 g sutureless ppv and ilm peeling with bb was performed on all patients.results:the mean age of patients was 63.34 9.6 years .
stage 2 macular hole was determined in 17 eyes ( 34% ) , stage 3 in 24 eyes ( 48% ) , and stage 4 in 9 eyes ( 18% ) .
the mean follow - up time was 13.6 1.09 months .
anatomic closure was detected in 46/50 eyes ( 92% ) , whereas , in four cases , macular hole persisted and a second operation was not required due to subretinal fluid drainage . at follow - up after 2 months , persistant macular hole was detected in one case and it was closed with reoperation . at 12 months ,
an increase in visual acuity in 41 eyes was observed , while it remained at the same level in six eyes . in three eyes visual acuity
decreased .
there was a postoperative statistically significant increase in visual acuity in stage 2 and 3 cases ( p < 0.05 ) , however , no increase in visual acuity in stage 4 cases was observed.conclusion:ppv and ilm peeling in stage 2 , 3 , and 4 macular hole cases provide successful anatomic outcomes , however , in delayed cases , due to photoreceptor loss , it has no effect on functional recovery .
bb , used for clarity of ilm , may be beneficial due to its low retinal toxicity . |
the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .
a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .
a random blood sample was obtained from the cross - sectional study participants recruited in boston .
bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .
presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .
current and former smokers were considered as one group and compared with those who never smoked .
all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration .
genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .
patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .
comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables , respectively .
because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies ,
a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event
was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,
the time variable was defined as the time between the enrollment date and the date of the last available clinical data .
the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,
the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as regression coefficients .
pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for study sample ) after excluding genotype - by - sample interactions .
genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .
the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) .
all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .
the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .
a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .
a random blood sample was obtained from the cross - sectional study participants recruited in boston .
bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .
presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .
current and former smokers were considered as one group and compared with those who never smoked .
all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . written informed consent was obtained from each study participant .
genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .
patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .
comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables , respectively .
deviations from hardy - weinberg equilibrium were investigated by exact test . because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies , a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event
was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,
the time variable was defined as the time between the enrollment date and the date of the last available clinical data .
the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,
the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as regression coefficients .
pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for study sample ) after excluding genotype - by - sample interactions .
genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .
the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) . a value of p < 0.05 was considered significant .
all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .
we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .
no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .
clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .
the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .
the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .
no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .
incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .
the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .
1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,
the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .
hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .
bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .
the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .
kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .
the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .
given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .
an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) . no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) .
because approximately 85% of these two cohorts consisted of nondiabetic individuals , we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .
indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .
thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .
2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) .
the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .
by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .
survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .
curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,
the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .
a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) . to seek replication of the gene - by - obesity interaction observed in patients with diabetes
, we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .
one sample was from the gargano area in italy , the other was from boston .
salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for study sample .
to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi and diabetes because all study participants were diabetic )
64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .
in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .
clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .
virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .
in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers ( = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .
3a ) , but not among kk individuals ( = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .
3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .
data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .
we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .
no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .
clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .
the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .
the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .
no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .
incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .
the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .
1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,
the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .
age at study entry ( hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .
bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .
the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .
kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .
the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .
given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .
an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) .
no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) . because approximately 85% of these two cohorts consisted of nondiabetic individuals
, we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .
indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .
thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .
2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) . the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .
by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .
survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .
curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,
the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .
a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) .
to seek replication of the gene - by - obesity interaction observed in patients with diabetes , we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .
one sample was from the gargano area in italy , the other was from boston .
salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for study sample .
to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi and diabetes because all study participants were diabetic ) were included as covariates . among obese subjects ,
64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .
in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .
clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .
virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .
in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers ( = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .
3a ) , but not among kk individuals ( = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .
3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .
data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .
our results indicate that the enpp1 k121q polymorphism predicts acceleration of major cardiovascular events in very high - risk patients .
the increased risk conferred by the q121 variant is independent of that of age , sex , bmi , diabetes , and cigarette smoking .
our findings are in agreement with a previous cross - sectional study of 445 mi survivors from central europe ( 29 ) .
by contrast , case - control genome - wide association studies reported that a single nucleotide polymorphism ( snp , rs7767502 ) , which is in perfect linkage disequilibrium with the enpp1 k121q polymorphism , was not associated with cad ( 610 ) .
several differences between our study and the genome - wide association studies , such as the prospective versus cross - sectional designs , the different end points under investigation , and the different baseline cardiovascular risk , with only the patients enrolled in our study being very high - risk as per selection criteria , might be responsible for this apparent discordance .
an additional important result of our study is that the effect of the q121 variant was modulated by obesity in diabetic patients among whom the risk of incident events was five times higher in q121 than in kk genotype carriers .
although not the aim of our study , one can infer that obese individuals ( fig .
2a ) as a whole tend to have a lower risk of future cardiovascular events than nonobese patients ( fig . 2b ; adjusted hr 0.68 [ 95% ci 0.4111.124 ] , p = 0.13 ) .
this paradoxic protective effect of obesity resembles that observed in patients with cad ( 41 ) , esrd ( 42 ) , heart failure ( 43 ) , and older age ( 44 ) , all conditions heavily over - represented in our samples . in this context , the q121 variant seems to eliminate the paradoxic protective effect of obesity .
an important finding was that the q121 variant - by - obesity interaction observed in the prospective study was replicated in a cross - sectional study on age at mi in diabetic patients .
information on the k121q genotypes tended to improve risk prediction in these patients when the improvement was measured by the idi , the approach that is currently favored to evaluate predictive ability increase conferred by a new marker when added to a well - performing model ( 39 ) .
thus , pending further validation in larger studies , one can hypothesize clinical implementation of the q121 variant as a marker of early cardiovascular events among obese diabetic patients .
given the increasing incidence worldwide of both obesity and diabetes ( 24 ) and the poor ability to stratify cardiovascular risk among diabetic patients , a large sector of society would be likely to benefit in the future from the availability of such a test .
the synergistic effect of the genetic marker and obesity in the modulation of cardiovascular risk resembles results repeatedly reported in the risk modulation of insulin resistance and related traits ( 2527,30,33,4549 ) .
placed in a broader perspective , this is an excellent example of genetic heterogeneity ( i.e. , different genetic effects being at play in different population subgroups ) and clearly illustrates how accounting for such heterogeneity may be critical to dissect the genetic architecture of multifactorial diseases .
understanding the mechanisms through which the q121 variant is associated with cvd is beyond the scope of this study .
however , one can speculate that the q121 variant exacerbates cardiovascular risk by inducing systemic insulin resistance ( 22,2527 ) and proatherogenic phenotypes ( 24,29,30,33 )
. it may also act by way of a direct detrimental effect on insulin - dependent endothelial function , as suggested by the observation that human endothelial cells carrying the q121 variant show impaired insulin receptor signaling and , most importantly , reduced release of nitric oxide ( 24 ) , a potent vasodilator whose deficiency is an established early step in the pathway development of atherosclerosis ( 50 ) .
one can hypothesize that the interaction between the q121 variant and obesity is sustained by the different sites of action on the insulin - signaling pathway .
although enpp1 acts at the insulin receptor level ( 21,23 ) , obesity acts by different mechanisms , mostly at a postreceptor level ( 51 ) .
it is , therefore , possible that postreceptor insulin - signaling abnormalities are necessary for the q121 variant to be fully effective in inducing insulin resistance and , eventually , related clinical outcomes .
the three cohorts of very high - risk individuals that we studied were quite different from each other : one comprised only patients with type 2 diabetes and cad , another included patients with a previous mi who did not have frank type 2 diabetes , and the third included only patients with esrds . despite such apparent phenotypic heterogeneity , the effect of the q121 variant was not heterogeneous across the three studies .
not only did this allow us to analyze the three cohorts together , increasing statistical power , but it also suggests that our findings may be generalizable to all high - risk patients , irrespective of their background clinical characteristics .
whether the predictive role of the q121 variant extends to situations characterized by a more moderate cardiovascular risk remains to be determined .
we acknowledge that , mainly because of the relatively small size of our samples , the significance level of our findings is still compatible with a false - positive result .
however , this seems unlikely given that the association between q121 was not heterogeneous across the three cohorts and , importantly , was further confirmed in cross - sectional studies as far as the interaction with obesity in diabetic patients is concerned .
we also acknowledge that due to the relatively small sample size of the studies that we analyzed , we can not exclude that the gene - by - obesity interaction that we observed among diabetic patients also occurs among nondiabetic individuals , as is the case for the modulation of insulin resistance ( 2527 ) .
therefore , our findings need further replication in larger samples before they can be considered as established . finally , because this study was entirely performed in individuals of european ancestry , we do not know whether our findings can be extended to populations of different race . in conclusion ,
pending confirmation in further larger studies , the q121 variant has the potential to become a clinical tool for identifying those very high - risk patients who are especially prone to major cardiovascular events and need , therefore , to be targeted with specific and even more aggressive preventive strategies . | objectiveinsulin resistance ( ir ) and cardiovascular disease may share a common genetic background .
we investigated the role of ir - associated enpp1 k121q polymorphism ( rs1044498 ) on cardiovascular disease in high - risk individuals.research design and methodsa prospective study ( average follow - up , 37 months ) was conducted for major cardiovascular events ( myocardial infarction [ mi ] , stroke , cardiovascular death ) from the gargano heart study ( ghs ; n = 330 with type 2 diabetes and coronary artery disease ) , the tor vergata atherosclerosis study ( tvas ; n = 141 who had mi ) , and the cardiovascular risk extended evaluation in dialysis ( creed ) database ( n = 266 with end - stage renal disease )
. age at mi was investigated in cross - sectional studies of 339 type 2 diabetic patients ( n = 169 from italy , n = 170 from the u.s.).resultsincidence of cardiovascular events per 100 person -- years was 4.2 in ghs , 10.8 in tvas , and 11.7 in creed . hazard ratios ( hrs ) for kq+qq versus individuals carrying the k121/k121 genotype ( kk ) individuals were 1.47 ( 95% ci 0.802.70 ) in ghs , 2.31 ( 95% ci 1.224.34 ) in tvas , and 1.36 ( 95% ci 0.882.10 ) in creed , and 1.56 ( 95% ci 1.152.12 ) in the three cohorts combined . in the 395 diabetic patients , the q121 variant predicted cardiovascular events among obese but not among nonobese individuals
( hr 5.94 vs. 0.62 , p = 0.003 for interaction ) .
a similar synergism was observed in cross - sectional studies , with age at mi being 3 years younger in q121 carriers than in kk homozygotes among obese but not among nonobese patients ( p = 0.035 for interaction).conclusionsthe enpp1 k121q polymorphism is an independent predictor of major cardiovascular events in high - risk individuals . in type 2 diabetes , this effect is exacerbated by obesity .
future larger studies are needed to confirm our finding . |
in the past two decades , molecular biology research has revealed the intimate mechanisms of epidemiologically significant diseases , such as cancer , infections and immunological disorders . as the next step beyond seeking the mechanisms involved ,
scientists are now increasingly making it possible to regulate human biological reactions . in recent years
, there have been breakthroughs in genetic engineering related to the inventory and methods necessary to physically construct and assemble biomolecular parts , such as synthetic rna - based regulatory systems .
synthetic biology relies on the engineering of biological systems that perform human - defined functions and on the synthesis of complex , biologically based systems that show functions that do not exist in nature . despite the possible advantages for clinical applications ,
more work remains to be done to elucidate the principles of biological design , and to overcome the scientific and technical challenges in designing and building more effective systems that are harmless to humans and therefore useful for clinical applications .
a recent study by chen et al . has produced a significant advance in solving such issues and therefore potentially bridging the gap between the bench and the bedside for synthetic rna - based regulatory systems .
the authors developed a modular device composed of a sensor ( an aptamer ) and a gene - regulatory component ( a hammerhead ribozyme ) and tested its ability to affect the expression of cytokines important for the function of t - lymphocytes in mouse and human systems .
first , it represents the logical continuation of years of experimental work performed by the same group , coming from a team that understands the way a synthetic rna - based regulatory system works and its immediate practical applications .
in fact , in a previous study , also published in proceedings of the national academy of sciences of the united states of america , the authors were the first to develop and set up universal rna - based regulatory platforms , called ribozyme switches , by using engineering design principles . in the present report
, the authors expanded the advantages of such biomodular platforms to a broader range of applications .
they were able to do so by the reliable de novo construction of modular , portable and scalable control systems that can achieve flexible regulatory properties , such as up- and down - regulation of target expression levels and tuning of regulatory responses to fit application - specific performance requirements .
second , the authors applied the synthetic rna regulatory device to a significant medical issue , the use of adoptive cell transfer ( act ) .
the act strategy uses t - cell - based cytotoxic responses to attack malignant cells ( or any other types of abnormal cells ) that escape the body 's natural surveillance by using t cells that have a natural or genetically engineered reactivity to a patient 's cancer cells . for this purpose ,
t cells have first to be naturally or genetically engineered to react against a tumor - specific antigen , then expanded and made more effective in vitro , and finally adoptively transferred into a cancer patient . however , the clinical efficacy of act , so far , has been limited .
there are many reasons for this , and insufficient persistence and reactivation of infused t cells are among the main ones .
conventional strategies for enhancing the persistence of transferred t cells include ablation of all white blood cells ( myeloablative methods ) , such as total body irradiation and administration of toxic levels of interleukin ( il)-2 .
however , myeloablation is associated with considerable morbidity , caused by decreased immune response and increased risk of infection .
report on a synthetic rna regulatory system , which marks a new era in adoptive t - cell therapy because of the increase in the amount and survival of infused t cells found with this system .
their system for the control of mammalian t - cell proliferation is based on a platform of assembled rna devices formed by a modular sensor ( aptamer ) and a gene - regulatory ( hammerhead ribozyme ) component .
this device converts a small - molecule input to an increased gene expression output , in this particular case cytokine production . in more detail , the authors fused a theophylline ribozyme switch to the 3 ' untranslated region of a tri - functional transgene ( cd19-tk - t2a - il15 ) encoding il-15 ( potent survival / proliferative cytokine of t cells ) , mutant hsv-1 thymidine kinase ( acting as a reporter and as a suicide protein in the presence of ganciclovir ) and cd19 ( a marker for fluorescence - activated cell sorting and immunomagnetic selection ) . using this system ,
they could strictly measure ( by monitoring the expression of cd19 ) and control ( by modulating the levels of the input molecule ) the biological response ( cell proliferation / viability ) .
in addition to all the in vitro evidence , the authors demonstrated that this system worked in vivo and effectively modulated the t - cell growth rate in mice in response to theophylline administration .
the growth rate was increased to 32% in the presence of theophylline over a 14 day study in mice .
they further investigated its possible clinical application by transducing primary human central memory t cells with this system . in vitro results showed that the population of live central memory t cells increased by 24% and that apoptotic cell population was decreased by 54% in the theophylline - responsive system .
finally , the presented gene regulatory system showed significant advantages over available gene regulatory techniques ( synthetic inducible promoters ) ; in particular , it provides a wide range of flexibility for clinical settings .
firstly , the ribozyme switches can be easily programmed to respond to different drug molecules .
secondly , the system can be stringently controlled and finely tuned by adding additional drug - responsive ribozyme switches ( up to four ) , therefore achieving lower basal gene expression levels .
thirdly , this system shows tight drug - mediated regulation of growth over an extended time period . taking all these features into consideration , it is feasible that this new synthetic rna - based regulatory system could have straightforward clinical utility .
it is likely that combining this new modular device framework with upcoming advances in synthetic biology will strongly support the tailoring of rna - based regulatory systems to diverse applications in various clinical and laboratory environments . yet applying these rna - based regulatory systems in clinical practice
in addition , there are many other factors that limit the use of adoptive t - cell therapy for cancer . for example
, the failure of adoptive immunotherapy against cancers lies in the absence of tumor - specific sources of t cells .
if such obstacles are not overcome , efficacy of these systems will be significantly limited in clinical practice .
also , recent data support the combined roles of protein - coding genes and non - coding rnas , such as micrornas , in the pathogenesis of frequent diseases ( such as cancer , immune and cardiac disorders ) .
one question for the future is whether such devices can be adapted for the regulation of the functions of non - coding rnas and micrornas .
the published research is good news , but it would be better to hold our cheers until the clinical trials are successfully completed , which we hope will be in the near future .
gac received his md and phd at carol davila university of medicine in bucharest , romania . after working on cytogenetics as an undergraduate student with dragos stefanescu in bucharest , he completed training in cancer genomics in massimo negrini 's laboratory at the university of ferrara , italy . in 2000
he became a postdoctoral fellow at the kimmel cancer center in philadelphia , in carlo croce 's laboratory .
since july 2007 he has been an associate professor in experimental therapeutics at the md anderson cancer center and studies the roles of micrornas and other non - coding rnas in cancer initiation and progression , as well as the mechanisms of cancer predisposition , and explores new rna therapeutic options for cancer patients .
skl graduated from yonsei university medical school , seoul , south korea with an md and phd and is an assistant professor in the department of gastroenterology , severance hospital , seoul .
the focus of his scientific research is understanding the roles of non - coding rnas in gastrointestinal cancers . in march 2009
, he began working in gac 's laboratory studying the roles of non - coding rnas , including micrornas in the initiation and development of gastrointestinal cancers , as well as the identification of new non - coding rna biomarkers .
gac is supported as a fellow at the university of texas md anderson research trust , as a fellow of the university of texas system regents research scholar and by the cll global research foundation .
work in gac 's laboratory is supported in part by nih , by dod , by developmental research awards in breast cancer , ovarian cancer and leukemia spores , and by a 2009 seena magowitz pancreatic cancer action network aacr pilot grant . | synthetic rna - based regulatory systems are used to program higher - level biological functions that could be exploited , among many applications , for in vivo diagnostic and therapeutic applications . chen and colleagues have recently reported a significant technological advance by producing an rna modular device based on a hammerhead ribozyme and successfully tested its ability to control the proliferation of mammalian t lymphocytes .
like all exciting research , this work raises a lot of significant questions .
how quickly will such knowledge be translated into clinical practice ? how efficient will this system be in human clinical trials involving adaptive t - cell therapy ?
we discuss the possible advantages of using such new technologies for specific therapeutic applications . |
the wilms ' tumor suppressor gene ( wt1 ) , located at chromosome 11p13 , has 10 exons , spans -50 kb , and encodes a zinc - finger - transcription factor presumed to regulate the expression of numerous target genes through dna binding ( 1 ) .
wt1 was initially identified as a gene inactivated in wilms ' tumor , although the estimated percentage of wilms ' tumors ( wts ) with wt1 mutations is only 10 - 15% .
investigations of wt1 have revealed the normal physiologic functions of this gene in embryogenesis , gonadogenesis and nephrogenesis ( 1 ) .
wt1 has been shown to play a role in kidney induction ( 2 ) and during later steps of nephrogenesis ( 3 ) , and there are suggestions that wt1 may play an important role in the maintenance of normal podocyte function ( 4 ) . besides wilms ' tumor , a number of human diseases have been shown to be associated with mutations of the wt1 gene ( 5 ) .
genital abnormalities are noted in three of these disorders , wagr syndrome ( wilms ' tumor , aniridia , genitourinary malformation , and mental retardation ) , denys - drash syndrome ( dds ) , and frasier syndrome ( fs ) , and the analysis of wt1 knockout mice also suggested a fundamental role of wt1 in gonad development ( 6 ) .
wilhelm and englert reported that the wt1 regulates early gonad development by activation of steroidogenesis factor 1 , sf1 , which encodes an orphan nuclear receptor that regulates the expression of several genes involved in steroidogenesis and gonadal development ( 7 ) .
dds traditionally encompasses patients with the triad of congenital nephrotic syndrome leading to end - stage renal failure ( esrf ) , xy pseudohermaphroditism and wilms ' tumor ( 8 , 9 ) .
subsequent reports described patients with incomplete forms of this syndrome ( 10 - 12 ) , and the definition of dds has now been widened to include xy individuals with mild to severe genital anomalies as well as xx individuals displaying nephropathy and wt ( 13 ) .
the nephropathies in dds are characterized by the histological finding of diffuse mesangial sclerosis ( dms ) , a finding also described in some cases of congenital nephrotic syndrome ( isolated diffuse mesangial sclerosis , idms ) .
analysis of genotype - phenotype correlation in idms suggests that at least some idms patients have a variant form of dds due to wt1 mutations ( 14 ) .
attempts to correlate the phenotype of dds and different wt1 mutations did not provide clear - cut results ( 15 ) , and no correlation could be established between any particular mutation and the occurrence of wilms ' tumor .
however , analysis of a large number of patients might provide clues to the role of each mutation , and might help in understanding the normal physiology of nephrogenesis and the prediction for tumorigenesis . in this report
, we present two cases of idms with different wt1 gene mutations that had previously been reported in association with dds .
she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .
laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .
a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .
a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .
analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .
the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation .
she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia
laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and
a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .
analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .
peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .
she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive .
she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .
laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .
a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .
a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .
analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .
the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation .
she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia
were detected and she was referred to us . on admission , generalized edema was noted .
laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and hematuria were detected .
a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .
analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .
peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .
she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive .
the mutation present in the second patient was previously reported in a dds patient with early onset esrf resulting from dms , ambiguous genitalia , and a 46 xy karyotype .
autopsy showed a streak gonad and nephroblastomatosis , but no wts were revealed ( 16 ) .
as in our second patient , this boy reached esrf within a few weeks . considering that esrf in dms
is reached within a few months or years ( 17 ) , early onset esrf in patients is noted as a feature of this mutation .
the mutation in the first patient was also previously reported in a dds patient ( 46 xy , female genitalia , streak gonad , dms , gonadoblastoma , without wt ) ( 18 ) .
it is well known that wt1 has many roles during gonadogenesis , and since wt1 is expressed in the same cell lineage as sry , the y - located testis determinant , wt1 may be acting upstream to sry during development of the genital ridge ; perhaps controlling sry expression , or interacting directly with sry during sex determination , or functioning immediately downstream of sry ( 19 ) .
wt1-null mutant mice of both sexes fail to develop kidneys and gonads , indicating that wt1 acts upstream of the sex - determining decision ( 6 ) .
frasier syndrome ( fs ) is another distinctive disease associated with wt1 mutation , characterized by male pseudohermaphroditism and nephropathy with late onset esrf , and a frequent association with gonadoblastoma ( 20 ) .
molecular analysis of a familial case of fs has been described , which provided a clue to wt1 's effects on gonadogenesis .
two sisters in their late teens showed signs of renal disease and each sister has a donor splice - site mutation that was predicted to result in an imbalance of the kts+/kts- isoforms .
one sister was 46 xy , with complete gonadal dysgenesis with normal female phenotype , while the other was 46 xx , with apparently normal ovarian development and function .
this suggests that either wt1 has a male - specific function in sex determination or that testis formation is much more sensitive to dosage effects than ovarian formation ( 21 ) .
our two cases are female with karyotype 46 xx , which might be the reason for the absence of gonadal abnormalities , and we suspect that if a patient is born with karyotype 46 xy , there may be a possibility of dds development . although male patients have been described ( 14 , 22 ) , idms most often occurs in females ; however , the percentage of patients with wt1 mutation - positive idms , the sex ratio and the associated risk of wilms ' tumor are still unknown .
a recent study of seven japanese idms patients reported a low rate of wt1 mutation ( 2/7 ) and a low risk of wilms ' tumor ( 0/7 ) ( 23 ) .
the mutations observed in these japanese patients were different from those observed in the previous reports of dds , and in the removed kidneys , nephrogenic nests were not found . therefore , it was concluded that in idms with wt1 mutations , the risk of wilms ' tumor might not be very high .
our report is limited to two patients , but both mutations were already associated with dds , and the early onset esrf in patients with the 396 asp ( gac ) > tyr ( tac ) mutation is distinctively similar .
this supports the concept that , at least in part , idms is a variant form of dds , and we suggest that idms patients require the same attention as dds patients because of the possible association with wilms ' tumor and/or gonadoblastoma .
hu et al . ( 24 ) recently reported two boys with incomplete dds with wt1 mutations who underwent prophylactic bilateral nephrectomy , and the removed kidneys showed nephroblastomatosis , which has malignant potential .
the authors suggested that missense mutations in exons 8 and 9 of wt1 can be regarded as risk factors for development of wilms ' tumor and proposed prophylactic bilateral nephrectomy and early renal transplantation .
it is still debatable whether prophylactic nephrectomies should be carried out in all children with dms before renal transplantation , and schumacher et al .
( 15 ) suggested that regular renal imaging might be necessary to evaluate the need for bilateral nephrectomy .
considering the clinical courses of the patients in the previous reports , our patients also might have had the possibility of malignant transformation , but we chose regular renal and gonadal imaging rather than prophylactic nephrectomy , and three - monthly ultrasonogram until death did not show evidence of malignancy in the second patient .
the first patient is well on peritoneal dialysis with no sign of malignancy on three - monthly screening and we are considering of nephrectomy at the time of transplantation .
the reason for the heterogeneity of the clinical features of patients with wt1 mutations is not yet clear , and we suggest that a classification based on genetic diagnosis rather than syndromatic diagnosis might be more applicable for the prediction of outcome , and routine genetic analysis to detect wt1 mutation should be applied to all idms patients .
if wt1 mutation is documented , karyotyping and serial abdominal ultrasonographies are warranted for the evaluation of tumor development in the gonad and kidney , and the analysis of genotype - phenotype correlations should be continued . | here we report two cases of isolated diffuse mesangial sclerosis ( idms ) with early onset end - stage renal failure .
these female patients did not show abnormalities of the gonads or external genitalia .
direct sequencing of wt1 pcr products from genomic dna identified wt1 mutations in exons 8 ( 366 arg > his ) and 9 ( 396 asp > tyr ) .
these mutations have been reported previously in association with denys - drash syndrome ( dds ) with early onset renal failure .
therefore we suggest that , at least in part , idms is a variant of dds and that investigations for the wt1 mutations should be performed in idms patients . in cases with identified wt1 mutations , the same attention to tumor development
should be required as in dds patients , and karyotyping and serial abdominal ultrasonograms to evaluate the gonads and kidney are warranted . |
titanium dioxide ( tio2 ) is a component of many sunscreens , soaps , shampoos , toothpastes , cosmetics , paper products , plastics , ink , paint , and building materials 1 in both its bulk form and its nanoform .
it is also used in human food as a colorant and inactive ingredient , where it can also be present in both forms 1 , 2 . from 1916 to 2011 , an estimated total of 165 050 000 metric tonnes of tio2 pigment were produced worldwide ( bulk form and nanoform combined ) , with a current annual estimated production of more than 6 million tonnes / yr 2 .
reviews of nanotio2 toxicology are available across various evolutionary groups of species 3 , 4 , 5 , 6 , 7 , 8 , often summarizing half maximal effective concentration ( ec50 ) , half maximal inhibitory concentration ( ic50 ) , and median lethal concentration ( lc50 ) values .
nanotio2 is also photoactive and produces reactive oxygen species ( ros ) on illumination 9 .
reactive oxygen species can be detrimental to many organisms , causing oxidative damage , cell injury , and ultimately death 10 .
recently , it has been argued that photoactivation of nanotio2 under natural levels of sunlight is sufficient to affect the output of lc50 and ec50 values in standard toxicology tests 11 , 12 .
the majority of studies investigating nanotio2 toxicity did not take photoactivation into account and performed tests either in dark conditions or under indoor commercial artificial lighting that did not simulate natural solar irradiation .
the aim of present study was to derive a phototoxicity ratio between the results of the nanotio2 experiments conducted in the absence of sunlight and conducted in the presence of solar or simulated solar radiation ( ssr ) . to achieve this aim , we searched the literature for studies that included nanotio2 experiments both with and without irradiance under the same experimental setup and otherwise identical conditions .
therefore , the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of natural sunlight or ssr .
such corrections also may be important for regulators and risk assessors when reviewing previously published data .
for example , one of the current challenges for conducting risk assessment of nanoparticles such as tio2 is lack of consistent toxicity data because of the varieties of materials and test conditions .
regulatory thresholds for nanotio2 do not exist currently ; however , regulation of nanoparticles discharge and monitoring in aquatic environment is anticipated in the future .
it is expected that regulators will use the phototoxicity ratio when deriving thresholds for nanotio2 because the majority of the published literature reported toxicity endpoints for nanotio2 in the absence of natural sunlight or ssr .
of course , the phototoxicity ratio value calculated in present study is not absolutely precise and correct for all environmental conditions and species ; it does , however , considerably reduce the possible error of data endpoints obtained in the absence of natural sunlight or ssr .
it also mitigates uncertainties in the risk assessment process by taking into account the photoactivation and phototoxicity of nanotio2 .
a comprehensive literature review was conducted to collect available toxicity endpoints for nanotio2 . the literature search ( september 2014 ) was performed within 4 databases web of science , scopus , google scholar , and the university of british columbia library database using the following keywords in various combinations : titanium dioxide , tio2 , nanoparticles , phototoxicity , photoactivation , ec50 , lc50 , ic50 , and lowestobservedeffect concentration ( loec ) .
abstracts of numerous hits were read , and downloaded papers were checked for useful information . only papers that reported results under the same environmental conditions for 2 different nanotio2 exposure groups ( with and without ssr ) in the form of ec50 , lc50 , ic50 , loec , or a ratio were selected .
thus , all included data were based on dose response curves , ensuring the highest possible quality .
the phototoxicity ratio ( pr ) was calculated in the form of a ratio :
pr = tio2 lc50 , ec50 , ic50 , loec without sunlight or ssrtio2 lc50 , ec50 , ic50 , loec with sunlight or ssra phototoxicity ratio greater than 1 means nanotio2 is phototoxic . in a few isolated cases ,
results were not given in the form of a number ; but it was possible to derive a number based on figures provided . because the papers reported only irradiance ( power of electromagnetic radiation per unit area ) intensity and not actual insolation ( total amount of solar radiation or ssr energy received on a given surface area during a given time ) , insolation value was calculated when possible .
insolation was calculated based on the irradiance ( w / m ) , actual duration of irradiance ( h ) , and total duration of the toxicity test . in cases
in which irradiance intensity was reported in units other than w / m , the data were converted for consistency . for the studies in which data existed only in the form of full spectrum insolation
, it was important to at least approximate the levels of ultraviolet a ( uva ) and ultraviolet b ( uvb ) used in the studies . at sea level ,
uva spectra is accountable for 5.7% of the total sunlight , whereas uvb is accountable for 0.3% of the total sunlight 14 .
thus , uva and uvb approximations were performed on the studies reporting full spectrum with factors of 0.057 and 0.003 for uva and uvb , respectively .
such conversions allowed us to determine whether uva and uvb levels used in the studies were of environmental relevance by comparing the data with published averaged uva and uvb levels over europe .
because the focus of the present study is environmental relevance , in all cases , data points were excluded from evaluation if testing conditions did not represent environmentally relevant exposure conditions , such as in vitro toxicity tests with cells .
nanotio2 toxicity was reported as greater than the highest exposure concentration with no negative toxic effects . in those cases ,
greater than value was from the control tio2 group that was not exposed to ssr or sunlight .
this might have led to a slight underestimation of the phototoxicity ratio value , which can be seen as a conservative approach . in all cases ,
the crystal structure of the nanotio2 particles , their primary particle size , and their hydrodynamic diameter were reported , and the data are presented in table 1 .
therefore , collected data are a mixture of both anatase and rutile crystal forms as well as various particle sizes . ecosystems generally contain a mixture of all sizes and types of crystal structures of anthropogenically introduced nanoparticles with which decision makers have to cope simultaneously ; thus , the aim of the phototoxicity ratio is to provide a distinct value within a muddle .
the coating of nanotio2 was not taken into account when evaluating phototoxicity of nanotio2 , since all of the collected studies have investigated exclusively bare nanotio2 .
review of nanotitanium dioxide ( tio2 ) phototoxicity to various species anatase ( a ) or rutile ( r ) .
derived data based on the presented data . calculated as 0.45 m distance ; 30% shadow angle ; and 250 w light source . not in total darkness .
ec50 = median effective concentration ; ic50 = median inhibitory concentration ; lc50 = median lethal concentration ; loec = lowestobservedeffect concentration ; uva = ultraviolet a ; uvb = ultraviolet b ; pr = phototoxicity ratio ; noec = noobservedeffect concentration ; na = data are not available .
data were checked for normality with the kolmogorovsmirnov test and were found not to be of normal distribution .
spearman rank correlation was performed between the phototoxicity ratio value and time duration of the reported toxicity test , time duration of irradiance , irradiance intensity , insolation , and the organism taxa to determine whether any of these variables drove the output value . in the case of organism taxa , for the purpose of analysis ,
a code of 5 different digits was assigned to bacteria , algae , invertebrates , fish , and amphibians .
kruskalwallis analysis of variance with a post hoc multiple comparison and/or mannwhitney u test were also performed where applicable .
validation of phototoxicity ratios in correction of toxicity tests endpoint values was performed on data obtained in absence of sunlight or ssr .
phototoxicity data summarized in table 1 ( obtained in the presence of sunlight or ssr ) , served as a control group .
results were log 10 transformed and then statistically compared with either log 10 ( data ) , log 10 ( data / median phototoxicity ratio ) , or log 10 ( data/75% phototoxicity ratio quartile ) .
these 3 groups of results originated from the same set of analyzed studies but were obtained in the absence of sunlight or ssr .
the literature search resulted in 25 usable references , from which 62 pairs of data were generated for calculation of a phototoxicity ratio ( table 1 ) . in total , experiments were performed on 20 different species , ranging from bacteria to amphibians .
applied total irradiance was between 0.46 w / m and 231 w / m ( mean , 35.63 w / m ; median , 17 w / m ) , and effective total insolation was between 0.013 wh / m and 200 wh / m ( mean , 17.44 wh / m ; median , 2.83 wh / m ) .
the recalculated and approximated insolation mean and median data are , respectively , 5.64 w / m and 1.7 w / m for uva , and 0.243 w / m and 0.015 w / m for uvb .
the majority of the studies have used the same nanotio2 products ( p25 degussa ) , resulting in a fairly similar size span of primary particle diameter .
phototoxicity ratio minimum and maximum values were 0.84 and 16 778 , respectively , and mean and median values were 407.5 and 3.7 .
the discrepancy between the mean and median was caused primarily by the data associated with the cladocera taxon .
when the data were analyzed for susceptibility of bacteria , algae , invertebrates , fish , and amphibians to phototoxicity , the invertebrates were significantly different compared with other groups .
nanotio2 was significantly more toxic to invertebrates after exposure to light compared with other groups , resulting in a greater phototoxicity ratio ( kruskalwallis followed by post hoc multiple comparison ) .
on the other hand , the spearman rank correlation test was not statistically significant for phylogeny and phototoxicity ratio ( decreased or increased phototoxicity of nanotio2 from species on the lower organism stadium , such as bacteria , toward more complex organisms , such as amphibians ) .
also , there was no correlation between phototoxicity ratio and irradiation intensity , duration of irradiation , or received insolation .
indeed , when exclusive cladocera data were analyzed against all other taxa ( figure 1 ) , the statistical difference was highly significant ( mannwhitney u test , p < 0.01 ) . because of the clear need for data segregation , separate descriptive statistics were performed for cladocera and noncladocera phototoxicity ratio values ( table 2 ) . on average ,
nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera ( median values , 3.3 and 24.7 , respectively ) after illumination .
descriptive statistics of phototoxicity ratio ( pr ) values significant statistical difference was observed between true
phototoxicity data and data obtained in the absence of sunlight or ssr ( figure 2 ) .
once the data were corrected by dividing data obtained in the absence of sunlight or ssr with a median phototoxicity ratio or a 75% quartile phototoxicity ratio value , there was no longer statistical difference compared with the data obtained in the presence of sunlight or ssr ( figure 2 ) .
however , we do not claim that values for the median phototoxicity ratio and the 75% phototoxicity ratio quartile are definite , because they will change over time as more data points become available from future studies .
comparison of uncorrected data ( top ) , corrected data with median phototoxicity ratio ( middle ) , and corrected data with 75% phototoxicity ratio quartile ( bottom ) obtained in the absence of sunlight or simulated solar radiation ( ssr ) with true data obtained in the presence of sunlight or ssr .
cladocera and noncladocera data were corrected separately with the group corresponding median or 75% quartile values .
the fact that the cladocera taxon was more sensitive to nanotio2 phototoxicity can not be explained by the intensity of irradiation or received insolation during testing , because such correlation was not statistically significant ( spearman rank correlation test ) . in cladocerarelated experiments ,
the original publications from which data were derived provided no evidence that cladocera were exposed to any specific grade , type , or size of nanotio2 particles to which other taxa were not exposed .
ultraviolet sensitivity of the taxon has to be ruled out as well , because appropriate exposure controls to uv were included and no increase in toxicity was detected . although uv is toxic and lethal to cladocera at higher exposure doses , numerous protection mechanisms prevent hazardous occurrences at lower doses 15 .
both uv and nanotio2 toxicity are based on ros , and oxidative stress was indicated in cladocera exposed to either uv 15 , 16 or nanotio2
17 .
however , this does not necessarily mean that uv and nanotio2 have the same toxicity mechanism .
whereas generation of ros and consequently oxidative stress following exposure to uv radiation requires endogenous photosensitizer molecules , generation of ros by nanotio2 under uv radiation is a direct photochemical process , and the substantial ros production can readily damage or kill cells or organisms such as cladocera .
why cladocera are more sensitive to irradiated nanotio2 remains unclear , and more targeted research is needed .
however , one possible explanation for the high sensitivity of cladocera to nanotio2 phototoxicity is that photoinduced ros on the surface of cladocera carapace may interfere with the respiratory gas exchange .
in fact , surface attachment of nanotio2 to cladocera carapace has been observed in previous studies 18 , 19 , and the inner wall of the carapace is a major site of respiratory gas exchange for cladocera 20 .
sunlight is composed of visible , uv , and infrared spectra . some of the analyzed studies reported irradiance values exclusively within the uv spectrum , whereas others reported values for the full spectrum .
thus , insolation data also were presented based on reported irradiance spectrum . when the data were segregated into what appeared to be insolation values for the full spectrum , the mean insolation was 25.9 wh / m , and the median was 5 wh / m . the studies that supposedly only reported values for the uv spectrum had a mean insolation of 8.72 wh / m , with a median of 2.83 wh / m . for the purpose of comparison ,
a solar constant ( irradiance of the sun when positioned at 1 astronomical unit compared with earth at zenith ) measured at the outer surface of earth 's atmosphere is approximately 1360 w / m
21 .
a significant amount of the solar constant is lost by the time sunlight reaches a location on the earth 's surface , depending on atmosphere , latitude , and time of day .
for example , average insolation of the visible spectrum during a decade of measurements over europe is between 5 wh / m and 302 wh / m in winter and between 285 wh / m and 430 wh / m in summer 22 .
therefore , both the mean and median ( 25.9 wh / m and 5 wh / m , respectively ) insolation used in the studies reporting only values for full spectrum are much less than the insolation values over europe .
the most likely culprits for tio2 phototoxicity are uva and uvb spectrum because those photons would have enough quantum energy ( uva , 3.103.94 ev per photon ; uvb , 3.944.43 ev per photon ) 23 versus energy of visible light photon ( 1.63.4 ev ) to overcome the band gap .
when uva and uvb level approximations were performed on the studies reporting full spectrum and combined with studies directly reporting uva and uvb , mean and median values were 5.64 w / m and 1.7 w / m , respectively , for uva and 0.243 w / m and 0.015 w / m , respectively , for uvb .
the actual uv spectrum insolation over europe is , on average , 0.7 wh / m to 37.7 wh / m in winter and 34 wh / m to 64.2 wh / m in summer for uva ; for uvb , the average is 0.001 wh / m to 1.08 wh / m in winter and 0.77 wh / m to 2.05 wh / m in summer 22 .
the mean and the median values for uva and uvb used in toxicity studies are well within the range of uva and uvb values over europe 22 .
therefore , the current experimental setups represent realistic and natural conditions , and the obtained results should not be doubted .
thus , levels used in experimental setups are credible for the purpose of risk assessment , since they do not exceed natural conditions .
it is important to note , however , that approximation to the uva and uvb values were based on the assumption that all of the irradiation lamps spectra used in the phototoxicity studies fully corresponded to sunlight spectrum .
although the technology has advanced significantly over the years , there is no absolute guarantee that all of the studies had the proper irradiation lamps .
furthermore , a significant amount of irradiation at sea level altitude is lost because of reflection and adsorption in the water column , according to the beerlambert law
iz = i0ekzwhere z is depth , e is natural logorithm , k is attenuation coefficient , and i
0 is the energy of the sunlight at the surface of the water .
although attenuation in pure water might not affect energy of uv light , reflectance of the water surface may , thus reducing the actual uv energy to which aquatic organisms are exposed . however
, an opposite effect may occur in shallow waters because of strong scattering of light , thus multiplying the uv exposure levels 25 .
therefore , although uv insolation levels currently used in nanotio2 phototoxicity studies are credible at the water surface level , it is still not clear whether they are credible for risk assessment below the water surface .
the fact that different studies used different exposure time and different irradiances only suggest that current scientific community does not really have a standardized toxicity test to check for the phototoxicity effects of nanoparticles .
therefore , we strongly recommend that universal agreement on irradiation time and irradiance in a standard nanomaterial phototoxicity test is necessary
. a validation test of phototoxicity ratio correction ( figure 2 ) showed that after correction with a median phototoxicity ratio value the corrected data are no longer statistically different from the real data obtained in the presence of sunlight or ssr .
data correction for the 75% quartile of the phototoxicity ratio was still not significantly different from the real data ( p = 0.052 ) but in general generated much lower endpoints ( higher toxicity ) , as expected . however , the value of 75% quartile application is that , compared with the median phototoxicity ratio , it can more successfully prevent false toxicity underestimation .
the use of phototoxicity ratio does not mean that the newly corrected data are the true representation of endpoints from toxicity tests , but rather that they are likely as close as possible .
the true correction of data can be achieved only by defining a function through regression analysis . however , because many variables such as particle size , hydrodynamic diameter , crystal structure , illumination time , irradiance , insolation , species , and organic matter content in test media will likely influence the phototoxicity of nanotio2 ( even if their effect is not statistically significant , they will contribute certain percentage of variability ) , generating such a function will be difficult .
therefore , the use of a phototoxicity ratio is an oversimplified method that can provide an approximate correction with lots of versatility .
one recent study 13 deployed a similar methodology to the present phototoxicity ratio approach to determine which is more toxic in the environment , nanosized or dissolved metals .
toxicity ratio was calculated between median lethal dose , lc50 , ec50 , and ic50 values of dissolved and nanoparticulated metals to provide corrections for threshold values in existing regulatory standards .
therefore , the ratio metric approach whether toxicity ratio , phototoxicity ratio , or nanoratio is an inexpensive , straightforward method that mitigates uncertainties for the purpose of risk assessment and management , assuming enough literature is available . in conclusion
, the present study found that nanotio2 is phototoxic to aquatic species , because the phototoxicity ratio values were substantially greater than 1 for the majority of analyzed studies .
existing literature on the subject is likely credible for the purpose of risk assessment because the insolation levels used in experimental setups did not exceed uv levels under natural conditions at the water surface .
a significant difference was observed between the phototoxicity ratios of 2 analyzed groups : aquatic species belonging to order cladocera , and all other aquatic species .
the order cladocera is very sensitive and prone to nanotio2 phototoxicity , at least in laboratorybased toxicity tests .
a median phototoxicity ratio value and a 75% quartile were chosen as the most practical approach for correcting nanotio2 toxicity endpoints obtained in the absence of sunlight or ssr . using a median phototoxicity ratio value in correction is a more conservative approach , whereas using the 75% quartile lowers the chance of underestimating toxicity and may be favored by risk assessors when analyzing previously published data .
the values for the phototoxicity ratio are not definite and may change as more data become available in the future .
all of the data used for calculation and statistical analysis are presented in table 1 of the present study , with corresponding references . | abstracttitanium dioxide nanoparticles are photoactive and produce reactive oxygen species under natural sunlight .
reactive oxygen species can be detrimental to many organisms , causing oxidative damage , cell injury , and death .
most studies investigating tio2 nanoparticle toxicity did not consider photoactivation and performed tests either in dark conditions or under artificial lighting that did not simulate natural irradiation .
the present study summarizes the literature and derives a phototoxicity ratio between the results of nanotitanium dioxide ( nanotio2 ) experiments conducted in the absence of sunlight and those conducted under solar or simulated solar radiation ( ssr ) for aquatic species .
therefore , the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of sunlight .
such corrections also may be important for regulators and risk assessors when reviewing previously published data .
a significant difference was observed between the phototoxicity ratios of 2 distinct groups : aquatic species belonging to order cladocera , and all other aquatic species .
order cladocera appeared very sensitive and prone to nanotio2 phototoxicity . on average
nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera ( median values 3.3 and 24.7 , respectively ) after illumination .
both median value and 75% quartile of the phototoxicity ratio are chosen as the most practical values for the correction of endpoints of nanotio2 toxicity tests that were performed in dark conditions , or in the absence of sunlight .
environ toxicol chem 2015;34:10701077 .
2015 the author
. published by setac . |
we report 2 patients infected with panton - valentine leukocidin ( pvl)positive mrsa t034 . each patient had a medical history typical of that reported for community - acquired mrsa of other lineages , which in most cases are pvl positive ( 8) . the first patient , a previously healthy 36-year - old male physiotherapist , sought medical care in march 2006 for a small abscess in his axilla . culture of the abscess grew mrsa .
presence of meca gene was confirmed by pcr ( 9 ) . during the next 2 months ,
his youngest child , adopted from china , had been found to be mrsa positive ( throat , perineum , and a small wound ) a month earlier during routine screening for adopted children . during subsequent screening of the family , the older sister , adopted from south korea ,
both parents were negative for mrsa at that time , which suggests that the father was newly infected when his abscess developed and that he had not acquired the strain abroad .
also , spa typing indicated that the children carried different strains from that of the father and from each other ( t286 , t1434 ) ( 10 ) .
subsequent screening of family members for mrsa on several occasions found only the father to be repeatedly positive .
the second patient , a 43-year - old male clerk , also previously healthy , sought medical attention during the summer of 2007 for a mrsa - infected elbow wound .
follow - up examination determined that he carried mrsa also in the perineum and in a chronic external otitis eczema .
the patients lived in geographically distinct areas in the western part of sweden and had no connection to each other . no animal contact
( e.g. , pets , farming ) was reported by the 2 patients , their family members , or other close contacts . both patient strains carried pvl , confirmed by identification of the luks - lukf genes ( 11 ) , and were resistant to digestion with restriction endonuclease smai when typing by pfge was attempted .
they produced -hemolysin according to phenotypic detection methods that used rabbit blood agar with hot cold analysis , which further indicated their animal origin ( 12 ) .
their drug - susceptibility profiles differed ; 1 was resistant to doxycycline and the other was resistant to ciprofloxacin , erythromycin , and clindamycin .
these strains carry pvl , a toxin partly responsible for the increased virulence of several of the mrsa clones in the community ( 8) . despite several recent publications concerning st398 mrsa ,
few have reported pvl in this lineage , which is believed to be of animal origin ( 2,4,6,13 ) .
most previous reports have described asymptomatic carriage in persons exposed to occupational hazards ( e.g. , veterinary personal and pig farmers ) ( 1,2,7 ) . however , severe clinical infections have been described ( 46 ) . in our patients these strains caused repeated infections that needed medical attention , even hospitalization .
since neither patient had even a remote connection to animals and we found no common source of infection , these strains may already be more common in our region than we had thought .
these case reports suggest that strains of this lineage may impose a threat in the community , even to patients with no obvious animal contact . | panton - valentine leukocidin ( pvl)positive methicillin - resistant staphylococcus aureus ( mrsa ) , sequence type 398 is believed to be of animal origin .
we report 2 cases of infection due to pvl positive mrsa , spa type t034 , in patients in sweden who had had no animal contact . |
the selective melatonin receptor agonism effect of ramelteon is useful for insomnia.1 ) here we wanted to present a refractory chronic migraine case , who had significant improvements in migraine after using ramelteon .
the precipitating factors were the stress , increase in physical activities and sudden cold weather .
the moderate or severe pain intensity was aggravated by or causing avoidance of increased physical activity .
the residual symptom between the migraine episodes were the head tense feeling , but not achieveing headache severity .
the computed tomography revealed no significant findings of other causes for headache , such as tumor or hemorrhage .
she ever received many kinds of medications , such as aspirin , nonsteroidal anti - inflammatory drugs , triptans , ergots , anticonvulsants and glucocorticoids .
however , the severity of migraine still remained moderate ( migraine disability assessment test [ midas ] scores , 19 ) and the associated symptoms were moderate headache , dizziness , nausea , and sensitivity to sound .
the migraine also exacerbated her insomnia problems , with fragmented sleep and inadequate sleep duration as 12 hours ( insomnia severity index [ isi ] score , 23 ) .
she did nt have any comorbid mental disorder except insomnia . due to the fear of abuse potential related to hypnotics
after 2 weeks treatment of ramelteon 8 mg / day , her sleep duration prolonged to 45 hours with less fragmentation .
in addition , her migraine severity started to decline ( midas scores , 11 ) .
after 6 months of ramelteon 8 mg / day treatment , the migraine severity continued to relieve ( midas scores , 6 ) with stable sleeping quality ( isi score , 14 ) .
in this case , we found significant improvements in migraine severity and insomnia after the use of ramelteon . since the patient did nt have insomnia before the first onset of migraine .
therefore we could speculate that the ramelteon had significant effects in migraine in such mild prolongation of sleep duration .
the melatonin might relieve the headache via the following possible mechanisms , such as anti - inflammatory effect , free radical scavenging , reduction of pro - inflammatory cytokine , membrane stabilization , nitric oxide synthase activity and dopamine release inhibition , gaba and opioid analgesia potentitation , glutamate neurotoxicity protection , neurovascular regulation,2 ) cytoprotection and antiallodynic action.3 ) the pineal gland , the primary source of serotonin and melatonin , might also play a significant role in the analgesic effect.4 ) however , the clinical trial of melatonin administration showed no significant improvements in migraine severity,5 ) which suggested the modulation of melatonin receptor might be a better option for the analgesic effects .
the modulation of serotonin system by the ramelteon would also relieve the pain sensation.2 ) in addition , the chemical structure of ramelteon was similar with that of indomethacin , which is a kind of non - steroidal anti - inflammatory medicine.2 ) in the study of animal model , the antinociceptive and antiallodynic actions of melatonin have been observed in different kinds of pathways , such as intrathecally or intracerebroventricular routes .
the use of ramelteon , a kind of mt1 and mt2 agonist , could also relieve the pain even lack of free radical scavenger effects.3 ) the state - of - art treatment of refractory chronic migraine also includes the ramelteon due to selective m1 receptor agonism.6 ) from the successful experience in this case , we can consider the use of ramelteon for refractory chronic migraine with insomnia . | the selective melatonin receptor agonism effect of ramelteon is useful for insomnia .
here we wanted to present a refractory chronic migraine case , who had significant improvements in migraine after using ramelteon .
the possible mechanism for the ramelteon in the migraine relief might be related to melatonin effects . |
it was previously suggested that atherothrombotic infarction ( ati ) and lacunar infarction ( li ) , as two different subtypes of ischemic stroke , might also differ in the set of the relevant risk factors , with ati being more associated to the atherogenic risk factors in contrast to li .
. however , decreased insulin sensitivity ( is ) , that is , insulin resistance , was observed both in ati and li , which was frequently accompanied with compensatory hyperinsulinemia in t2d patients as well as in nondiabetics [ 2 , 3 ] .
simultaneously , it has been shown that impaired balance between products of oxidative stress and the level of antioxidant enzyme activities might be the important mechanism underlying the occurrence of ischemic stroke . moreover , it was elucidated that the brain has only moderate content of glutathione - dependent enzymes , for example , glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) , together with the fact that the intact antioxidant defense could provide first line of protection from initiation and exacerbation of ischemic cerebral injury .
in addition , changes in enzymatic antioxidative defense mechanisms in patients with stroke are still controversial .
previous results implied that the majority of antioxidant enzyme activity was significantly reduced in acute ischemic stroke , possibly as a consequence of increased oxidative stress while the recent finding suggested increased levels of glutathione dependent enzymes as an adaptive mechanisms during acute cerebral ischemia . finally , due to novel facts
, oxidative stress can be an important component for astrocytic cell death following metabolic stress . until now , experimental studies provided evidence of an association between ischemic stroke and increased oxidative stress [ 8 , 9 ] , but data in humans are still heterogeneous and limited .
therefore , our study was aimed to determine is levels and three different types of antioxidant enzyme activities gsh - px , gr , and sod , in t2d with ati and li .
in this study we included a total of 93 patients with t2d , ascribed to the following groups : t2d patients with ati ( group a , n = 30 ) , and t2d with li ( group b , n = 30 ) , and t2d without ischemic stroke ( group c , n = 33 ) .
simultaneously , we involved a total of 93 nondiabetics , matched with the t2d patients regarding gender and age , and also comprising the following groups : nondiabetics with ati ( group d , n = 30 ) , nondiabetics with li ( group e , n = 30 ) , and nondiabetics without stroke ( group f , n = 33 ) . t2d was diagnosed in accordance with the criteria of the world health organization .
diagnosis of ati and li was done by a neurologist due to clinical features and brain imaging methods such as cranial computerized scan and/or magnetic resonance imaging in two consecutive examinations , during the first 7 days from the appearance of ischemic stroke .
the patients with ati or li were included in the study provided that they had not shown signs of cardioembolic cerebral infarction , or coronary heart disease based on a history of myocardial infarction with definite elevation of serum cardiac enzymes or coronary angiography .
t2d patients were treated with insulin therapy , and/or ingestion of antioxidant supplements and drugs , which might affect free radical and antioxidant activity potential ; likewise patients who had other endocrine disease or autoimmune diseases , renal or hepatic failure , current infections , neoplasms , polycythemia , or rheumatic diseases were also excluded as well as the patients with history of trauma or operation within the last 3 months .
no patient had uncontrolled hypertension , severe alcohol consumption , acute infection , or an inflammatory disease during the last 4 weeks .
all the patients , with or without ati and li , showed the similar level of their physical activity .
in addition , they were required not to smoke at least 12 hr before the tests were performed .
the patients were fully informed about the study and gave the inform consent to participate .
the study was conducted at the clinic for endocrinology , diabetes and metabolic diseases and at the clinic for neurology , clinical centre of serbia , faculty of medicine , university of belgrade , and was approved by the institutional ethics committee .
the interview , physical examination , metabolic test , and evaluation of antioxidant enzyme activities were performed in all the patients included in the study , for each patient within the same day .
hypertension was diagnosed according to world health organisation criteria ( systolic / diastolic blood pressure 140/90 mm hg ) or by the use of antihypertensive agents .
the metabolic tests were implemented at least after 6 months from the occurrence of the ischemic stroke .
the evaluation of insulin sensitivity was done by frequently sampled intravenous glucose tolerance test ( fsigt ) with minimal model analysis .
briefly , before testing , each patient was required to be at a 12 hr fasting state . during the fsigt ,
0.3 g / kg body weight of glucose was injected and the blood samples for plasma glucose ( pg ) and plasma insulin ( pi ) determination were taken immediately before and 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 14 , 16 , 20 , 23 , 24 , 25 , 27 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 160 , and 180 minutes after intravenous glucose stimulation .
insulin was injected as a continuous infusion 4 mu / kg / min between minutes 20 and 25 in order to avoid the effect of the potentially blunted insulin response .
the insulin sensitivity index ( si ) was calculated from the results of pg and pi levels by computerized minimal model analysis , using the minmod program ( kindly provided by dr .
determination of the antioxidant enzymes sod , gsh - px , gr was conducted using the commercial assays ( produced by randox laboratories ltd . , uk ) , based on spectrophotometer determination methods as described previously .
pg was determined by glucose oxidase method using a beckman glucose analyser ( beckman instruments , fullerton , ca ) .
pi was tested by radioimmunoassay ( inep , zemun , rs , double antibody kits ) .
total cholesterol , hdl cholesterol , and triglyceride concentrations were determined with the chromatography method using commercial kits ( produced by boehringer mannheim ) .
the continuous variables within each subtype of ischemic stroke were analyzed with analysis of variance ( anova ) with a post hoc bonferroni test .
all analyses were performed with the spss statistical package ( version 16.0 for windows ) .
the clinical characteristics and biochemistry parameters of t2d patients and nondiabetics with ati or li as different subtypes of ischemic stroke are shown in table 1 .
the age , gender , duration of diabetes , and duration from the onset of ischemic stroke were similar in t2d patients and nondiabetics with different subtypes of ischemic stroke , together with hba1c levels , implying satisfactory metabolic control before metabolic investigation was done . however , ldl - c level was significantly higher in t2d patients with ati compared to the t2d patients with li and t2d patients without stroke and also in nondiabetics with ati compared to nondiabetics with li and healthy controls .
there is no difference in prevalence of hypertension in patients with t2d and ati or li and t2d without ischemic stroke , while it was significantly higher in nondiabetics with ati or li than in healthy controls .
we found that si levels were significantly lower in t2d patients with ati ( group a ) and li ( group b ) compared to t2d patients without ischemic stroke ( group c ) ( 1.14 0.58 and 1.00 0.26 versus 3.14 0.62 min / mu / l 10 , resp .
also , the results showed significantly lower si levels in nondiabetics with ati ( group d ) and li ( group e ) compared to healthy controls ( group f ) ( 3.38 0.77 and 3.03 0.72 versus 6.03 1.69 min / mu / l 10 , resp .
simultaneously , pi levels were higher in t2d patients , ati ( group a ) , and li ( group b ) than in t2d patients without ischemic stroke ( group c ) ( 20.94 4.31 and 20.07 0.88 versus 16.06 0.91
mu / l , respectively , p < 0.001 ) and in nondiabetics with ati ( group d ) or li ( group e ) in comparison to healthy controls ( group f ) ( 15.57 1.86 and 15.59 1.26 versus 7.54 2.03
mu / l , respectively , p < 0.001 ) ( figure 2 ) .
when we evaluated antioxidant enzyme activities in t2d patients with ati ( group a ) and li ( group b ) and without ischemic stroke ( group c ) and in nondiabetics with ati ( group d ) and li ( group e ) and healthy controls ( group d ) , we detected the levels of the gsh - px and gr activity being significantly lower in group a and b versus c and in group d and e versus f ( gshpx : a : 21.96 3.56 versus b : 22.51 1.23 versus c : 25.12 1.67 u / ghb , p < 0.001 ; d : 24.75 3.02 versus e : 25.57 1.92 versus f : 28.56 3.91 u / ghb , p < 0.001 ; gr : a : 44.37 3.58 versus b : 43.50 2.39 versus c : 48.58 3.67 u / ghb , p < 0.001 ; d : 24.75 3.02 versus e : 25.57 1.92 versus f : 28.56 3.91 u / ghb , resp . , p < 0.001 ) ( figure 3 ) , while the sod levels did not differ between the investigated groups ( a : 769.57 72.36 versus b : 768.97 34.50 versus c : 789.18 60.28 , d : 795.23 48.28 versus e : 797.80 69.21 versus f : 813.88 45.80 mu / mghb , resp .
this analysis identified that decreased insulin sensitivity si and the decreases of gr were related to both ati and li in t2d patients .
simultaneously , this model identified decreased insulin sensitivity si and the level of gr and gsh - px in nondiabetics with ati , but predominantly decreased insulin sensitivity si in nondiabetics with li ( table 2 ) .
in this study , we directly measured the is level , together with three different types of antioxidant enzyme activities , gsh - px , sod , and gr , in t2d patients and nondiabetic individuals with two different subtypes of ischemic stroke in type 2 diabetics .
our results have shown decreased is level in t2d patients with two different subtypes of ischemic stroke , ati and li , compared to t2d without stroke , while we could not demonstrate the difference between the subtypes , and the same pattern of is changes was found in the nondiabetics . to our knowledge
, there are scarce data regarding the changes in is in t2d with ischemic stroke subtypes .
previous study also suggested that insulin resistance measured using different methodology , the short insulin tolerance test , is independently associated with markers of atherosclerosis detected on carotid arteries in t2d patients .
simultaneously , an association has been documented between insulin resistance and other markers at the large vessels , such as the pulsatility index on cerebral arteries . on the other hand
, it has been shown that insulin resistance , evaluated by the homeostasis model assessment ( homa ) index , was higher in diabetic in contrast to nondiabetic patients with li being a small vessel disease .
additionally , it has been suggested that diabetes , hypertension , and metabolic syndrome which share insulin resistance as a common mechanism , contribute to li occurrence [ 1820 ] .
however , detailed mechanisms of the possible link between the presence of ischemic stroke subtypes and insulin resistance remain to be clarified .
in addition , when we measured the level of insulinemia in these patients , the significantly higher level of insulinemia was detected in both groups of t2d patients , with ati and li .
the increases in insulin levels might be primarily consequence of the simultaneous presence of insulin resistance in the relevant groups .
however , the increases in insulin might contribute to the appearance of the ischemic stroke subtypes .
insulin , as a growth factor , might interfere with the beneficial effects of nitric oxide ( no ) on the vasculature .
moreover , insulin infusion during euglycemic insulin clamp was able to suppress endothelium - dependent vasodilation in large arteries , which is reported to be based on increased availability of endothelin-1 , leading to downregulation of nad(p)h oxidase and superoxide anion production , and endothelial dysfunction is proposed to play an important role in the pathogenesis of cerebral small vessel disease [ 23 , 24 ] .
also , our results demonstrated lower values of antioxidant enzymes in t2d patients with ati or li than in type 2 diabetics without ischemic stroke .
the patients with ati and li did not differ regarding the level of antioxidant enzymes .
these findings could be explained by the fact that in diabetes there is already reduced capacity of antioxidant protection , which is further significantly disturbed in the acute phase of ischemic stroke [ 4 , 5 ] . in our data , in patients with ati and li , we found the decreases in glutathione - dependent enzymes activity in contrast to other types of antioxidant enzyme activity , for example , sod .
these results imply a prolonged and severe depression of gluthatione dependent antioxidative defense mechanisms irrespective of stroke subtypes .
since it has been documented that free radicals are extremely difficult to measure directly , antioxidant enzymes have been proposed to represent indirect markers of oxidative stress .
numerous , but mostly experimental , studies provided evidence of an association between ischemic stroke and decreased antioxidant enzyme activity , although this possible association in humans has been less investigated [ 9 , 25 ] . moreover ,
the analyses of treatment with agents in stroke showing an ability to prevent further depression of antioxidant protection and scavenging reactive free radicals were reported to fail to restore gsh - px and gr activities [ 26 , 27 ] .
generally , a recently study that aimed to assess total antioxidant capacity and oxidative stress in diabetic and nondiabetic acute stroke patients with 2 different stroke subtypes , large and small vessel disease strokes , concluded that oxidative stress and counterbalancing antioxidant capacity are more pronounced in diabetic acute stroke patients than in nondiabetics .
the study provided different data in comparison to the previous investigations showing decreased gsh - px activity in both diabetic and nondiabetic patients with coronary heart disease when compared to controls [ 17 , 29 , 30 ] .
however , the results from our study have shown the diminished activity of both gsh - px and gr in t2d patients with ati and li , which is consistent with previous reports of decreased gsh - px levels patients with stroke [ 6 , 8 , 31 , 32 ] .
the levels of sod are found to exhibit great variations in the previous study in patients with the stroke [ 6 , 8 , 26 , 29 , 3137 ] .
our results could not detect the differences in the sod levels in different groups of patients , and thus they are in line with data showing no changes in respect to sod level in both diabetics and nondiabetics irrespective of different subtypes of ischemic stroke [ 33 , 34 , 37 ] . the tentative explanation for the inconsistent findings regarding sod might reflect the predominant role of intracellular versus extracellular fraction of sod in free radical scavenging . in our study , the detected antioxidant enzyme activities were not affected by other important factors potentially influencing the enzyme levels , for example , by hyperglycemia , aging , duration of diabetes , and presence of macrovascular complications , due to the fact that in all groups of t2d patients had similar levels of metabolic and satisfactory metabolic control and that patients were matched in respect of age , duration of diabetes , and the prevalence of macrovascular complications .
the multiple regression analysis applied to our data has demonstrated that decreased is together with the decreases in gr are related to both ati and li in t2d patients . in nondiabetics , the decreases in si levels and the diminished gr
the analysis reveals the potential difference in the mechanisms underlying the onset of the two subtypes of the stroke in t2d patients compared to nondiabetics .
the results imply that higher levels of insulin resistance combined with lower levels of gr , detected in t2d patients compared to nondiabetics , were underlying the onset of li together ati in t2d in contrast to the findings in nondiabetics . in this context ,
our results are consistent with the findings that li represents the most frequent ischemic stroke subtype in t2d .
taken together , our data imply that insulin resistance exerts its pathogenic influence on the level of gluthatione dependent antioxidant enzymes , especially in t2d .
in conclusion , our results suggest that the presence of different subtypes of ischemic stroke is associated with insulin resistance and diminished antioxidant enzyme activity in both subtypes of ischemic stroke in t2d .
the results also imply that atherogenic influence of decreased is in the different subtypes of stroke in t2d might be exerted through a significantly reduced glutathione dependent antioxidant enzyme activity , while the mechanisms relating the effects of insulin resistance and decreased antioxidant enzyme activity remain to be clarified . | we analyzed ( a ) insulin sensitivity ( is ) and ( b ) glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) antioxidant enzyme activity in type 2 diabetic ( t2d ) patients with atherothrombotic infarction ( ati ) ( group a ) , lacunar infarction ( li ) ( b ) , or without stroke ( c ) and in nondiabetics with ati ( d ) , li ( e ) , or without stroke ( f ) .
ati and li were confirmed by brain imaging is levels were determined by
minimal model ( si index ) , and the enzyme activity by spectrophotometry . in t2d patients ,
si was lower in a and b versus
c ( 1.14 0.58 , 1.00 0.26 versus 3.14 0.62 min1/mu / l 104 , p < 0.001 ) and in nondiabetics in d and e versus f ( 3.38 0.77 , 3.03 0.72 versus 6.03 1.69 min1/mu / l 104 , p < 0.001 ) .
also , gsh - px and gr activities were lower in a and b versus c ( gsh - px : 21.96 3.56 , 22.51 1.23 versus 25.12 1.67 ; gr : 44.37 3.58 , 43.50 2.39 versus 48.58 3.67 u / ghb ; p < 0.001 ) and in d and e versus f ( gsh - px : 24.75 3.02 , 25.57 1.92 versus 28.56 3.91 ; gr : 48.27 6.81 , 49.17 6.24 versus 53.67 3.96 u / ghb ; p < 0.001 )
. decreases in si and gr were significantly related to both ati and li in t2d .
our results showed that decreased is and impaired antioxidant enzymes activity influence ischemic stroke subtypes in t2d .
the influence of insulin resistance might be exerted on the level of glutathione - dependent antioxidant enzymes . |
the formation of spores is a survival mechanism of microorganisms when exposed to unfavorable environmental conditions ( e.g. , heavy metal stress , nutrient limitations ) leading to a dormant or resting
growth state [ 1 , 2 ] . a variety of bacteria identified in diverse habitats including soil is able to form endospores .
these physiological groups include aerobic heterotrophs ( e.g. , bacillus , paenibacillus , brevibacillus , geobacillus , thermoactinomyces , and sporolactobacillus ) , anaerobes ( clostridium , anaerobacter , and desulfotomaculum ) , microaerophiles ( sporolactobacillus ) , halophiles ( sporohalobacter ) , and phototrophs ( heliobacterium , heliophilum ) [ 3 , 4 ] . bacterial spores are characterized by a series of unique chemical features which can facilitate their identification in natural environments . besides the high content of minerals ( particularly calcium ) , spores contain high amounts of dipicolinic acid , dpa .
dpa is uniquely found in bacterial spores in amounts of up to 25% of the spore dry weight and depends on the bacterial species [ 6 , 7 ] . in solution ,
a complex is formed in the presence of terbium which shows a very strong and distinctive fluorescence spectrum .
originally , dpa was used to detect very low concentrations of terbium ( iii ) . on this basis ,
methods for the detection of bacterial endospores have been developed [ 1013 ] : by the addition of terbium , the dpa content was determined .
however , terbium - dpa fluorescence might be interfered by a series of compounds , especially when dpa has to be determined in complex samples such as sediments or soils .
it has been reported that the presence of phosphorus compounds ( especially ortho - phosphate ) reduced terbium fluorescence by as much as 98% .
the addition of aluminium compounds ( especially aluminium chloride , alcl3 ) , however , ameliorated the interference caused by the quenching substances . from a series of organic compounds (
benzoate , tryptophan , tyrosine , phenylalanine , glucose , malate , riboflavin , nad , and tryptone ) only the latter two ( especially tryptone ) reduced fluorescence significantly .
carbohydrates ( e.g. , starch , dextrine ) were reported not to interfere with the terbium fluorescence .
inorganic compounds such as calcium carbonate , sodium chloride , potassium chloride , ammonium sulphate , ammonium nitrate , and sodium nitrate did not lead to a reduction of the fluorescence , but only dipotassium phosphate did .
the aim of this study was to adopt the fluorescence - based method to determine the spore content in soils sampled from various locations .
in particular , we were interested in the differentiation between different types of soil such as grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments , the relationship of soil parameters ( carbon - to - nitrogen ratio ) on the occurrence of bacterial spores , and the distribution of spores in relation to sampling depth .
different bacillus species ( b. megaterium , b. subtilis ) were cultivated in liquid medium containing ( in g / l ) : glucose ( 3.6 ) , ammonium chloride ( 2.5 ) , magnesium sulfate ( 0.2 ) , calcium chloride ( 0.07 ) , iron sulfate ( 0.01 ) , edta ( 0.01 ) , potassium dihydrogen phosphate ( 0.6 ) , dipotassium hydrogen phosphate ( 0.9 ) , and yeast extract ( 1.0 ) .
erlenmeyer flasks ( 250 ml ) containing 100 ml of growth medium were inoculated and incubated for 10 to 15 days ( 150 rpm , 30c ) . to initiate and stimulate sporulation ,
bacteria were subsequently transferred to a sporulation medium ( identical composition , but without glucose and less ammonium chloride [ only 1 g / l ] ) .
after additional 30 days of incubation until vegetative cells were not present anymore after inspection by microscopy spores were harvested by centrifugation , immediately frozen in liquid nitrogen followed by lyophilization .
samples from different locations were collected using a stainless steel soil corer ( 15 mm in diameter ) , which was sterilized before each sampling .
cores with a maximum length of 25 cm were obtained , cut in sections of 5 cm , transferred to sterile screw cap falcon tubes ( 20 ml ) , and stored on dry ice .
after return to the laboratory , samples were immediately lyophylized or stored at 80c until further processing .
sampling sites were located in the surroundings of zurich ( switzerland ) : grassland soil , meadow ( municipalities of mnnedorf ; uerikon ; stfa ; and dbendorf ) , allotment garden ( university of zurich , irchel campus ) , pasture ( university of zurich , irchel campus ; municipality of wdenswil ) , forest soil ( municipality of stfa ) , and aquatic sediments ( river glatt in dbendorf ) . lyophilized aliquots of approximately 1 g were transferred to an eppendorf micro test tube ( 2 ml ) and ground ( by adding a 6 mm glass bead ) in tissuelyser ( retsch , haan , germany ) for 5 1 min .
elemental composition ( carbon , hydrogen , nitrogen ) of soil was performed with a chn-932 elemental analyzer ( leco corp . , st .
composition ( in % of dry soil ) varied between 2.2 and 15.4 , 0.2 and 1.4 , and 0.2 and 2.0 for total carbon , total hydrogen , and total nitrogen , respectively .
phosphate in aqueous soil extracts ( 250 mg soil in 5 ml sodium acetate buffer ; 0.2 m , ph 5 ) was determined using commercially available kits ( lck 348 and 349 ; hach lange ag , hegnau , switzerland ) .
10 mg of dry spore powder was resuspended in 10 ml sodium acetate buffer ( 0.2 m , ph 5 ) .
soil samples were thawed and 50 mg were suspended in 0.9 ml sodium acetate buffer and 0.1 ml aluminium chloride ( alcl3 , 0.5 m ) .
samples were microwaved ( berghof microwave digester mws-1 , with built - in in situ infrared temperature control ) in teflon tfm screw cap digestion vessels .
temperature and power were set to 140c and approximately 680 w ( 80% ) , respectively .
alternatively , dpa was released from spores by autoclaving the samples in screw cap glass test tubes for 15 minutes at 121c .
the identical dpa extraction protocol was applied for soil samples . however , microscopy was not possible due to the presence of mineral particles interfering the observation .
after cooling for 30 minutes , 100 l of the spore suspensions were mixed with 100 l terbium chloride solution ( tbcl3 , 30 m ) in white 96-well microtiter plates ( in 8 replicates ) .
fluorescence was immediately measured using a plate reader ( spectramax m2 , bucher biotec , basel , switzerland ) with the following settings : time - resolved fluorescence ( delay 50 s , interval 1200 s ) at an excitation wavelength of 272 nm , emission wavelength of 545 nm , and 10 endpoint readings per sample at 30c .
the number of spores in the soil samples was determined using standard addition method with spores of b. subtilis .
microwave treatment of spore suspensions and soil samples led to a fast release of dpa ( figure 1 ) . within two minutes , maximum release was obtained .
highest spore numbers up to 4 10 spores per gram dry soil were found in agriculturally used land ( meadow , pasture ) , less in forest soil .
the interference of different compounds present in soil ( e.g. , phosphate ) might lead to quenching of the fluorescence signal .
this drawback has been overcome by the addition of aluminium chloride as already shown for the determination of bacterial spores in aquatic sediments .
concentration of ortho - phosphate in soil extracts ( 22.5 m ) was reduced by the addition of aluminium chloride to concentrations below the detection limit ( < 1.2 m ) .
concomitantly , a de - colorization of the extract was observed suggesting the removal of humic acids which have also the potential to form complexes with terbium and quench the fluorescence signal .
the method based on terbium fluorescence for the detection of total numbers of bacterial endospores in soils is fast and easy .
a transect ( approximately 100 m in length ) through a grass field with different land use management ( unused meadow , allotment garden , and pasture ) gave spore numbers in the range of 5 to 9 10 spores per gram of dry soil ( figure 3 ) .
spore counts were not related to the type of land use : in allotment garden soil , counts were not significantly different from soil samples taken from a pasture ( p = 0.423 ; t - test ) . regarding the different sampling sites ,
our results show that grassland soils ( meadow , allotment garden , and pasture ) contains much more bacterial spores than forest soils and fluvial sediments .
spore content was related to the carbon - to - nitrogen ratio ( figure 4 ) . at c / n ratios > 20 only low spore counts ( 0.5 10 spores per gram of dry soil ) were detected as compared to c / n ratios < 20 .
it has been demonstrated in pure cultures of bacillus thuringiensis in a stirred bioreactor that low carbon - to - nitrogen ratios of 4 : 1 resulted in high spore counts .
in contrast however , spore formation in streptomyces coelicolor was stimulated under nitrogen - limiting conditions .
in particular , c / n ratios between 50 and 100 promoted sporulation , whereas c / n rations < 40 did not allow spore formation .
our results showed that in soils with extremely high c / n ratios , spore content was low .
the importance of c / n ratio was stressed by gao and coworkers regarding the sporulation of fungi , although fungal spores do not contain dpa . a carbon - to - nitrogen ( c / n ) ratio of 20 stimulated spore formation by fungi such as penicillium camembertii .
the fungus colletotrichum coccodes produced highest spore counts at a c / n ratio of 5 to 10 , whereas at a ratio of 40 , spore formation was significantly lower .
similarly , in plectosporium tabacinum optimal spore formation was found when c / n rations were between 5 and 10 .
the distribution of spores in marine sediments ( determined as dpa ) showed only a low correlation with the content of total organic carbon and varied with the sediment type .
highest numbers have been found in organic - rich black sediments , lowest number in sandy sediments .
it was hypothesized from anthrax outbreaks , that the high numbers of bacillus spores might be related to soils rich in organic matter that is , to a high c / n ratio .
these soil environmental conditions are suggested to support the presence and viability of b. anthracis spores .
depth distribution of spores from an area currently used as allotment garden ( cultivation of flowers and vegetables ) showed highest numbers in a horizon of 5 to 10 cm ( figure 5 ) .
the two methods evaluated ( microwaving , autoclaving ) for the mobilization of dpa from bacterial spores gave similar results .
however , microwaving was less time - consuming , whereas autoclaving allowed faster throughput of samples .
in summary , microwave treatment of soil samples followed by the measurement of fluorescence after addition of terbium proved to be a fast and easy method to assess the content of bacterial spores .
our study might provide a basis for the detection of hot spots of endospores in soil . | spore formation is a survival mechanism of microorganisms when facing unfavorable environmental conditions resulting in dormant states .
we investigated the occurrence of bacterial endospores in soils from various locations including grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments .
bacterial spores are characterized by their high content of dipicolinic acid ( dpa ) . in the presence of terbium , dpa forms a complex showing a distinctive photoluminescence spectrum .
dpa was released from soil by microwaving or autoclaving .
the addition of aluminium chloride reduced signal quenching by interfering compounds such as phosphate .
the highest spore content ( up to 109 spores per gram of dry soil ) was found in grassland soils .
spore content is related to soil type , to soil depth , and to soil carbon - to - nitrogen ratio .
our study might provide a basis for the detection of hot spots of bacterial spores in soil . |
alzheimer 's disease ( ad ) is a common neurodegenerative disease in the central nervous system .
the pathological features of ad include senile plaques formed by extracellular amyloid ( a ) aggregation , neurofibrillary tangles formed by abnormal accumulation of hyperphosphorylated tau protein , synaptic impairments , and neuronal loss in the cerebral cortex and hippocampus .
a aggregation in the brain is central to the pathological changes of ad and leads to a series of pathological events , which further promote a aggregation , resulting in cascade amplification .
the endoplasmic reticulum ( er ) , a dynamic membranous organelle , is involved in protein synthesis , posttranslational modification , folding , calcium storage , lipid metabolism , and steroid hormone synthesis .
specific stress conditions such as hyperglycemia , hyperlipidemia , hypoxia , chemical toxicants , and genetic mutations can induce the accumulation of unfolded or misfolded proteins in the er and changes in er functions , resulting in endoplasmic reticulum stress ( ers ) . in turn , ers stimulates the unfolded protein response ( upr ) and restores cellular homeostasis while sustained ers leads to cell apoptosis .
many studies have shown that ers is involved in the development and progression of neurodegenerative diseases such as ad , parkinson 's disease , huntington 's disease , and amyotrophic lateral sclerosis although the underlying mechanisms still remain blurred .
the activation of the upr pathway requires the activation of three sensor proteins : inositol - requiring enzyme 1 ( ire-1 ) , activating transcription factor 6 ( atf6 ) , and protein kinase rna - like er kinase .
these sensors serve to promote the expression of chaperone protein , glucose - regulated protein ( grp ) 78 , to repair misfolded or unfolded proteins , to accelerate er - associated protein degradation ( erad ) , and to aggravate the phosphorylation of eukaryotic translation initiation factor 2a .
sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of the transcriptional activator ccaat / enhancer - binding protein homologous protein ( chop ) and caspase-12 activation .
activated ire-1 interacts with tumor necrosis factor receptor - associated factor 2 ( traf2 ) and apoptosis signal - regulating kinase 1 ( ask1 ) via the cytosolic enzyme domain to form ire-1-traf2-ask1 complexes , which in turn stimulate the c - jun amino - terminal kinase ( jnk ) pathway to promote cell apoptosis , thus preventing the damaging impact of misfolded and secreted proteins on tissues and organisms .
genetic mutations in ad lead to a overexpression in the brain and subsequent neurotoxicity , which leads to pathogenesis .
the five familial alzheimer 's disease ( 5fad ) mice were established by overexpressing the five familial - inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida ] + v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter .
this mouse model is characterized by many pathological features similar to ad , including amyloid plaque deposition , gliosis , neuronal degeneration , neuronal loss , and cognitive deficits at the age of 45 months .
more importantly , this mouse model has been documented to show the earliest signs of intracellular a aggregation in neurons in 1.5-month - old mice , which indicates these mice as a suitable candidate to investigate the early events of ad . in this study
, we investigated the time - ordered changes of pro - apoptotic and anti - apoptotic factors to determine the role of the upr signaling pathway in cognitive decline of 5fad mice
. the outcome may clarify the role of ers in the pathological progression of ad .
the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .
marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .
experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .
they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study
were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health
the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .
the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .
the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .
mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) .
then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .
brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%
paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .
immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .
the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .
biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .
the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .
the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis , 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .
the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .
the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .
cells that were lightly stained or had irregular shapes were excluded from quantification .
brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .
the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .
alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .
triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .
louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .
the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .
supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .
subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .
horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .
quantitative data were expressed as the mean standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .
escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .
the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .
marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .
experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .
they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study
were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health
the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .
morris water maze tests were performed as described previously . the swimming behavior of mice was evaluated four times a day for 5 days .
the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .
the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .
mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) . then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .
brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%
paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .
immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .
the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .
biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .
the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .
the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis ,
6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .
the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .
the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected . cells that were lightly stained or had irregular shapes were excluded from quantification .
brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .
the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .
alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .
isolated mouse cortical tissues were added into tissue lysates ( 1% triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .
louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .
the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .
supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .
subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .
horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .
quantitative data were expressed as the mean standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .
escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .
to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .
we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,
when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .
immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .
a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .
furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .
levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .
cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .
the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .
the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .
escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *
p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .
( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .
( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .
( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .
scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid ; v : the fifth layer of the frontal cortex .
grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,
grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,
grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .
interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .
grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,
grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .
furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile , the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .
these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .
significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice .
( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) . ( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .
( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .
( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .
syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,
however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .
although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,
western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .
of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p
the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .
punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .
furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .
meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .
these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .
the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,
caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .
this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p < 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] .
significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .
( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .
( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <
0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .
( c ) western blots analysis showed the expression of p - jnk / jnk .
( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .
( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . however , it remains unknown if jnk signaling is activated to trigger chop .
the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .
these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together
, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .
to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .
we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,
when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .
immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .
a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .
furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .
levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .
cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .
the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .
the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .
escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *
p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .
( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .
( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .
( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .
scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid ; v : the fifth layer of the frontal cortex .
grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,
grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,
grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .
interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .
grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,
grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .
furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile ,
the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .
these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .
significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice . ( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) .
( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .
( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .
( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .
syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,
however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .
although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,
western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .
of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p < 0.05 ) [ figure 3a ] .
the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .
punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .
furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .
meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .
these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .
the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,
caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .
this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p
< 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] . significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .
( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .
( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <
0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .
( c ) western blots analysis showed the expression of p - jnk / jnk .
( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .
( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis .
the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .
these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together
, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .
in 5fad mice , we observed that neuronal loss and cleaved caspase-3 increase at the age of 12 months , cognitive deficit at the age of 7 months and 12 months .
interestingly , at the age of 2-month - old 5fad mice , the related factors involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased compared with those in age - matched wt mice .
these findings suggest that 2-month - old 5fad mice exhibit enhanced ers - associated upr pathway , consistent with intracellular a aggregation in neurons .
oakley et al . reported , in 5fad brain , accumulated intraneuronal a42 starting at 1.5 months of age , the presence of cerebral amyloid plaques and gliosis at 2 months of age , decreased synaptic markers including synaptophysin and postsynaptic density 95 ( psd95 ) accompanied by cognition deficit at 4 months of age , and pyramidal neuron loss in cortical layer 5 at 9 months of age .
consistently , in our study , the deposit of intra- and extra - neuronal a increased with age in 5fad brain ; neuronal loss in cortical layer 5 was observed at 12 months of age ; however , before significant neuron loss , impaired memory of 5fad mice at 7 months of age was monitored in the morris water maze .
our previous study has also shown that cognitive impairment present in 5fad mice was accompanied by structural degradation of synapses and decreased expression of synaptophysin and psd95 in the brain at the age of 67 months . under physiological conditions , approximately one - third of proteins in the er are assembled abnormally and do not form mature proteins .
aberrant proteins are bound by chaperone proteins , such as grp 78 , to be repaired or transported into the er - associated ubiquitin - proteasome system for degradation via the erad pathway , which serves as an accurate quality control system .
erad promotes the interaction between ubiquitin and proteins waiting for degradation via e1 , e2 , and e3 .
an impaired proteasome system leads to accumulation of aberrant proteins and increased risk of cell death .
sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of chop and caspase-12 activation .
ers characterized by increased ers - specific apoptosis is obvious in postmortem examinations of brain tissues of ad patients . in vitro experiments have also confirmed that a induces the cellular ers response directly , indicating that abnormal aggregation of a is responsible for ers induction .
other studies have shown that astrocytes and macrophages also induce ers . however , in our study , the co - immunostaining of grp 78 and chop with astrocytic or neuronal markers showed that ers in cortical tissues occurred mainly in neurons .
therefore , the ers - associated proteins examined here represent neuronal ers and reveal the impact of the upr on neuronal functions under a-induced stress conditions . in this study , we selected mice at 2 , 7 , and 12 months of age to investigate the changes of ers - associated proteins in 5fad mice and wt mice .
our results showed that the expression of grp 78 and syvn1 changed without statistical significance but displayed an early - increase and subsequent - decline tendency from 2 to 12 months in wt mice .
interestingly , at the age of 2 months , the expression of grp 78 and syvn1 in 5fad brains was significantly higher than that in wt mice ; however , at the age of 12 months , grp 78 level in 5fad brains significantly declined when compared with that of 2-month - old 5fad mice .
meanwhile , the expression of chop and cleaved caspase-12 increased continuously with age , either in 5fad mice or in wt mice . of note , the level of chop and cleaved caspase-12 in 5fad mice was obviously higher than that in wt mice .
altogether , we speculate that a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via pire1a - xbp1s and atf6a pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) .
interestingly , no significant differences in pjnk / jnk levels were found between 5fad mice and wt mice at different periods , which indicates that the pjnk / jnk pathway is not involved in a-induced ers . under sustained stress conditions caused by the a-associated toxic effects ,
the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .
the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death [ figure 4 ] .
schematic drawing shows that activated upr pathways of ers induced by intraneuronal -amyloid in 5fad mice .
a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via p - ire-1-xbp1s and atf6 pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) . under sustained stress conditions caused by the a-associated toxic effects ,
the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .
the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death .
upr : unfolded protein response ; ers : endoplasmic reticulum stress ; a : amyloid ; 5fad : transgenic mice with five familiar alzheimer 's disease ; p - perk : phosphorylated protein kinase rna - like er kinase ; p - eif2 : phosphorylated eukaryotic translation initiation factor 2 ; atf4 : activating transcription factor 4 ; chop : ccaat / enhancer - binding protein homologous protein ; p - ire-1 : phosphorylated inositol - requiring enzyme 1 ; xbp1s : spliced x box - binding protein 1 ; atf6 : activating transcription factor 6 ; grp 78 : glucose - regulated protein 78 ; erad : endoplasmic reticulum - associated protein degradation ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; traf2 : tumor necrosis factor - receptor - associated factor 2 ; ask1 : apoptosis signal - regulating kinase 1 ; jnk : c - jun amino - terminal kinase . in conclusion ,
current data reveal that intracellular a aggregation induces obvious ers in neurons at the early stage of 5fad brains .
imbalanced regulation of grp 78 or syvn1 and chop or cleaved caspase-12 may be involved in the resistance to the restoration of er homeostasis in a-secreting neurons . if the development of ers can be delayed or reduced appropriately or the protective functions of the upr can be enhanced exogenously , the capacity of the er to tolerate abnormal proteins can be increased to delay cell damage and disease progression .
targeting at ers to control the pathophysiological changes at the early stage of ad may be a better strategy for the prevention and treatment of ad .
this work was supported by grants from the national natural science foundation of china ( no .
xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs .
this work was supported by grants from the national natural science foundation of china ( no .
xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs .
| background : amyloid ( a ) deposits and the endoplasmic reticulum stress ( ers ) are both well established in the development and progression of alzheimer 's disease ( ad ) .
however , the mechanism and role of a-induced ers in ad - associated pathological progression remain to be elucidated.methods:the five familial ad ( 5fad ) mice and wild - type ( wt ) mice aged 2 , 7 , and 12 months were used in the present study .
morris water maze test was used to evaluate their cognitive performance .
immunofluorescence and western blot analyses were used to examine the dynamic changes of pro - apoptotic ( ccaat / enhancer - binding protein homologous protein [ chop ] and cleaved caspase-12 ) and anti - apoptotic factors ( chaperone glucose - regulated protein [ grp ] 78 and endoplasmic reticulum - associated protein degradation - associated ubiquitin ligase synovial apoptosis inhibitor 1 [ syvn1 ] ) in the ers - associated unfolded protein response ( upr ) pathway.results:compared with age - matched wt mice , 5fad mice showed higher cleaved caspase-3 , lower neuron - positive staining at the age of 12 months , but earlier cognitive deficit at the age of 7 months ( all p < 0.05 ) .
interestingly , for 2-month - old 5fad mice , the related proteins involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased when compared with those in age - matched wt mice ( all p < 0.05 ) .
moreover , ers occurred mainly in neurons , not in astrocytes.conclusions:these findings suggest that compared with those of age - matched wt mice , ers - associated pro - apoptotic and anti - apoptotic proteins are upregulated in 2-month - old 5fad mice , consistent with intracellular a aggregation in neurons . |
according to the world health organization , diabetes mellitus ( dm ) is one of the most common chronic diseases in the world , affecting 9.3 % of all adults older than 25 years .
diabetes poses a high risk for the development of cardiovascular diseases ( cvd ) , because it heralds an accelerated process of atherosclerosis and an increased risk for atherothrombotic complications .
cvds are the leading causes of death in diabetic patients and the substantial rise in diabetes prevalence will ultimately lead to an increase in the burden of diabetes - related cvd in coming decades . among the cardiovascular diseases ,
ischemic heart disease is the most frequent [ 5 , 6 ] and is associated with the highest morbidity and mortality in patients with type 2 dm . despite the advances in the treatment of coronary artery disease ( cad ) , morbidity and mortality are still high , especially in patients with type 2 dm .
moreover , despite the evolution in the treatment of hyperglycemia , patients with both cad and diabetes have worse clinical outcomes , irrespective of the treatment applied .
although dm alters the healthy functioning of arteries and blood compounds , some clinical studies suggest that myocardial responses to ischemic insults may be deficient in diabetic patients [ 11 , 12 ] , leading to higher myocardial damage and risk of complications .
thus , recent studies have focused on the understanding of such myocardial responses , aiming at discovering the mechanisms of myocardial protection to achieve less aggression and a better prognosis .
a substantial effort has been put forth to investigate any promising cardioprotective strategy to effectively reduce myocardial infarct size .
and this matter is of particular importance to diabetic cad patients . by 1986 , in a landmark experimental study , murry and colleagues demonstrated that a short antecedent period of ischemia could result in a great reduction in myocardial infarct size .
for the first time , a method for reducing myocardial cell death other than reperfusion had been discovered .
since its discovery , this phenomenon , termed ischemic preconditioning ( ip ) has been extensively studied .
currently , ip is the intrinsic myocardial mechanism that provides the greatest protection regarding the reduction in myocardial ischemic damage .
it has demonstrated a 75 % reduction in the infarcted area in animal models that have undergone the preconditioning protocol .
ip is assumed as a mechanism of myocardial protection in which brief episodes of sublethal myocardial ischemia followed by reperfusion trigger multiple intracellular pathways that ultimately result in greater myocardial resistance to a subsequent intense ischemic injury .
experimentally , this phenomenon was demonstrated by the reduction in the infarcted area by short episodes of ischemia prior to a pronounced ischemic insult . although ip was initially thought to result from the opening of collateral vessels and higher coronary flow , some interesting studies have shown that the phenomenon occurs irrespective of coronary flow changes [ 17 , 18 ] .
such studies also demonstrated that ip can be observed by sequential exercise tests ( sets ) , in which the improvement in ischemic parameters in the second of 2 sets were confirmed by invasive measurements of myocardial oxygen consumption .
moreover , the clinical observation that patients with chronic ischemic heart disease frequently describe attenuation or even cessation of angina symptoms if they rest and restart the exercise is termed warm - up or walk - through angina . this phenomenon has been related to ip and documented in studies using sets [ 19 , 20 ] . following the demonstration of ip , the cellular mechanisms underlying the phenomenon began to be investigated .
although some cellular pathways have not been fully elucidated , there is a growing understanding of some phenomena and currently it is assumed that ip may be modulated by external factors like age , diseases [ 2224 ] , as well as some specific classes of medications [ 2527 ] .
currently , the opening of k - atp channels plays a fundamental role in the cellular cascades of ip .
it is also assumed that medications that bind to this channel may interfere with the ip mechanism .
thus , some classes of medications like the oral hypoglycemic agents may also block such channels in extrapancreatic sites , such as in the heart .
it has been shown by some authors that these medications may cause the blockage of myocardial ip . however , despite the fact that some oral hypoglycemic agents may interfere with ip , it is still uncertain whether the intrinsic , complex , intracellular alterations of diabetes itself may affect the cellular mechanisms of this cardioprotective phenomenon .
some of these studies have shown that diabetes does not interfere with ip [ 31 , 32 ] , but others have shown negative influences [ 33 , 34 ] .
the great variability in these results is especially due to differences in study protocols , most notably the variability in animal models studied , in the protocols to induce diabetes , the duration of the disease , as well as the variability in myocardial injury protocols .
on the other hand , studies in humans are scarce and their results have also been conflicting [ 12 , 13 , 35 ] .
thus , in this study , we aimed at identifying ip in symptomatic multivessel cad patients and compared the results among patients with and without type 2 dm .
this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .
briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .
the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .
multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70 % obstruction in 2 or more coronary artery territories .
the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 .
treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm , associated or not with thoracic discomfort .
exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction < 0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3 months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .
after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5 days before sets .
diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests .
patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .
a telephone contact was available 24 h a day in case of questions or worsening symptoms . before treadmill test initiation ,
all patients underwent 2 sets , symptom limited , with a 30-min interval between them .
tests were conducted during the same period each day , 1 h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .
a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5 min during the recovery phase , at the onset of 1.0 mm st - segment depression , during arrhythmias , and when it was clinically relevant .
the level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . in case of disagreement
only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0 mm ) .
criteria for interrupting the exercise test were st - segment depression 3.0 mm , st segment elevation 2.0 mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .
the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0 mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0 mm , and exercise duration in seconds .
the improvement in ischemic parameters ( t-1.0 mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .
other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .
both arrhythmias and st - segment morphology were graded according to their density and severity .
according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia ,
the quartiles were defined as quartile 1 : < 90 mg / dl , quartile 2 : 90100 mg / dl , quartile 3 : 101125 mg / dl , and quartile 4 : 126 mg / dl . for glycosylated hemoglobin ,
the quartiles were defined as quartile 1 : < 5.7 % , quartile 2 : 5.76.3 % , quartile 3 : 6.46.9 % , and quartile 4 : 7.0 % .
briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . by the differences in t-1.0 mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30 % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group )
, 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .
continuous variables were compared using an unpaired student s t test or mann whitney test , when appropriate .
data are expressed as mean standard deviation or as absolute frequencies and percentages .
all tests were 2-sided , and a value of p < 0.05 was considered significant .
this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .
briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .
the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .
multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70 % obstruction in 2 or more coronary artery territories .
the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 .
treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm , associated or not with thoracic discomfort .
exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction < 0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3 months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .
after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5 days before sets .
diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests .
patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .
a telephone contact was available 24 h a day in case of questions or worsening symptoms . before treadmill test initiation ,
all patients underwent 2 sets , symptom limited , with a 30-min interval between them . the modified bruce protocol was applied .
tests were conducted during the same period each day , 1 h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .
a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5 min during the recovery phase , at the onset of 1.0 mm st - segment depression , during arrhythmias , and when it was clinically relevant .
the level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . in case of disagreement ,
only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0 mm ) .
criteria for interrupting the exercise test were st - segment depression 3.0 mm , st segment elevation 2.0 mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .
the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0 mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0 mm , and exercise duration in seconds .
the improvement in ischemic parameters ( t-1.0 mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .
other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .
both arrhythmias and st - segment morphology were graded according to their density and severity . according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia , the quartiles were defined as quartile 1 : < 90 mg / dl , quartile 2 : 90100 mg / dl , quartile 3 : 101125 mg / dl , and quartile 4 : 126 mg / dl . for glycosylated hemoglobin ,
the quartiles were defined as quartile 1 : < 5.7 % , quartile 2 : 5.76.3 % , quartile 3 : 6.46.9 % , and quartile 4 : 7.0 % .
the sample size calculation has been previously published elsewhere . briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . by the differences in t-1.0 mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30 % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group ) , 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .
continuous variables were compared using an unpaired student s t test or mann whitney test , when appropriate .
data are expressed as mean standard deviation or as absolute frequencies and percentages .
all tests were 2-sided , and a value of p < 0.05 was considered significant .
of 2,140 patients with stable cad followed at our institution , 361 met the inclusion criteria .
the main reasons for non - inclusion and exclusion are shown in fig . 1 .
thus , a total of 174 patients completed the 2 sets and had ip assessed .
the study population comprised 86 patients with dm and 88 without this diagnosis ( fig .
the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .
cad = coronary artery disease ; ckd = chronic kidney disease ; cva = cerebrovascular accident ; ecg = electrocardiogram ; ef = ejection fraction ; et = exercise test ; dm = diabetes mellitus ; ip = ischemic preconditioning ; set = sequential exercise test ( s ) study flow chart . of
the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .
cad = coronary artery disease ; ckd = chronic kidney disease ; cva = cerebrovascular accident ; ecg = electrocardiogram ; ef = ejection fraction ; et = exercise test ; dm = diabetes mellitus ; ip = ischemic preconditioning ; set = sequential exercise test ( s ) the main demographic , clinical and biochemical characteristics of the 2 groups are presented in table 1 . despite the higher prevalence of previous myocardial infarction in the diabetic population and lipid profile ( higher ldl - cholesterol levels in nondiabetic patients ) , both groups had homogeneous characteristics .
the duration of diabetes was 11.5 8.8 years ( mean sd ) , with a median of 10 years ( interquartiles ranges of 6 and 15 years ) .
table 2 shows the medications used in the two groups of patients.table 1main demographic , biochemical , and clinical characteristics of the study populationtotal n = 174diabetics ( n = 86)non - diabetics ( n = 88 )
p valueage64.1 6.864.3 8.60.84male n ( % ) 73 ( 84.9)76 ( 86.3)0.83hypertension64 ( 80)74 ( 84.1)0.54smokers7 ( 8.8)7 ( 8.6)previous smokers38 ( 48.1)33 ( 40.7)0.6non - smokers34 ( 43.0)41 ( 50.6)previous ami36 ( 46.1)17 ( 22.7)0.004cabg28 ( 34.6)23 ( 26.1)0.25pci24 ( 30.4)28 ( 32.2)0.87ef0.61 0.060.62 0.060.2tri - vessel disease38 ( 52)43 ( 51.2)1.0bi - vessel disease35 ( 48)41 ( 48.8)1.0lad disease63 ( 87.5)74 ( 86.0)1.0collateral circulation34 ( 50.7)38 ( 46.9)0.64collateral grade 2/330 ( 88.2)32 ( 84.2)0.74weight ( kg)74.5 11.073.2 12.70.51height ( m)1.66 8.11.66 9.10.75bmi26.5 2.826.4 3.20.54fasting glycemia143.5 47.099.0 9.40.0001a1c7.35 1.615.63 0.300.0001bun41.9 13.440.1 9.40.32creatinine1.04
0.231.07 0.200.37total cholesterol156.5 34.5168.9 36.30.02ldl cholesterol90.5 27.4102.0 32.70.01hdl cholesterol38.0 9.640.3 9.00.12triglycerides147.2 88.3132.6 72.30.24data are expressed as means standard deviation or as absolute and relative risks
ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoproteintable 2classes of medications used by the two groups of diabetic and nondiabetic patientsaspirin / clopidogrelstatinsbeta - blockersacei / arbcalcium blockersdiureticsoadinsulinsdm93.2 % 93.2 % 89.8 % 78.4 % 35.2 % 37.5 % 88.5 % 26.5 % non - dm96.5 % 96.5 % 86.2 % 78.2 % 36.8 % 34.5 % --
dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs main demographic , biochemical , and clinical characteristics of the study population data are expressed as means standard deviation or as absolute and relative risks
ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoprotein classes of medications used by the two groups of diabetic and nondiabetic patients
dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs among the 86 diabetic patients , 62 ( 72 % ) had an improvement in t-1.0 mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68 % ) had an ischemic improvement consistent with ip ( fig . 2 , p = 0.62).fig .
2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s ( table 3 , p = 0.12).table 3t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6
126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4 108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test .
* the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg ( table 4 , p = 0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr ( baseline)79.0 15.186.3 17.07.3 11.0hr ( peak)139.0
18.2149.7 19.36.8 12.6bp ( peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr ( baseline)79.0 14.784.4 14.95.4 8.0hr ( peak)141.7 14.3143.0 13.51.3 7.4bp ( baseline)153.3 22.6147.1 19.76.2 14.7bp ( peak)183.7 23.2180.6 22.23.12 14.7non - dm / ip + hr ( baseline)78.0
13.187.1 13.99.1 7.9hr ( peak)139.6 15.4144.3 14.74.7 5.7bp ( baseline)146.7 20.6138.7 18.38.0 11.3bp ( peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr ( baseline)82.5 12.987.1 12.64.6 7.1hr ( peak)139.3 12.4139.6 13.00.35 5.2bp ( baseline)158.6 26.2147.9 26.110.7 14.6bp ( peak)204.6 25.3194.8 28.18.9
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20 39 s versus 17 36 s , respectively , p = 0.60 ) .
the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50 % versus 63 % of patients demonstrated improvement in arrhythmias , respectively ; p = 0.41 ) .
the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5 % versus 48.3 % , respectively , p = 0.41 ) .
when the total group of patients ( n = 174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table 6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n = 122)ip - ( n = 52 )
p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig .
3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .
ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia
among the 86 diabetic patients , 62 ( 72 % ) had an improvement in t-1.0 mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68 % ) had an ischemic improvement consistent with ip ( fig . 2 , p = 0.62).fig .
2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s ( table 3 , p = 0.12).table 3t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6 126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4
108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation
. dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg ( table 4 , p = 0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation
. dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip
+ hr ( baseline)79.0 15.186.3 17.07.3 11.0hr ( peak)139.0
15.1142.1 14.73.2 5.6bp ( baseline)156.5 18.2149.7 19.36.8 12.6bp ( peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr ( baseline)79.0 14.784.4 14.95.4 8.0hr ( peak)141.7 14.3143.0 13.51.3 7.4bp ( baseline)153.3 22.6147.1 19.76.2 14.7bp ( peak)183.7 23.2180.6 22.23.12
14.7non - dm / ip + hr ( baseline)78.0 13.187.1 13.99.1 7.9hr ( peak)139.6 15.4144.3 14.74.7 5.7bp ( baseline)146.7 20.6138.7 18.38.0 11.3bp ( peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr ( baseline)82.5 12.987.1 12.64.6 7.1hr ( peak)139.3
12.4139.6 13.00.35 5.2bp ( baseline)158.6 26.2147.9 26.110.7 14.6bp ( peak)204.6 25.3194.8 28.18.9 16.5data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test
the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20 39 s versus 17 36 s , respectively , p = 0.60 ) .
the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50 % versus 63 % of patients demonstrated improvement in arrhythmias , respectively ; p = 0.41 ) .
the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5 % versus 48.3 % , respectively , p = 0.41 ) .
when the total group of patients ( n = 174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table 6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n = 122)ip - ( n = 52 )
p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig
3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip = ischemic preconditioning
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .
ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia
considering that diabetes is an independent risk factor for the occurrence of major cardiovascular events and mortality [ 5 , 6 ] , it is reasonable to consider that diabetes might damage the protective mechanism of ip in cad patients , leading to worse outcomes .
however , in this study , the presence of type 2 dm did not have any deleterious effects on the myocardial protective mechanism termed ischemic preconditioning . in this context
, our study showed that patients with type 2 dm demonstrated ip in similar frequency and intensity compared with nondiabetic patients and , interestingly , our data indicated an improvement in ischemic parameters associated with diabetes .
the analyses of the data showed a better ischemic response evaluated by rpp in diabetic patients .
thus , this study adds clinical information on some questions that have emerged from contradictory experimental studies .
the improvement in myocardial oxygen consumption , observed by the analysis of rpp , was more pronounced in patients with compared to those without diabetes , indicating better adaptation of the myocardium of diabetic patients after an ischemic insult . moreover , diabetic patients had an improvement in t-1.0 mm greater than that in nondiabetic patients , although it did not reach statistical significance .
analyzes of the total exercise time also confirmed the main results of the study , as times were similar among patients with and without dm .
similarly , the st - segment deviation morphology did not differ among diabetic and nondiabetic patients . because myocardial ischemia is a frequent cause of arrhythmias during treadmill stress tests
, we also assessed the occurrence and complexity of arrhythmias and their improvement during set .
thus , the improvement in the occurrence of arrhythmias also confirmed the main findings of the study , as the occurrence did not differ among diabetic and nondiabetic patients .
interestingly , analyzes of the percentage of patients who demonstrated ip among different quartiles of fasting glycemia and glycosylated hemoglobin showed similar rates of ip demonstration .
this analyzes infer that the differences in the intensity of glycemic control in our population did not prevent the demonstration of this cardioprotective phenomenon .
however , despite our consistent findings , the information from this study is contrary to lee s et al . and ishihara s et al . , who have also studied ip in humans .
studied diabetic and nondiabetic cad patients during percutaneous coronary interventions , and evaluated the action of hypoglycemic agents on ip .
patients underwent consecutive balloon coronary inflations . during the second sequential balloon inflation , patients had less thoracic discomfort , less myocardial lactate production , and lower st - segment deviation .
moreover , the authors observed that diabetic patients treated with glimepiride had higher lactate production compared to nondiabetic patients treated with glimepiride . despite this finding , an important limitation of this study is that there was not a direct comparison of diabetic and nondiabetic patients who had no drug interference in ip evaluation .
studied patients hospitalized due to an acute myocardial infarction and evaluated the effects of preinfarct angina , on the release of cardiac markers of necrosis , ventricular function , and in - hospital death and compared the results among diabetic and nondiabetic patients .
the authors found that in the nondiabetic population , patients with preinfarct angina had a lower release of cardiac markers of necrosis , better recovery of ventricular function , and lower in - hospital mortality , compared to patients with no preinfarct angina . on the other hand , when they analyzed diabetic patients , these variables were not different among patients with and without preinfarct angina .
thus , the authors inferred that diabetes prevented the appearance of ip . despite their findings ,
first , this was a retrospective study , which included a small number of diabetic ( n = 121 ) compared to nondiabetic patients ( n = 490 ) .
many studies have shown that some of these drugs may block ip , and it has been speculated that this interference with ip mechanisms may partially explain the worse prognosis of diabetic patients hospitalized due to an acute myocardial infarction . on the other hand ,
other experimental studies that evaluated in vitro human myocardial tissue [ 41 , 42 ] showed results that match those of the present study .
they evaluated the release of cardiac biomarkers of necrosis and the percentage of tissue viability . among other findings
, the authors found a similar intensity in myocardial protection among diabetic and nondiabetic patients .
additionally , cleveland et al . evaluated the contractile function of isolated right atrial trabeculae from cad patients , resected during coronary artery bypass graft surgery .
they showed that the diabetic group that underwent the preconditioning stimuli had similar improvement in contractile function compared to the nondiabetic group .
moreover , a small study conducted by bilinska and colleagues with diabetic patients treated with glibenclamide , gliclazide , and diet compared the demonstration of ip in these groups with that in nondiabetic patients .
besides the main findings of the study , which were related to the effects of sulfonylureas in the warm - up phenomenon , they showed that the group of diabetic patients on diet ( n = 15 ) had similar improvement in ischemic parameters compared to nondiabetic patients ( n = 17 ) .
of note , this was a secondary result , and the small sample size did not permit to make a definitive conclusion .
this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .
the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .
, the response of the myocardium to an ischemic insult may probably play an important role in prognosis .
one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .
contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .
this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .
the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .
, the response of the myocardium to an ischemic insult may probably play an important role in prognosis .
one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .
contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .
in this study , diabetes mellitus did not substantially affect myocardial ip in symptomatic cad patients . | backgroundthe influence of diabetes mellitus on myocardial ischemic preconditioning is not clearly defined .
experimental studies are conflicting and human studies are scarce and inconclusive.objectivesidentify whether diabetes mellitus intervenes on ischemic preconditioning in symptomatic coronary artery disease patients.methodssymptomatic multivessel coronary artery disease patients with preserved systolic ventricular function and a positive exercise test underwent two sequential exercise tests to demonstrate ischemic preconditioning .
ischemic parameters were compared among patients with and without type 2 diabetes mellitus .
ischemic preconditioning was considered present when the time to 1.0 mm st deviation and rate pressure - product were greater in the second of 2 exercise tests .
sequential exercise tests were analyzed by 2 independent cardiologists.resultsof the 2,140 consecutive coronary artery disease patients screened , 361 met inclusion criteria , and 174 patients ( 64.2 7.6 years ) completed the study protocol . of these , 86 had the diagnosis of type 2 diabetes .
among diabetic patients , 62 ( 72 % ) manifested an improvement in ischemic parameters consistent with ischemic preconditioning , whereas among nondiabetic patients , 60 ( 68 % ) manifested ischemic preconditioning ( p = 0.62 ) .
the analysis of patients who demonstrated ischemic preconditioning showed similar improvement in the time to 1.0 mm st deviation between diabetic and nondiabetic groups ( 79.4 47.6 vs 65.5 36.4 s , respectively , p = 0.12 ) .
regarding rate pressure - product , the improvement was greater in diabetic compared to nondiabetic patients ( 3011 2430 vs 2081 2139 bpm x mmhg , respectively , p = 0.01).conclusionsin this study , diabetes mellitus was not associated with impairment in ischemic preconditioning in symptomatic coronary artery disease patients .
furthermore , diabetic patients experienced an improvement in this significant mechanism of myocardial protection . |
most malignant heart tumors are sarcomas : myxosarcoma , liposarcoma , angiosarcoma , fibrosarcoma , leiomyosarcoma , osteosarcoma , synovial sarcoma , rhabdomyosarcoma , undifferentiated sarcoma , reticulum cell sarcoma , neurofibrosarcoma , and malignant fibrous histiocytoma .
arrhythmias can develop from intraatrial invasion , which can also lead to massive pericardial effusion and tamponade .
if the tumor mass is located in the left atrium or ventricle , embolic infarction might occur .
a 55-year - old man presented signs of progressive cardiac decompensation during the last 4 months due to tumorous obstruction of the mitral valve . in 2014 the patient underwent resection of a suspected myxoma ( 824124 mm ) in the left atrium .
however , in the histological workup , the tumor mass , resected from the posterior mitral annulus , was diagnosed as chondrosarcoma . in 2014 ,
our colleagues in the referring center noticed nodal calcification in the myocardium and the mitral valve .
the tumor board in 2014 decided on palliative care with poor sensitivity to chemotherapy and radiation and high risk for organ toxicity .
ct scan and magnetic resonance imaging ( mri ) showed no signs of malignancy outside the cardiac tissue , and the chondrosarcoma found was the primary tumor .
during the course of 12 months , the patient was in a clinically stable state with combined mild mitral valve regurgitation and stenosis and good left ventricular pump function , with ejection fraction ( ef ) 63% . in january 2016 ,
the patient first presented with recurring tumor mass on the posterior mitral leaflet ( pml ) max .
23 mm , with moderately decreasing pump function ( ef 58% ) . in april 2016
, the patient showed increasing peripheral edema and orthopnea , with no signs of angina pectoris , and nyha iii - iv .
the patient presented absolute arrhythmia with atrial fibrillation , pulse 109/min , and blood pressure 82/52 mmhg .
echocardiography showed pleural effusions on both sides and distended vena cava inferior and hepatic veins .
upon admission to our center , the patient presented a 403532 mm mass , originating from the pml ( figure 1 ) .
a cardio mri could not be performed , as the patient was unable to lie down flat due to major orthopnea .
instead , we decided to perform a cardio ct , which showed atrial roof and interatrial septum invasion .
it was not perfectly clear on the ct scan whether the tumor was infiltrating the ascending aorta .
a cerebral scan showed no signs of cerebral embolisms or metastasis . due to clinical instability
serum creatinine levels at that point were 1.56 mg / dl and bilirubin 1.10 mg / dl .
after careful review of the scan images , we decided on the implantation of a total artificial heart , as the sarcoma had already invaded the whole atrium and the complete mitral valve annulus .
a mitral valve replacement was not feasible because there would be no myocardium or endocardium to fixate the stitches in .
implantation of a self - expanding stent valve would also require an intact mitral valve annulus .
furthermore , autotransplantation was rejected for the same reasons . the severe mitral valve stenosis precluded use of a left ventricular assist device ( lvad ) or biventricular assist device ( bivad ) . the new approach introduced by bruckner et al .
has never been performed in our center and needs experience in the surgical technique as the construction of the new atria presents major challenges because they easily collapse when not prepared properly .
we decided on the syncardia total artificial heart ( tah ) ( syncardia , tucson , az ) .
as the ct scan showed no metastases and the sarcoma presented as a recurrence in the left atrium , we hoped that the patient could be listed for htx following recovery after tah implantation . in a similar setting , reich et al .
successfully transplanted a patient after tah implantation , with an occult intracardial malignancy , after a cancer - free period of 14 months . before the operation
, the patient and next of kin were informed that it would be an ultimo ratio intervention because we could not 100% foresee the invasion of the myocardium and the intraoperative circumstances .
zhang et al . demonstrated a 50% survival rate after initial surgical treatment of primary cardiac sarcomas , depending on histological grade of the tumor .
surgical treatment is the most common approach to improve survival . even as a palliative measure , reducing the tumor mass through an operation relieves orthopnea and ameliorates signs of decompensation if the patient stabilizes after the intervention . after re - sternotomy
following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk were distally resected , preserving both the aortic and pulmonary valve .
the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .
due to extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .
functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .
to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution
, the patient was transferred to the icu under moderate catecholamine support and no ventilation .
five days after the initial operation , we planned a secondary thorax closure . during the intervention
cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .
neurological examination showed the patient was comatose , with slight head movement to the left side . a right - sided facial paralysis was diagnosed . under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .
. the patient showed signs of prolonged low - output syndrome with beginning multiorgan failure .
neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .
histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .
chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .
hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading .
cardiopulmonary bypass was established via the ascending aorta and venae cavae cannulation . following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk
the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .
the atrial cuffs were prepared and broad felt strips for suture support were placed . due to
extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .
functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .
to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution
, the patient was transferred to the icu under moderate catecholamine support and no ventilation .
five days after the initial operation , we planned a secondary thorax closure . during the intervention
cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .
neurological examination showed the patient was comatose , with slight head movement to the left side .
under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .
neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .
histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .
chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .
hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading .
chondrosarcomas are cartilaginous tissue sarcomas . our patient was first diagnosed with primary cardiac chondrosarcoma .
after initial resection , relapse of the cardiac mass in the left atrium was diagnosed as early as 14 months later .
after excision of the heart for tah implantation , our pathologists found an undifferentiated high - grade spindle cell sarcoma , fnclcc g2 . with histological grading through the fnclcc classification , survival , by major correlation
cell differentiation , mitotic rate , and percentage of necrosis are evaluated in a systematic score .
since the 4 edition of the who classification of tumors of soft tissue and bone , there is a new category of tumors that can not be classified in earlier - defined categories .
there were no signs of assist malfunction during icu stay . with atrial infiltration and major excision of healthy tissue , implantation of tah
traditional bivad implantation as bridge - to - transplant as a short - term solution was not an option due to severe mitral valve stenosis from the atrial mass and mitral valve infiltration .
the left atrial tumor was successfully removed . like bruckner et al . , we acknowledge that tah implantation as bridge - to - transplantation is surgically possible even with atrial wall involvement .
surgical treatment is still the criterion standard for localized cardiac sarcomas , with various perioperative survival rates , ranging from 6 to 17 months . with complete resection of the tumor mass
there is no valid data on the benefits of adjuvant chemotherapy or radiation therapy on malignant cardiac soft - tissue sarcomas
. limitations of radiotherapy are cardiac sensitivity to radiation injury and adverse effects on global cardiac functions .
our patient showed good recovery after initial gross resection in 2014 for 14 months without any adjuvant therapy .
bleeding complications due to continuous anticoagulation for device functioning remains a major risk factor in circulatory assist device programs . in a study in 13 patients with tah , ramirez et al . found that bleeding complications occur generally between postoperative days 1 and 3 .
patients with mechanical assist devices are prone to suffer from thrombotic events [ 1316 ] , device malfunctioning , wound infections , and bleeding complications .
this intervention was the second attempt to help the patient with a long - term solution after first recurrence of an atrial tumor .
the surgical options and possibilities were discussed at length with the patient and his family before the tah implantation .
a multidisciplinary team and truthfully informed next of kin are hugely important when taking decisions in such a high - risk case . | backgroundtotal artificial heart ( tah ) implantation in patients with aggressive tumor infiltration of the heart can be challenging.case reportwe report on a patient with a rare primary undifferentiated high - grade spindle cell sarcoma of the mitral valve and in the left atrium , first diagnosed in 2014 .
the referring center did a first resection in 2014 .
in the course of 17 months , computer tomography ( ct ) scan again showed massive invasion of the mitral valve and left atrium .
partial resection and mitral valve replacement was not an option .
we did a subtotal heart excision with total artificial heart implantation . in this report
we discuss complications , risk factors , and perioperative management of this patient.conclusionspatients with aggressive tumors of the heart can be considered for tah implantation . |
cannabis is a prominent political , health , and law enforcement issue in north america that is currently receiving a great deal of attention .
as we watch the effects of the legalization of cannabis in colorado , washington , alaska , oregon , and the district of columbia , other jurisdictions are debating the future of this illicit drug .
how can we prevent commercialization promoting increased rates of use within a legalization model ? where does marijuana for medical purposes fit into the picture , if at all ?
a critical piece of information often missing from these debates or not addressed adequately is the scientific evidence about the effects of cannabis use on a person 's overall health .
this concern is even more pressing when it comes to the cognitive , physical , and mental health harms to adolescents who use cannabis .
it is critical we ask these questions because adolescence is a time of rapid development that helps lay the foundation for success later in life .
conversely , it can also set the stage for experiencing challenges in adulthood , as youth are disproportionately more likely than adults to experience greater harms from drug use .
their brains are undergoing rapid and extensive development that can be negatively affected by cannabis use .
there is certainly cause for concern about the amount and frequency of cannabis use among youth . according to the 2014 monitoring the future ( mtf ) survey in the us , 35.1% of high school seniors reported pastyear use of cannabis , with 5.8% reporting daily or near daily use .
comparable prevalence rates are reported in canada , with 22% of 1519yearolds and 26% of 2024yearolds reporting pastyear use of cannabis in 2013 according to the canadian tobacco , alcohol and drugs survey . moreover
, a 2013 unicef report noted that canada 's youth are the highest users of cannabis when compared to students in other developed countries .
the mtf survey also indicates a growing perception among young people that cannabis is a relatively harmless drug , and there is strong evidence that perception of harm is inversely related to rates of use . knowing that youth are disproportionately heavy users of cannabis
, what specific concerns should this raise about short and longterm consequences and how do we incorporate these concerns in the debate ?
recent evidence shows that early and frequent use of cannabis has been linked with deficits in shortterm cognitive functioning , reduced iq , impaired school performance , and increased risk of leaving school early all of which can have significant consequences on a young person 's life trajectory.1 of note , the impact of cannabis use on iq has been the topic of recent debate among experts in the field and further research aimed at understanding this relationship will provide much needed clarity .
heavy cannabis use in adolescence is also a risk factor for psychosis , a risk that is heightened if users have a preexisting vulnerability to that condition.1 cannabis use can also lead to dependence , with evidence from longitudinal studies in the us estimating the risk of developing cannabis dependence to be one in six among those users who initiated in adolescence,2 a rate higher than that reported among adults .
recent data from the 2012 canadian community health survey reveal that more than one in 20 young canadians aged 1524 met the criteria for cannabis abuse or dependence during the past year . there is also growing evidence from the human literature that early and frequent use of cannabis can alter structural aspects of the developing brain , including those that are responsible for memory , decisionmaking , and executive functioning.1 however , we do not yet know the full extent of the impact of early cannabis use on changes in brain function and behavior of young people , which underscores the need for further investment in research in this area . beyond longerterm consequences , cannabis can also acutely impair cognitive and motor functions like perception , coordination , and balance for hours after use .
this impairment presents a safety hazard for drivers getting behind the wheel , as well as for other motorists and pedestrians .
in fact , the risk of car crashes doubles for drivers who get behind the wheel after using cannabis and this risk is further elevated if cannabis use is combined with other substances , most notably alcohol .
recent data from the national fatality database indicate that cannabis was the most common illicit drug present among fatally injured drivers aged 1524 in canada between 2000 and 2010 .
the drug abuse warning network , which monitors the health impact of drugs in the us , estimated that in 2011 , there were nearly 456,000 drugrelated emergency department visits in which cannabis use was mentioned in the medical record .
data from the canadian institute for health information reveal that from 2006 to 2011 , youth aged 1524 spent the largest number of days in a hospital for a primary diagnosis of mental and behavioral disorders due to the use of cannabinoids . furthermore , the number of days spent in a hospital increased by almost 40% during the same time period.3
these findings are particularly troubling because recent research from canada has revealed that young people are confused about cannabis and do not have the knowledge they need about the risks associated with this drug to make more informed decisions.4 some have expressed questionable beliefs about the effects of cannabis , indicating that it helps to improve their focus at school and can prevent or even cure cancer .
youth also expressed mixed beliefs as to whether cannabis improves or impairs driving performance , and felt that cannabis and driving was not as dangerous as drunk driving .
youth talked about how cannabis is natural and so they did not really think of it as a drug .
the findings from this reviewed research highlight the complexity of the issues surrounding youth consumption of cannabis . youth are confused with the mixed messages they are receiving from discussions ranging from legalization to the use of cannabis for medical purposes , which points to the need for a coordinated , comprehensive , factual , and consistent approach to increasing awareness of this drug and its impact .
as primary care is an important intervention point for youth experiencing harms related to cannabis , there is an urgent need to develop practical , evidenceinformed tools to assist healthcare providers in the early identification of cannabis use and cannabis dependence , as well as tools to deliver interventions to treat potential problems .
examples of such tools may include screening instruments to screen for cannabis use that warrants clinical intervention , as well as brief intervention strategies aimed at addressing the problematic substance use revealed during the screening process .
there is also an urgent need for further research aimed at improving the understanding of differences between the short and longterm effects of cannabis use , particularly on the developing adolescent brain .
such research should include mechanistic studies regarding alteration of cellular structure and function related to cannabis to determine the impact on adolescent brain development .
of particular concern are the impacts of the dramatic increase in concentrations of tetrahydrocannabinol ( thc ) , the main psychoactive ingredient of the cannabis plant , over the past 25 years .
the average potency of federally seized cannabis in the us has steadily increased from 3.5% in 1985 to 13.2% in 2012.5 in light of such an increase in potency , some historical findings about the health and developmental effects of cannabis use might not be relevant when trying to predict effects on contemporary users .
more research is also needed to shed light on the influence of cannabis policy on public health , public safety , and other outcomes .
the current understanding of the impact of cannabis policy on market forces , such as healthcare costs , lost productivity costs , and tax revenues , for example , is very limited , as is overall understanding of how perception , use , and outcomes interrelate around this drug . in an effort to help clear the smoke , the canadian centre on substance abuse is currently undertaking a comprehensive review and synthesis of the literature on the effects of cannabis use during adolescence that will highlight key areas for action in policy , practice , and research
. this muchneeded work will provide clarity to misperceptions about cannabis , and make sense of some conflicting evidence about the effects of cannabis use and the impact of early or regular use during a time when the brain is still developing . by integrating neuroscience with the behavioral and social context of cannabis use by youth
, this report will provide a muchneeded resource for those working with youth and those involved in making decisions about policies , programs , or practices related to cannabis .
aj porathwaller has provided expert testimony at the standing committee on health 's ( hesa ) recent report on marijuana 's health risks and harms , october 2014 . | cannabis is the most commonly used illicit substance among youth .
recent policy developments and ongoing debate related to this drug underscore the urgent need to clear the smoke and better understand what the scientific evidence says about the health and behavioral effects of cannabis use , particularly on youth whose brains are undergoing rapid and extensive development . |
hypertension ( htn ) is the most common comorbidity in the world with significant public health implications [ 1 , 2 ] . the overall u.s .
prevalence of hypertension among adults ages 18 years in 20052008 was 30.9% and was highest among persons ages 65 years ( 69.7% ) and non - hispanic blacks ( 44% ) .
the american heart association estimates that the incurred costs of hypertension are more than $ 93.5 billion per year , and that cardiovascular disease and stroke for which htn is the predominant risk factor , account for 17% of the total annual health expenditures in the united states .
hypertension and its sequelae , heart failure ( hf ) , are a progressive disease .
evidence shows that less than half of patients with heart failure survive five years ( after diagnosis ) , and less than a quarter of them live ten years after their initial diagnosis .
framingham heart study showed that hypertensive patients were more likely to develop heart failure ( 142 cases of hf detected during the first 16 years of followup ) than those who were normotensive .
the lifetime risk for development of hf among people with blood pressure ( bp ) > 160/90 mm hg is double that of those with bp < 140/90 mm hg .
heart failure in comparison to the most prevalent gender malignancies ( bowel cancer in men and breast cancer in women ) , was associated with worse long - term survival .
over a million hospitalizations , contributing to more than 250,000 deaths , and escalating billions of dollars in health care expenditure is the norm .
the disastrous duo , htn and hf , should be recognized , and a tailored focus on management in special populations , high - risk ethnic minority groups especially blacks who are disproportionately burdened should be developed . among racial groups ,
african - american adults have the highest rates ( 44% ) of hypertension in the world and are more resistant to treatment .
in particular , black women have the highest prevalence and the lowest blood pressure control .
the relative incidence of hf is 50% higher in african americans , 3% of whom have hf , compared with 2% of the general population [ 13 , 14 ] .
the disease occurs at an earlier age , and usually at a more advanced stage along with increased hospitalization and mortality .
other causes of hf include coronary artery disease , valvular heart disease , diabetes , left ventricular hypertrophy and cardiomyopathies .
overt clinical hf resulting from diastolic left ventricular dysfunction may be clinically indistinguishable from that resulting from systolic dysfunction
. hf with preserved left ventricular function is observed in 30% to 50% of adult cases of hf .
coronary artery disease and myocardial infarction is a principal cause of systolic left ventricular dysfunction followed by hypertension .
a variety of neurohormonal systems , especially the renin - angiotensin aldosterone and sympathetic nervous systems are activated in response to the left ventricular dysfunction and such activation leads to abnormal ventricular remodeling . the inexorable progression to more severe stages of left ventricular dysfunction
can be significantly reduced by effective therapy with neurohormonal blockade including angiotensin converting enzyme inhibitors ( aceis ) , beta blockers ( bbs ) , and aldosterone antagonists .
current understanding of the pathophysiology of hf has revolved around neurohormonal activation [ 19 , 20 ] .
this endothelial dysfunction may result from insufficient nitric oxide ( no ) secondary to either reduced endothelial production of no or to increase no inactivation [ 17 , 19 , 21 ] . in hf , a pronounced increase in angiotensin ii , with modestly reduced no is seen among whites . however , african americans with hf may exhibit greater no reduction ; conversely , angiotensin ii might be less elevated among whites .
important studies are investigating gene variation among the races that may influence the risk of developing hypertension and its complications , such as single nucleotide polymorphisms in the genes encoding for 1-adrenergic receptors and a2c - adrenergic receptors .
african americans who are homozygous for polymorphisms in both the 1-adrenergic and a2c - adrenergic receptor genes are at a 10-fold greater risk for heart failure , which is overexpressed in african - americans .
groups with overexpression of transforming growth factor -1 ( tgf-1 ) , a cytokine involved in cardiac fibrosis and remodeling , vascular and renal disease , production of the vasoconstrictor , endothelin-1 , and stimulation of renin release , have a worse prognosis .
other genetic polymorphisms in african americans that may influence heart failure risk include elevated production of endothelial nitric oxide synthase , aldosterone synthase , and the g protein 825-t allele .
there are two major approaches to classifying the patient 's hf stage or clinical severity .
the most common is the new york heart association ( nyha ) classification , mainly based on functional capacity and subjective level of symptoms severity .
this scheme is fluid and its class fluctuates in the same patient depending on their subjective assessment and clinical status .
the other approach , adopted by the american college of cardiology / american heart association ( acc / aha ) guidelines , emphasizes disease progression
. classified as stages a , b , c , and d , each of these stages has definable characteristics and recommended interventions . to emphasize the progressive nature of hf and the need to prevent its development or progression to end - stage disease ( stage d ) [ 18 , 23 ] ,
current hf guidelines define patients in stage a as those at increased risk for hf without evidence of structural heart disease or symptoms of hf .
stage a represents a critically important group because many of the risk factors including hypertension can be effectively controlled and by doing so may prevent development of cardiovascular diseases including hf .
patients are in stage b when they present with evidence of structural heart disease but without current or prior symptoms of hf .
stage c is recognized by the presence of structural heart disease and current or prior symptoms of hf .
stage d patients are those who have refractory heart failure requiring frequent hospitalization and specialized interventions .
several studies have shown the efficacy of treatment of hypertension in preventing hf [ 2427 ] .
treatment of hypertension reduces the incidence of hf by about 50% . in recent years
, significant advances have been made in increasing awareness , treatment , and control of blood pressure .
several large , randomized clinical trials have shown that specific classes of medication have mortality benefit in patients with heart failure [ 2834 ] . in the most recent guidelines for the diagnosis and management of hf in adults , the american college of cardiology foundation ( accf)/american heart association ( aha ) recognized the need for specific recommendations for special population .
lifestyle modifications have been shown to reduce blood pressure , increase drug efficacy , and decrease cardiovascular risk .
long - term primary prevention of hypertension includes weight reduction in overweight / obese individuals .
weight loss intervention was associated with a 21% reduction in the incidence of hypertension over a 36-month period .
dietary practices , reducing intake of dietary sodium to < 1.5 g nacl [ 3 , 36 ] , low potassium , magnesium , and saturated fat intake , increased calcium intake , decreased consumption of alcohol , have demonstrated greater bp reduction in blacks
. these practices are most successful when reinforced with comprehensive education and counseling [ 39 , 40 ] .
exercise training , regular aerobic physical activity for at least 30 minutes per day most days , has been shown to reduce recurrent cardiac events in patients with lv dysfunction from ischemic heart disease and is recommended under close medical supervision . culturally cognizant and sensitive modalities should be implemented to help guide improved compliance of therapeutic lifestyle changes ( tlc ) in special populations [ 43 , 44 ] . despite lifestyle modifications ,
most patients with hypertension need pharmacologic therapy for better bp control . in the united states ,
pharmacological treatment of hypertension before development of structural heart disease or symptoms of hf is mainly based on studies looking at prevention of end - organ damage .
specific classes of medication are recommended for patients in stages b - d that have shown benefit in reducing mortality and number of hospitalizations .
the reader should refer to the major guidelines for full treatment recommendations of hf in the general population [ 18 , 23 , 46 ] .
. however , ethnic minorities who are at greater risk for developing hf remain underrepresented in most clinical trials . according to a recent paper by mitchell et al .
treatment of hf in african americans a call to action , most of the available evidence is based on limited representation of african americans in these trials or posthoc analysis of limited evidence .
these papers once again summarize the available evidence in hf management in african americans and call for more therapeutic trial focused in these high - risk population .
the african american heart failure trial ( a - heft ) was the first heart failure large trial to focus specifically on the african - american population and evidence represented approximately 50% of all african americans ever studied in heart failure trials .
this is randomized placebo - controlled double blind study of fixed dose isosorbide dinitrate / hydralazine ( isbd / hyd ) ( figure 2 ) . in an effort to summarize efficacy of available treatments , psaty et al
the database included 192,478 patients from 42 trials randomized to 7 treatment strategies including placebo .
low - dose diuretics proved the most effective first - line treatment for preventing cardiovascular disease and mortality including hf .
the allhat trial showed that diuretics are as effective as calcium channel blockers or acei in preventing cardiovascular disease outcomes including hf .
the allhat study also has suggested that thiazide - diuretic therapy is useful in preventing disease progression .
although , allhat trials and other trials showed diuretics is the first - line of antihypertensive therapy and is still the most commonly prescribed antihypertensive medications , this is challenged by recent studies as published in february jacc and presented in the european society of htn meetings .
however , the diuretics used in the allhat trials was chlorthalidone a long acting thiazide ( shown to effectively lower bp and improve survival ) , not really the same as hydrochlorothiazide ( hctz ) which is the most commonly prescribed antihypertensive thiazide at a dose of 12.525 mg .
( st luke roosevelt hospital , new york , ny ) , published the analysis of 18 trials on hctz where the antihypertensive effect of hctz at the dose that is most often used12.5 to 25 mg a day is consistently inferior to that of most other antihypertensive drug .
the other limitation of diuretics as first line therapy in african americans is not only not adequate but also , has multiple side effects like dm , electrolyte abnormalities and urecemia , to mention a few .
however , there is still a role of diuretics in combination with neurohormonal ( renin angiotensin aldosterone axis ) blockade and when there is compelling indication . according to the recent consensus statement of the international society on hypertension in blacks ( ishb ) , chlorthalidone
furthermore , the ishb consensus statement reinforces , patient above goal bp ( sbp / dbp15/10 ) with ( goal bp < 130/80 ) end organ damage ( hf , lvh , kidney disease ) or without end organ damage ( goal bp < 135/85 ) should be on at least on combination pharmacotherapy .
use of beta blockers ( bb ) as treatment for hypertension has come under scrutiny .
conducted a meta - analysis of 13 trials , which included 105,951 patients showing that atenolol did not improve outcomes in terms of stroke and cardiovascular events .
in stage b chf ( nyha class i ) , defined by the presence of reduced left ventricular ejection fraction ( lvef ) < 40 percent in otherwise asymptomatic individuals , aceis and bbs are recommended .
iii ) manifest left ventricular dysfunction and overt symptoms ; in these individuals , aceis and bbs are also indicated .
bbs are also recommended in hf because of clinical studies demonstrating decreased morbidity and mortality , and improvement in hf symptoms .
aldosterone antagonists may provide additional benefit in patients with severe left ventricular dysfunction , usually late stage c ( nyha class iii
low - dose spironolactone ( 12.525 mg daily ) , when added to standard therapy , decreased mortality by 34 percent . in the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) ,
eplerenone reduced mortality by 15% in patients following a recent mi with lvef < 40% , 90% of whom had hf symptoms .
patients were excluded who had a serum creatinine of < 2.5 mg / dl and hyperkalemia .
the v - heft i and ii are a large - scale trial that compared different vasodilators with placebo and among themselves .
v - heft i showed , the mortality rate was significantly lower for the total cohort ( african americans and whites ) in isdn / hyd arm compared to patients treated with prazosin or placebo added to digitalis and diuretics ( figure 1 ) . in v - heft ii , though isbd / hyd was statistically inferior to enalapril in effect on mortality , patients taking isbd / hyd experienced similar mortality rate in patients taking isbd / hyd in v - heft - ii as those in v - heft - i taking isbd / hyd .
posthoc analysis of v - heft i showed isbd / hyd significantly reduced mortality in african american patients but not in white patients .
the reanalysis of v - heft ii , demonstrated the annual mortality rate african american ( 12.9 ) patients receiving isbd / hyd was 12.9% compared with 12.8% for african americans receiving enalapril .
due to difference in outcomes in african americans in v - heft , the african american heart failure trial ( a - heft ) definitive study was designed .
the a - heft was terminated earlier than anticipated completion date due to a significant ( 43% ) reduction in mortality in patients receiving combination ( isdn / hyd ) compared to the placebo arm . adding the combination of a fixed - dose of isdn / hyd ( 20 mg/37.5 mg ) to a standard medical regimen for hf , including acei and bbs ,
is recommended in order to improve outcomes for patients self - described as african americans , with nyha functional class iii or iv chf .
furthermore , genomic evidence from the grahf ( genetic risk assessment of heart failure in african americans ) arm of the african american heart failure trial ( a - heft ) showed that blacks with the more common tt phenotype ( 61% ) of the aldosterone synthase gene ( cyp11b2 ) showed greater responsiveness to the combination of isosorbide dinitrate and hydralazine . the vast majority of blacks will have no identified cause for their htn .
past studies elucidating the pathophysiological mechanisms have shown conflicting information and further investigations exploring the complex relationships and interactions are warranted .
there are , however , several secondary forms of htn in blacks that are prominent and preventable .
we will focus on the most common , preventable and treatable secondary form of hypertension , obstructive sleep apnea ( osa ) .
the prevalence of osa in hypertensive populations is estimated to range between ( 3040% ) and in heart failure at 53% ; apnea - hypopnea index ( ahi ) 10/h ) .
it is an independent risk factor for htn , nondipping of blood pressure and increased bp variability and follows a dose - dependent relationship .
a prospective study showed that the odds of developing hypertension over 48 years was 2.89 times more likely for participants who had ahi 15/h compared to ahi 0 , independent of known confounders .
cross - sectional data has shown 2.38-times increased likelihood of having heart failure in association with osa , independent of confounders ( n = 6424 ) [ 67 , 68 ] .
osa has been shown to act through various mechanisms , independent of blood pressure , in promoting left - ventricular ( lv ) hypertrophy , diastolic , and systolic dysfunction , and overt heart failure .
these mechanisms include the hemodynamic consequences of repetitive increases in left - ventricular transmutable wall pressure and over time impaired vigil heart rate modulation leading to lv hypertrophy and ventricular remodeling .
additionally , osa patients without overt heart failure have more impaired diastolic relaxation than those without osa .
emerging evidence illustrates patients with osa have more active atherosclerotic disease with a greater degree of vessel involvement and more vulnerable plaques than do patients without the disease , resulting in greater cardiovascular burden .
the definite treatment for osa , long - term adherence to continuous positive airway pressure ( cpap ) therapy , has shown to suppress sympathetic activity , lower blood pressure , reduce right ventricular volume , and improve systolic function in patients with hf .
systematic reviews , meta - analyses , and randomized controlled trials have shown statistically significant net reduction in blood pressure with cpap , especially in patients with increased ahi events [ 7883 ] . a recent 3-year study ( n = 340 ) by durn - cantolla et al . reported that participants assigned to cpap therapy for 3 months had mean 24-hour ambulatory blood pressure decreased by 2.1 mmhg ( 0.4 to 3.7 ) mmhg ( p = .01 ) for systolic and 1.3 ( 0.2 to 2.3 ) mm hg ( p = .02 ) for diastolic blood pressures .
similar evidence from randomized trials showed up to 9% increase in lvef and increased quality of life ( qol ) and functional capacity among patients treated with 13 months of cpap therapy .
furthermore , among patients with no overt failure , cpap therapy has been shown to reverse impaired diastolic relaxation .
there is a need for further randomized clinical trials with cpap therapy among high - risk ethnic minorities especially blacks to assess whether treatment of osa has similar long - term effects and to evaluate the effect on htn and hf morbidity and mortality .
taking into consideration , lifestyle and pharmaceutical interventions , our changing health care community needs to emphasize the essential nature of the patient - provider relationship and establish trust and collaboration . in the past , this has been accomplished through physician , patients , family member , and other caretaker awareness , increased education of both provider and patients , and establishment of community outreach programs .
these measures have contributed to an increase in compliance and a reduction in the risk of hospitalizations [ 23 , 87 ] .
we recommend an additional emphasis on the implementation of specialty care in patients with heart failure in collaboration with the primary care provider .
the dramatic improvement in the management of hf and hypertension over the last 50 years has allowed us to start to target specific populations and provide more evidence - based treatment that will lead to an improvement in mortality .
there are disparities in the prevalence , treatment , and control of hypertension and the incidence and morbidity and mortality of heart failure between blacks , a diverse and heterogeneous population , and whites .
the management team needs to take into consideration new evidence and develop tailored strategies for effective treatment , especially for hypertension , one of the main causes of hf and many other cardiovascular complications in african africans . | hypertension ( htn ) is the most common co - morbidity in the world , and its sequelae , heart failure ( hf ) is one of most common causes of mortality and morbidity in the world .
current understanding of pathophysiology and management of htn in hf is mainly based on studies , which have mainly included whites . among racial groups ,
african - american adults have the highest rates ( 44% ) of hypertension in the world and are more resistant to treatment .
there is an emerging consensus on the significance of racial disparities in the pathophysiology and treatment options of hypertension and heart failure .
however , african americans had been underrepresented in all the trials until the initiation of the a - heft trial . since the recognition of obstructive sleep apnea ( osa ) as an important medical condition ,
large clinical trials have shown benefits of osa treatment among patients with htn and hf .
this paper focuses on the pathophysiology , causes of secondary hypertension , and treatment of hypertension among african - american patients with heart failure .
there is increasing need for randomized clinical trials testing innovative treatment options for african - american patients . |
radiological investigations and therapeutics have become an integral part of the management of critically ill patients in the intensive care unit ( icu ) .
patients admitted to the icu routinely undergo bedside imaging procedures such as chest radiographs for diagnosing heart , lung and other pathology , and for confirmation of the position of devices like endotracheal tubes , nasogastric tubes , central venous catheters and intercostal drains .
a review article has suggested that among icu patients , up to 65% of daily chest radiographs and 70 - 75% of chest computerized tomography ( ct ) scans reveal significant or unsuspected abnormalities that may lead to a change in the patient 's management .
further , critically ill patients are frequently transported to the ct scan as well as digital subtraction angiography suites for diagnostic and therapeutic procedures such as angiography , embolization and stenting . in most situations ,
the icu team is responsible for the transport and management of the patient in the radiology department for these procedures .
there is , therefore , potential radiation exposure to healthcare workers , particularly for those working for long periods of time in the icu .
epidemiological data indicates that the exposure to even low - dose radiation may be a cause for concern because such exposure can result in leukemia , thyroid malignancies and other cancers .
nonneoplastic effects of radiation include genetic mutation and developmental malformation in children whose mothers were exposed to radiation during pregnancy .
a number of studies have looked at radiation exposure in critically ill patients undergoing repeated radiological procedures .
one study has specifically focused on the pediatric age group probably in view of the high ratio of exposure to patient size and the potential for long - term sequelae as the radiation effects have a longer period to become manifest . however , the available literature on the extent of radiation exposure to icu personnel is scarce and relates mainly to the level of scattered radiation within the icu .
the conclusion of these studies is that the level of radiation exposure is extremely low and does not pose a hazard to icu personnel .
none of these studies have considered the additional radiation exposure to icu personnel involved in the management of critically ill patients in the radiology department . in our icu in a tertiary care cancer referral center , resident doctors working on 12 h shifts in the icu are also responsible for the transport and management of icu patients when they undergo diagnostic and therapeutic procedures in the radiology department .
we , therefore , carried out this study to determine the total radiation exposure to icu resident doctors involved in the course of their duties .
we conducted a prospective , observational study in the icu and postanesthesia care unit ( pacu ) of a 500-bedded tertiary care cancer referral center in mumbai , india from september 2012 to february 2013 .
all resident doctors who gave voluntary written consent to participate in the study were included .
since the study did not involve patient contact , the requirement for obtaining consent from patients was waived by the ethics committee .
the study was carried out in accordance with the principles of good clinical research practice .
the resident doctors provide 24 h cover , working in 12 h shifts , with four doctors in each shift . in each shift
the icu has a total of 14 beds as shown in figure 1 . on another floor
is the pacu with 23 beds , subdivided into 14 postoperative recovery room beds and 9 intensive care beds [ figure 2 ] .
residents provide care for patients in the icu and pacu and also accompany patients from these locations for radiological procedures in the ct scan or interventional radiology suites .
location of thermoluminescent dosimeters are shown with filled triangles layout of postanesthesia care unit showing location of thermoluminescent dosimeter in pacu ( tld surrounded by beds ) and other in long lobby as control tld to detect levels of radiation , we used thermoluminescent dosimeters ( tlds ) which are a type of radiation detector .
the tld measures cumulative dose of ionizing radiation exposure by measuring the amount of visible light emitted from a crystal in the detector when the crystal is heated .
tlds can measure a wide dosimetric range ( from 10 gray to 10 grays ) of radiation exposure and are routinely used as personal dosimeters because they are small in size , convenient to use and not expensive .
a tld was given to each of the four resident doctors ; they were handed over to the next team during shift changeover .
in addition , three tlds were kept in nursing stations in icu , and one tld was placed in the pacu .
one tld was kept in the doctors ' duty room which was within the premises of the icu , and the last was kept as a control in the office of the department of anesthesia , critical care and pain , remote from icu and pacu , and where no radiological procedures were performed .
the layout of icu and pacu is represented by figures 1 and 2 respectively and locations of tlds are marked with filled triangles .
the participating resident doctors were instructed to wear the tlds at all times during their duty hours .
staff not required to be present during radiographic procedures were kept at least 3 m away from the patient ( a distance at which exposure from scattered radiation is considered negligible ) .
it was ensured that patients who were shifted from icu to the radiology suite were accompanied by one icu resident and that the resident was wearing his tld throughout .
residents , if required to be directly involved in the radiologic procedure in suites , wore lead aprons , and the tld was underneath the lead apron . bedside chest
x - rays in the icu were done by siemens multimobil 2.5 ( manufactured by siemens ltd , goa , india ) , ct and ct guided biopsies were performed on siemens somatom emotion 16 ( manufactured by siemens shanghai medical equipments ltd , shanghai china ) and ge light speed 16 ( manufactured by general electric co. , milwaukee , usa ) respectively .
interventional radiology procedures were carried out using ge innova 4100 iq ( manufactured by ge medical systems , france ) .
the study was carried out in two phases of 3 months each . at the end of the first phase ,
the tlds were handed over to department of nuclear medicine for analysis and another set of tlds was issued at the same time . during the period of the study , we maintained a database of procedures performed both in the icu and in the radiological suites along with the bed number and the patient 's hospital registration number .
the primary outcome of this study was to quantify the cumulative radiation exposure to the resident doctors working in the icu over a period of 6 months in the course of their duties .
the secondary outcomes were to measure the cumulative scattered radiation exposure in various areas of the icu calculated as the average of readings of icu tld badges over 6 months and the estimated cumulative radiation exposure to the resident doctors per year .
the control tld placed in the department of anesthesia served as a measure of baseline atmospheric ionizing radiation , and all other values are reported above this baseline .
the results of dosimetric analysis of residents tlds during the two phases are shown in table 2 .
readings from tlds placed in the nursing station and doctors ' duty room during both phases were immeasurable .
the mean values in the first and last 3 months were 0.052 and 0.069 msv respectively , though the highest individual value approached 0.1 msv .
since this was recorded in a 3 month period , the projected reading for similar exposure throughout the year , even assuming a 24 h duty period , would be 0.4 msv , which is well below the safety limit of 20 msv / year .
revolutionary progress in the field of medical imaging has given a big leap to advances in medical diagnostics and therapeutics .
this development has also infiltrated the field of critical care medicine , and radio - diagnosis and interventional radiological procedures now play a key role in the management of critically ill patients . while this advancement offers the advantages of rapid bedside diagnosis , and cost - effective and minimally invasive treatment options to critically ill patients , it carries the danger of exposure of patient and physician to radiation .
the detrimental effects of exposure to even low - level ionizing radiation have always been known ; however , there is renewed concern because of its wide - spread use in medical radio - diagnosis and therapeutics in critically ill patients .
it is , therefore , natural that there may be concerns about the long - term effects of radiation exposure to doctors working for long periods of time in the icu .
in addition , where icu doctors are responsible for transport of patients to the radiology suite , the extent of radiation exposure is increased .
it is reassuring that the results of our study confirm that the extent of radiation exposure to critical care physician during the course of his duty is well within acceptable limits .
there is considerable literature on occupational hazards of radiation exposure among radiologists ; however , it is difficult to directly extrapolate the conclusions of these studies to the icu .
working conditions in the icu do not mimic those in radiology suites - working hours tend to be long , there is a need to accompany mechanically ventilated and hemodynamically unstable patients inside the radiology suite for procedures , and icu doctors may sometimes take radiation safety norms lightly .
a study performed in a trauma icu ( ticu ) has concluded that radiation exposure is not a significant occupational hazard for the ticu personnel .
similarly , another study looked into the radiation exposure to icu nurses and found that the exposure was well below the permissible level .
the strength of our study is that it was planned in a more pragmatic way - apart from bedside radiological procedures , we also took into account the radiation exposure to icu residents accompanying the patient to radiological suites for diagnostic and therapeutic procedures
. ionizing radiation from fluoroscopy in the ct scan or interventional radiology suites may be significant and were not considered in the previous studies .
we also tried to measure the amount of scattered radiation within the icu and the pacu , which contributes to overall radiation exposure .
even after taking these additional sources of radiation exposure into account , we found that the cumulative radiation exposure was negligible .
the number and types of bedside and out - of - icu radiological procedures can vary on a day - to - day basis according to the case - mix of the icu population , and this may affect the overall radiation exposure ; however , this study was carried out over a period of 6 months , and the data obtained would have been adequately representative .
the other limitation relates to the generalizability of the study ; differences in the types of cases , working pattern of resident doctors , quality and maintenance of radiological equipment between hospitals may restrict the applicability of these results to other hospitals .
however , given the large margin of safety that our study has shown , it is unlikely that exposure levels would be dangerously high in any other setting .
though , with advances in technology , the number and types of radiological procedures performed on patients are likely to increase . furthermore , there is a growing trend toward using radionuclide - based positron emission tomography scans for diagnostic procedures in critically ill patients especially when they are admitted to icu during their diagnostic work - up .
some of these patients may continue to emit radiation long after their procedures are completed .
though none of these patients featured in our study , it will add to the radiation exposure to doctors .
the results of this study do not in any way underrate the need to follow safety precautions , while carrying out radiological procedures in critically ill patients .
the levels of exposure found in this study should be interpreted bearing in mind that standard protection norms were used by all personnel involved in the study . with these precautions in place ,
radiation exposure to doctors managing critically ill patients in the icu is minimal and acceptable .
literature on radiation exposure among icu doctors is scarce . in addition to bedside radiological procedures , the risk of exposure may be increased if the same doctors accompany icu patients for out - of - icu radiological investigations .
however , we found that if standard safety precautions were followed , cumulative radiation exposure to icu resident doctor was well within permissible limits and was not the cause of concern and hence routine personal dosimetric monitoring is not needed for residents in icu .
however , in view of changing practice , there is a need to repeat such audits periodically to monitor radiation exposure . | background and aims : with the expanding use of diagnostic and therapeutic radiological modalities in critically ill patients , doctors working in intensive care units ( icus ) are increasingly exposed to ionizing radiation .
this risk of radiation exposure occurs not only during bedside radiologic procedures , but also when icu physicians accompany patients to radiology suites .
the aim of this study was to quantify levels of radiation exposure among medical professionals working in the icu.materials and methods : the study was carried out prospectively over 6 months in the icu of a tertiary - referral cancer hospital .
two teams consisting of 4 icu resident doctors each were instructed to wear thermoluminescent dosimeters ( tlds ) during their duty shifts .
standard radiation protection precautions were used throughout the study period .
tlds were also placed in selected areas of the icu to measure the amount of scattered radiation .
tlds were analyzed at the end of every 3 months.results:the readings recorded on tlds placed in the icu were almost immeasurable .
the mean value of residents ' radiation exposure was 0.059 msv , though the highest individual reading approached 0.1 msv .
the projected maximum yearly radiation exposure was 0.4 msv.conclusions:if standard radiation safety precautions are followed , the cumulative radiation exposure to icu resident doctors is well within permissible limits and is not a cause of concern .
however , with the increasing use of radiological procedures in the management of critically ill patients , there is a need to repeat such audits periodically to monitor radiation exposure . |
despite the huge burden of hepatitis b worldwide , with an estimated 350 million people chronically infected , very few patients in low- and middle - income countries are currently receiving antiviral treatment .
this is largely to blame on lack of access to viral load quantification , because this is considered a mandatory component of the diagnostic work - up in all international liver society guidelines . without a viral load measurement ,
it is virtually impossible to establish whether a hepatitis b surface antigen ( hbsag ) positive individual has chronic hepatitis b , characterized by a level of hepatitis b virus ( hbv ) dna > 200020 000 iu / ml and continued necro - inflammation in the liver .
these patients are at high risk of progression to cirrhosis and hepatocellular carcinoma in the absence of antiviral treatment , whereas inactive carriers , characterized by hbv dna levels < 2000 iu / ml , have an excellent prognosis in the absence of treatment .
the situation for hepatitis c is somewhat different because the main obstacle to treatment has been expensive and toxic treatment regimens . with the release of new direct acting antivirals for hepatitis c
, opportunities are opening up in less developed countries where the disease burden is often high .
an estimated 130150 million people are chronically infected with hepatitis c virus ( hcv ) globally , and the prevalence is higher in certain areas such as north africa and central and east asia . however , prior to starting treatment of hepatitis c , hcv rna measurements are required to establish the diagnosis of chronic hcv infection , which is a major obstacle in many places in the world .
lack of access to viral load testing and antiviral treatment of hbv and hcv in resource - limited settings is a silent epidemic with major consequences .
the world health organization ( who ) estimates that about 1 million people die from chronic hbv and hcv infection each year , which places viral hepatitis on the top 10 leading causes of mortality globally .
first , serological tests are used to screen for hepatitis markers and exclude co - infections .
thereafter , a new battery of tests is needed to distinguish past or inactive infection from chronic hepatitis .
this diagnostic work - up involves molecular biology laboratories and advanced target amplification methods such as polymerase chain reaction ( pcr ) assays .
these tests are typically performed at large referral laboratories , because they require sophisticated equipment , highly specialized laboratory personnel and strict quality control measures . in resource - limited settings ,
furthermore , stringent requirements for storage and shipment of plasma to the referral laboratory are barriers in settings with limited infrastructure .
the solution to this challenge would be to develop reliable , cheap , and easy - to - use assays that can be performed at the site of patient care ( point - of - care assays ) .
numerous rapid diagnostic tests ( rdts ) for hepatitis b and c are commercially available , most of which provide a test result within 530 minutes ; however , the diagnostic accuracy varies from excellent to very poor . a recent meta - analysis by shivkumar and colleagues found that the sensitivity of hbsag rdts used to screen for hepatitis b infection varied from 42 to 100% and the specificity from 0 to 100%
. the same authors also reviewed anti - hcv rdts used for hepatitis c screening and found sensitivities ranging from 0 to 100% and specificities from 81.6 to 100% .
hence , although high - quality rdts can be used with confidence in screening for hbv and hcv , health authorities should warn against the use of unreliable ( and often very cheap ) rdts with poor sensitivity and specificity .
even though serological point - of - care assays for hepatitis b and c are widely available , the same is not the case for virological analyses . however ,
if we look over the fence into the hiv landscape , there has been an active development of point - of - care kits for viral load quantification over the past few years .
because of major international investments in the fight against hiv / aids , there is an attractive commercial market for such kits .
the first products were recently launched , including the alere q analyzer ( alere inc . ,
waltham , ma , usa ) and the samba ( diagnostics for the real world , sunnyvale , ca , usa ) , both of which have undergone field testing in sub - saharan africa and shown excellent performance . as there are no similar funding mechanisms for viral hepatitis in resource - limited settings ,
commercial actors have not shown the same interest in low - tech diagnostics for hbv and hcv ; however , many of the technological advances for hiv can be modified to detect other viruses . currently , there is one product , truenat ( molbio diagnostics , goa , india ) , for hbv dna quantification which is advertized as a point - of - care assay .
the truenat kit provides a pcr result within an hour ; however , it seems rather complex , involving several manual steps and multiple reagents , making it less ideal for outside laboratory settings .
. an independent evaluation of the truenat kit under field conditions would be of major interest .
the us - based company wave 80 biosciences is developing a point - of - care kit for hbv dna quantification as well as a qualitative assay for hcv rna detection , building on their existing hiv viral load assay eoscape - hiv ( wave 80 biosciences , san francisco , ca , usa ) .
eoscape - hiv is a fully automated , cartridge format system which is said to be robust and easy to operate .
however , eoscape - hiv is still not commercially available , so the timing for the release of the hbv and hcv kits is still uncertain .
the uk biotechnology company epistem received a prestigious grant award in 2013 to develop an hcv point - of - care device for viral load testing and genotyping .
this novel assay will build on their existing genedrive ( epistem , manchester , uk ) device , which is a rapid , easy to use , sensitive , handheld pcr platform already ce - ivd marked for other genotype tests , such as mycobacterium tuberculosis identification and antibiotic resistance testing .
far too often advanced technology has been shipped to low - income countries with good intentions , but ended up in a storeroom because of insufficient training , lack of maintenance , and shortage of reagents .
partly , this has to be blamed on the manufacturers , who rarely offer training or service agreements to low- and middle - income countries .
it is important , therefore , that point - of - care assays should be designed specifically to operate under basic conditions with minimal maintenance requirements and not just be high - tech solutions forced to fit low - tech settings .
the international nongovernmental organization mdecins sans frontires published their desired specifications for a point - of - care hiv viral load assay a few years ago , and the same specification would also apply to an ideal hbv or hcv assay : no need for specialized laboratory facilities closed system to avoid contamination long shelf life in tropical climate no need for cold chain transportation or refrigerated storage affordability is an important issue if treatment for viral hepatitis is to be scaled - up globally .
both the initial cost for the machine , but also the consumables thereafter must be priced reasonably . with regard to hbv , repeated tests over time are usually required , and the accumulated cost of viral load testing can be high . for hepatitis c , on the other hand , one viral load measurement prior to treatment and another 24 weeks after treatment would be sufficient if patients are being treated with the new direct acting antivirals .
sustainability is another major concern with all technological devices , and it should be a prerequisite that the manufacturers provide adequate training and service agreements locally . furthermore , sensitivity of the assay should be high enough to ensure treatment for those who need it , and even more importantly , specificity must be close to 100% to avoid unnecessary and expensive treatment in uninfected individuals .
as described by greenman and colleagues in the current issue of jvh , dried blood spots ( dbs ) can be a feasible and reliable alternative to point - of - care assays for viral hepatitis .
the main advantage of dbs is that it solves the problem of storage and shipment of samples in places with poor infrastructure .
dbs can be stored for weeks at ambient temperature without clinically significant degradation of nucleic acids .
a drawback with dbs is the delayed reporting back of results . with viral hepatitis , however , this might be less of a concern , as viral load quantification is part of the pretreatment work - up of each patient and not the day - to - day monitoring of treatment effect ( as in hiv ) .
and in viral hepatitis , the decision to start treatment is rarely a matter of urgency .
use of dbs is limited by the small amount of plasma per blood spot and less efficient nucleic acid extraction , which gives a reduced sensitivity in samples with low - level viraemia . with regard to hepatitis c
, this rarely has any practical consequences , as most untreated patients have viral loads ( far ) above 1000 iu / ml . for hepatitis b ,
however , the situation is rather different as hepatitis b e - antigen ( hbeag ) negative hepatitis is now the main type of chronic hepatitis b worldwide .
these patients typically have fluctuating viral load levels in the lower to medium range , and it is often difficult to distinguish them from inactive carriers . a lack of precision in dbs could therefore jeopardize the management of these patients
. previous studies of hbv dna quantification in dbs have shown inconsistent results . a recent study by mohamed and colleagues showed that dbs yielded viral loads 0.65 log10 lower than plasma , and an older study by jardi and colleagues found that viral load levels were 1 log10 lower in dbs compared to plasma . on the contrary , lira and
colleagues found no significant difference between dbs and plasma ( 0.21 log10 lower in dbs ) .
thus , more studies are needed to evaluate the precision of dbs for hbv dna quantification , especially in the lower range around the decision threshold of 200020 000 iu / ml . in conclusion ,
dbs can be a valuable tool for the pretreatment evaluation of patients with hepatitis c , but might be more troublesome in hepatitis b due to a reduced sensitivity in the lower range of viral loads .
management of viral hepatitis is going through a revolution with the launch of new direct acting antivirals for hepatitis c treatment .
the cost of nearly 100 000 usd per patient , however , keeps these drugs out of reach for most patients worldwide .
recently , the pharmaceutical company gilead announced that they work with generic drug manufactures in india to produce high - quality , low - cost sofosbuvir for developing countries , raising expectations that this game - changing drug might become available in low- and middle - income countries .
drug availability , however , is not the only issue . in the absence of viral load measurements ,
doctors in resource - limited settings are left virtually blindfolded in the management of their patients with hepatitis .
point - of - care viral load assays for hepatitis b and c have the potential to bridge this gap and prove valuable tools for expansion of treatment globally
. however , assay performance under standardized conditions in europe or north america does not necessarily reflect real - life application in sub - saharan africa , and it is crucial to carry out independent field testing of these instruments before large scale use can be recommended . finally , sustainable funding mechanisms for diagnostics and treatment of viral hepatitis must be established .
scaling up of hiv treatment globally would have been impossible without major donor programs such as pepfar , unitaid and the global fund to fight aids , tuberculosis , and malaria .
then our colleagues in resource - limited settings might be able to treat viral hepatitis in the not so distant future . | viral hepatitis claims one million lives each year .
scaling up treatment for hepatitis b and c in resource - limited settings is not possible without access to reliable diagnostic tools .
this article gives an overview of current technologies and the pipeline for easy - to - use assays for serological and virological analyses , which can be performed at the site of patient care ( point - of - care assays ) .
furthermore , the utility of dried blood spots for hepatitis b and c viral load testing is discussed .
in addition to simple and reliable diagnostics , there is a need for a sustainable funding scheme and generic production of antiviral drugs to reduce the burden of viral hepatitis worldwide . |