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immunizations are common procedures in the life of most children , preventing diseases that inflict disability or death . the human papillomavirus ( hpv ) vaccination aims to protect against the development of cancer caused by hpv .
administration of the vaccination is recommended before sexual debut in order to maximize the effectiveness of the immunization .
however , immunizations are the most common iatrogenic intervention posing a risk of pain for children , and the children 's cognitive as well as emotional needs ought to be taken into consideration when planning for caring interventions related to immunizations . according to galvin and todres ,
caring can be divided into three domains : the head , the hand , and the heart .
the head involves existing knowledge of the context ; the hand demonstrates the nurse 's practical processes of caring ; and the heart represents the nurse 's ability to meet individual needs . engaging the heart in caring means taking individuals ' health - related experiences into consideration .
children report that they can feel threatened when in contact with the health care system , and needle - related procedures in particular make them afraid .
taddio et al . found that 63% of healthy children aged 6 to 17 years reported having a fear of needles .
when afraid , children ask for extra support to endure medical procedures and consequently require extra caring interventions .
however , providing extra caring interventions for every child could be considered too time consuming and , furthermore , undermine the children 's own coping skills .
consequently , identifying those who need extra support would be advantageous . according to galvin and todres ' knowledge , it is important to consider the context of the pain .
the neuromatrix theory includes emotional and cognitive aspects of the way pain signals are interpreted in the central nervous system .
every previous psychological incident in life creates a neurosignature in the brain , and this signature is recalled for the individual when a similar context recurs .
this knowledge encourages nurses to examine past experiences of similar contexts , as earlier pain memories may affect the child in a negative way .
earlier pain experiences of immunizations may affect the child 's coping , and these experiences have to be highlighted when planning for and performing a procedure .
previous experiences of anxiety explain the child 's pain memory and his or her reaction the next time procedural pain occurs .
this highlights the need for nurses to explore children 's anxiety related to needle procedures , as this plays an important role in the child 's development of pain memories in connection with immunizations .
carter emphasizes that nurses need to listen to and notice the things children perceive as important and engage with children 's pain on their own terms .
this means being prepared to care for children with the heart , mind , and self , as well as using all the tools and technology available .
this is in accordance with galvin and todres emphasis on the head , the hand , and the heart as equally important domains of caring .
consequently , nurses have to be aware of the fact that some children need extra support , that is , using the heart , relating to the way pain memories can be developed , that is , using the head . to be able to meet the individual child 's needs ,
it may be beneficial to predict the need for extra support , that is , using both the head and the hand , facilitating individualized caring interventions , that is , using the heart , for each child undergoing needle - related procedures , such as immunizations .
galvin and todres highlight that a nurse needs to use the heart to conduct empathic responding .
however , it should be noted that confirming the child 's emotions using pain - related words may influence the processing of the central nervous system associated with the cognitive dimension of pain .
for this reason , strategies that reduce children 's emotional response in conjunction with needle - related procedures are of utmost importance .
patient - centred care means involving the patient in decisions about their own health issues and is regarded as important for good quality .
the un convention on the rights of the child stresses the need for society to enable children to express their own views on all matters affecting themselves .
nurses are therefore expected to take the child 's perspective and establish whether children experience pain unpleasantness in conjunction with immunizations .
self - reports tend to take the child 's perspective , and they should be the first choice .
self - reports in these circumstances are valuable for capturing children 's pain unpleasantness , but the level of interpretation needed to understand self - reports can vary .
the facial affective scale ( fas ) developed by mcgrath et al . gives children from the age of five an opportunity to report their affective reactions related to pain by marking one of nine faces presented in an ordered sequence from
happiest feeling possible ( 0.04 ) to saddest feeling possible ( 0.97 ) ( figure 1 ) .
an advantage of the fas is that this scale has demonstrated a discrepancy between pain unpleasantness and pain intensity , consequently catching the emotional component of pain .
the assessment of the individual child 's fas score is a method that facilitates the use of the child 's perspective , as it will encourage a discussion of pain unpleasantness between the child and the nurse on the child 's level of knowledge and experience .
this knowledge may facilitate these children learning coping strategies , such as distraction , before they undergo the vaccination .
additionally it has been shown that pictures , such as faces , facilitate children being able to understand and communicate the meaning of pain unpleasantness in contrast to pain intensity . however , whether the fas can predict children 's experience of pain unpleasantness is unknown .
the aim of this study was to evaluate whether the fas can predict pain unpleasantness in girls undergoing immunization .
the participants in the study were females aged 11 to 12 years from three different schools located in western sweden who were fluent in the swedish language .
immunization against hpv is part of the swedish vaccination programme for girls and is given on three occasions .
the primary outcome in this study is whether the fas scores of expected pain unpleasantness predict the fas scores of experienced pain unpleasantness .
the fas scores assess a child 's emotional or affective reaction to a pain experience and range from
it only takes approximately half a minute to administer and assess the fas score .
anxiety scores can be assessed by a visual analogue scale ( vas - a ) with the anchor 's no anxiety and the worst known anxiety .
the vas - a has shown validity in assessing perioperative anxiety in children aged 716 years and has shown significant correlations with the staiy ( state - trait anxiety inventory for youth ) . in the present study , the vas - a was used to assess and evaluate the concurrent validity of the fas .
stress affects the body and the brain in many ways and appears when a person experiences a threat .
the verbal rating scale for stress ( vrss ) reports the recent experience of stress on a verbal rating scale ( 05 ) .
a test of vrss showed no statistical evidence for systematic disagreement or random disagreement , which indicates that it is a valid and reliable scale . in the present study ,
each child reported fas scores three times in conjunction with each vaccination , that is , a total of nine times ( figure 2 ) .
before each vaccination all participants received a letter sent to their homes , and they were asked to report their expected feelings prior to the vaccination .
the level of pain unpleasantness was reported on the fas , conducted approximately two weeks before the first vaccination .
the fas scoring before the subsequent vaccination occasions was conducted approximately two weeks after the previous vaccination .
after further two weeks , those who did not return their self - report received a reminder .
the girls assessed their expected pain unpleasantness on the fas at least two weeks before the vaccination to minimize influence between the different measurement points .
the participants also reported their experienced pain unpleasantness in relation to each of the three vaccinations , just before and during the immunization .
the scores during the immunizations were collected immediately afterwards . to further test the concurrent validity of the fas , two other instruments were used in conjunction with each immunization .
the girls reported their anxiety on a vas - a , and the scores during the immunizations were collected immediately afterwards . in addition , the girls reported their vrss score after each immunization ; this score reflected the experience of stress during the immunization .
the fas has been validated in earlier studies using parametric statistics , which led to the selection of equal statistics in this study .
the study population was deemed sufficient compared with heden et al . , who calculated the clinical significance using parametric statistics .
the fas scores before each immunization were compared with the fas scores during each immunization using linear regression analysis .
the linear regression analysis was repeated for each immunization , that is , three times .
the dependent variable was the output , that is , the experienced pain unpleasantness , and the independent variable was the expected pain unpleasantness .
a beta score close to 1 indicates a strong connection between independent and dependent variables .
the comparison between experienced pain unpleasantness ( fas scores ) before the immunizations and the experienced pain unpleasantness ( fas scores ) during the immunizations was tested by a paired t - test .
the concurrent validity was tested by a pearson correlation test that compared the fas scores and the vrss scores as well as the fas scores and the vas - a scores during the immunizations .
written information was provided to both children and parents , and verbal information was given in school .
voluntariness as well as the right to withdraw from the study at any time without any explanation and consequences was highlighted .
the study was approved by the regional ethical review board ( dnr : 466 - 12 ) .
two invited girls declined to participate and one girl dropped out of the offered immunization and consequently from this study . in the end , 37 study participants , aged 11 to 12 years , participated in this study .
some of the study participants declined to report their expected pain unpleasantness on the fas ; 31 study participants ( 84% ) reported their expected pain unpleasantness before the first immunization , 28 study participants ( 76% ) before the second immunization , and finally 32 study participants ( 86% ) before the third immunization .
all study participants assessed their pain unpleasantness before and during each vaccination and anxiety and stress during each vaccination .
most study participants reported a moderate level of expected pain unpleasantness on the fas ( mean 0.56 ) before all three immunizations ( table 1 ) .
the level of expected pain unpleasantness differed between the study participants ( table 2 ) .
the study participants reported their pain unpleasantness just before the first ( mean 0.64 ) , second ( mean 0.58 ) , and third vaccinations ( mean 0.52 ) .
thirty - seven study participants reported their experienced pain unpleasantness , anxiety , and stress during each immunization ( table 3 ) .
the study participants also reported moderate experienced pain unpleasantness on the fas during all three immunizations .
however , the fas scores did not increase significantly from directly before to immediately after , which reflected the feelings during the first ( p = 0.05 ) , second ( p = 0.06 ) , or third ( p = 0.56 ) occasions of immunization .
the comparison of the expected pain unpleasantness ( fas scores ) before the first vaccination and the experienced pain unpleasantness ( fas scores ) during the first vaccination showed beta 0.59 ( p = 0.001 ) , the expected pain unpleasantness ( fas scores ) before the second vaccination , and the experienced pain unpleasantness ( fas scores ) during the second vaccination showed beta 0.75 ( p < 0.001 ) , and finally the expected pain unpleasantness ( fas scores ) before the third vaccination and the experienced pain unpleasantness ( fas scores ) during the third vaccination showed beta 0.64 ( p < 0.001 ) .
the pearson correlation test showed significant correlations between the fas , vas - a , and vrss during all three of the immunizations ( table 4 ) .
the self - reporting instrument , fas , acceptably predicted adolescent study participants ' level of pain unpleasantness when undergoing immunizations . since needle - related fear has been found to be as common as in 63% of children and youth , nurses are challenged to reach those requiring extra support .
the finding of this study brings hope for achievements in the future for nursing care for needle - related procedures such as immunizations . following galvin and todres , skills in the practical processes of caring as well as knowledge about the context
knowledge about how to predict pain unpleasantness during hpv vaccination enables nurses to plan for caring interventions and strive to reduce the potential negative experience .
additionally , the study revealed that fas showed a good to high level of concurrent validity with both the vas - a and vrss measures , suggesting that the fas captures a global rating of the child 's emotions rather than specific stress or anxiety .
this is in accordance with perrott et al . defining the fas as an instrument describing children 's emotions globally and ranging from a happy to a sad condition .
the study participants ' fas scores did not significantly change over time , making it possible to use this instrument to predict pain unpleasantness .
the fas may then be used for screening to identify children who will experience immunization unpleasantness .
this facilitates providing these children with extra support and can perhaps help them to cope better with the procedure during immunizations .
moreover , if nurses use a validated assessment scale to find children who need extra support it will minimize the risk of nurses using their own preunderstanding of pain instead of the children 's perspectives , which is in accordance with the un convention on the rights of the child .
despite the fact that some study participants did not expect or experience pain unpleasantness , the number scoring substantial pain unpleasantness ( fas score 0.75 ) should not be ignored .
similarly , an estimated one - sixth of children aged 12 assessed high fas scores both before and during a needle - related procedure .
the results in the current study also showed mean values of the fas scores that pointed out pain unpleasantness as an issue to highlight .
if the fas score shows that a child is in need of extra support , this support can be implemented before the pain unpleasantness appears .
it is an advantage if an instrument is easy to use and can actually predict pain unpleasantness .
this study showed that the fas instrument has the potential to meet this requirement . while the fas discriminates pain unpleasantness from pain intensity and additionally assesses pain unpleasantness in conjunction with procedural pain ,
. it may be difficult to learn new strategies in direct conjunction with the procedure causing pain unpleasantness .
children who undergo immunizations can feel uncomfortable , that is , experience a fear of needles . however , not all children are afraid of immunizations .
forsner et al . found that children 's emotional experiences when undergoing a venepuncture varied greatly , and some of the children 's narratives even suggested that they
love shots . those children who can easily manage the situation are perhaps not candidates for extra caring interventions .
nevertheless , the consequences of missing a child who really is in need of extra support are significant , involving , for instance , unnecessary suffering for the child and furthermore requiring time - consuming interventions in order to help the child to cope with upcoming situations concerning needle - related procedures .
carter stresses that besides nursing skills and technique , nurses need to be empathetic in order to understand children 's feelings .
similarly emphasize that caring means letting the fearing child 's emotion into the heart in order to make caring creativity available .
however , it should not be forgotten that using pain - related words in communication with the child may have a negative influence on the experienced pain . beyond using the heart in caring , galvin and todres
emphasize that nurses need to use the head and the hand to offer children optimal caring .
the ability to screen and identify children who need extra support makes it easier for nurses to fulfil caring activity and prevent unnecessary suffering .
the current study showed that the fas could be used as a screening method and , fortunately , does not require that much time .
the fas is thereby a way for nurses to use both their head and their heart and to respond to the individual child 's own experience .
nurses need to identify those children who experience pain unpleasantness and offer them caring interventions , which may prevent further negative pain memories .
nurses administer immunizations to children and thereby contribute to child health , but , unfortunately , some children are afraid of needle procedures .
this fact provides good reason for nurses to help children who predict a high level of pain unpleasantness . to achieve this ,
nurses need knowledge of interventions that reduce pain unpleasantness , according to galvin and todres : using their heads .
this means that nurses gain knowledge that they can apply in their caring of children undergoing immunizations .
furthermore , they need to actually use this knowledge and offer individualized caring intervention , that is , use their hands .
there are several evidence - based interventions to offer children who are in need of extra support during immunization .
an important start to prevent children 's pain unpleasantness is to inform them about what will happen .
the knowledge about what will happen gives the child a sense of control , and this can often be an important key to success when children undergo needle - related procedures . additionally , combining local anaesthesia with both sensory and procedural information
consequently , preparations before immunizations are important and facilitate children having influence on their own health care .
furthermore , offering children distraction , such as guided imagery , has been found to be a pain - reducing intervention .
music medicine and video games have also been found to help children cope with procedural pain . however , it is important to ask each child about his or her desire to cope with the situation .
some children benefit more from watching the needle - related situation than from being distracted , and children 's coping styles have consequently been categorized as either
however , when given distraction training and offered the option to choose at follow - up , both
distracters and attenders preferred to use distraction . warning or sympathizing using language that refers to negative experiences may not facilitate children feeling better , but there is value in focusing on pain unpleasantness in children . the ability to discuss with children how they will manage the pain unpleasantness during the vaccination ,
the nurse ought to discuss and decide in consultation with the child how the practical processes should be carried out in conjunction with the immunizations .
in this way , the nurse listens to the child and takes the child 's perspective , as prescribed in the un convention on the rights of the child .
the assessment with the fas takes hardly any time , and future research could look at the feasibility of incorporating this as a standard instrument in clinics .
the study ought therefore to be repeated with a larger population . on the other hand
, the study design was strengthened by the fact that there were no dropouts during the immunizations and , in addition , it permitted each child to repeat the measurement on the fas up to nine times .
the fact that only girls participated in the study could also be considered a weakness , but , to date , only girls have received hpv immunizations in sweden .
it would in any case be valuable to repeat the study design with boys . finally ,
since the findings are limited to young adolescents , further studies should focus on whether children in other age groups are able to predict own pain unpleasantness on the fas .
the fas is valid for predicting , as well as measuring , pain unpleasantness in 11 - 12-year - old girls who undergo immunizations .
this instrument consequently has the potential to identify children who are in need of extra support to cope with the situation , facilitating individualized prevention and caring interventions for children undergoing immunizations . | needle fear is a common problem in children undergoing immunization . to ensure that the individual child 's needs are met during a painful procedure it would be beneficial to be able to predict whether there is a need for extra support .
the self - reporting instrument facial affective scale ( fas ) could have potential for this purpose .
the aim of this study was to evaluate whether the fas can predict pain unpleasantness in girls undergoing immunization .
girls , aged 11 - 12 years , reported their expected pain unpleasantness on the fas at least two weeks before and then experienced pain unpleasantness immediately before each vaccination .
the experienced pain unpleasantness during the vaccination was also reported immediately after each immunization .
the level of anxiety was similarly assessed during each vaccination and supplemented with stress measures in relation to the procedure in order to assess and evaluate concurrent validity .
the results show that the fas is valid to predict pain unpleasantness in 11 - 12-year - old girls who undergo immunizations and that it has the potential to be a feasible instrument to identify children who are in need of extra support to cope with immunization . in conclusion
, the fas measurement can facilitate caring interventions . |
after the initial description of the role of the pulmonary veins in initiating and maintaining atrial fibrillation ( af ) , increasing clinical evidence of efficacy and safety of left atrial ablation has been gathered . consequently , catheter ablation of af is performed in increasing numbers and centers [ 3 , 4 ] . despite this increase in experience and the rapid development of novel tools to facilitate the procedure and improve success rates , the procedure remains complex mainly due to ( a ) the need for transseptal access , ( b ) the variable left atrial anatomy , and ( c ) the possible risk of serious periprocedural complications , including cerebral embolism .
a major concern is the development and embolism of intracardiac thrombi during the procedure with the possible risk of stroke .
therefore , the use of heparin with an activated clotting time ( act ) of 300400 s is mandatory and endorsed by current guidelines . despite this , the reported rate of peri - interventional cerebrovascular accidents ranges from 0 to 7% .
the administration of heparin is commonly started after safe transseptal access has been obtained in order not to aggravate a possible , transseptal , puncture - related pericardial effusion .
this however results in a short dwell time of foreign material within the left atrium without effective anticoagulation , which may be sufficient for thrombus formation and which thus may account for the still evident risk of embolism .
therefore , we sought to evaluate the possible benefits of early versus late heparin administration in regard to intracardiac thrombus formation as assessed by intracardiac echocardiography ( ice ) .
intracardiac echocardiography can be safely used throughout invasive procedures to visualize intracardiac structures , monitor transseptal puncture and intracardiac catheters as well as energy delivery , early detect complications , and possibly avoid adverse events [ 68 ] .
the value of ice in monitoring the left atrium for intracardiac thrombi has previously been demonstrated and is therefore used in this study to investigate the presence of thrombi depending on peri - interventional anticoagulant treatment .
in this observational study , we included 60 consecutive af ablation procedures in 55 patients ( 5 re - do procedures ) , which were performed with the use of ice .
one day prior to the ablation procedure , transesophageal echocardiography ( tee ) was performed in all patients to rule out intracardiac thrombi . emptying velocity of the left atrial appendage
all patients underwent pulmonary vein isolation without additional lesion sets after written informed consent was obtained .
oral anticoagulation was stopped prior to the ablation procedure until the international normalized ratio dropped below 1.5 .
vascular access was obtained via the left femoral vein for insertion of a decapolar , 4f coronary sinus catheter ( inquiry , st .
jude medical , usa ) , and the 10f ice catheter ( acunav , siemens , germany ) .
the right atrium , interatrial septum , left atrium , mitral and aortic valves , left atrial appendage , and the left and right pulmonary veins were visualized by ice . subsequently
, transseptal sheaths were advanced via the right femoral vein into the right atrium , and , after transseptal access , into the left atrium .
catheters for mapping and ablation were advanced via the transseptal sheaths into the left atrium .
we used either a carto guided approach ( n = 26 ) with a circular mapping catheter ( lasso , biosense webster , diamond bar , usa ) and an open irrigated tip ablation catheter ( navistar thermocool , biosense webster , diamond bar , usa ) , or a duty - cycled circular mapping and ablation catheter ( n = 34 , pulmonary vein ablation catheter , ablation frontiers , usa ) .
heparin was administered after transseptal access as a bolus of 100 u / kg in the first 13 procedures ( group i , late heparin ) and in all following 47 procedures ( group ii , early heparin ) as a bolus of 40005000 u after puncture of the femoral veins but before transseptal access , followed by another bolus after transseptal access with a cumulative target dose of 100 u / kg .
heparin was then continuously administered to achieve an act of 300400 s. act was measured 15 min after transseptal access and every 1530 min thereafter .
catheters were guided by an electroanatomical map whenever available , by intermittent fluoroscopy and by continuous monitoring of the ice images .
special attention was paid to verify correct and stable catheter positions in the ice images and to continuously monitor for possible side effects such as pericardial effusion , intracardiac thrombi , and showers of bubbles suggestive for tissue overheating .
endpoint of the procedure was pulmonary vein isolation as determined by loss of pulmonary vein potentials and entrance block .
subsequent comparison was performed using the mann - whitney test for variables with non - gaussian distribution or the unpaired t - test for gaussian distribution , respectively .
patients in group i were younger ( 56.6 13.7 versus 65.9 9.9 years , p = .01 ) .
other baseline factors such as gender ( 76.9% versus 61.7% males , p = .51 ) , type of af ( 46.2% versus 65.9% paroxysmal , p = .21 ) , chads2-score ( 1.5 1.1 versus 1.5 1.1 , p = .91 ) , history of cerebrovascular event ( 30.8% versus 12.8% , p = .20 ) , hypertension ( 76.9% versus 70.2% , p = .74 ) , diabetes ( 0% versus 12.8% , p = .32 ) , and coronary artery disease ( 15.4% versus 27.7% , p
echocardiographic parameters such as left atrial diameter ( 46.9 9.0 versus 45.1 7.0 mm , p = .65 ) , left ventricular ejection fraction ( 51.2 9.8 versus 51.7 8.8% , p = .74 ) , left ventricular end - diastolic diameter ( 51.4 5.6 versus 52.7 9.4 mm , p = .98 ) , wall thickness of septum ( 10.8 1.2 versus 10.7 1.5 mm , p = .61 ) , posterior wall ( 10.5 1.9 versus 10.8 1.3 mm , p = .62 ) , and maximal emptying velocity of the left atrial appendage ( 41.2 11.6 versus 48.5 17.1 cm / s ) also did not differ between groups . in 2/13 ( 15.4% )
group i patients as compared to 0% of group ii patients , intracardiac thrombi were detected ( p = .04 ) .
classification of thrombus was made because of independent mobility and tissue characteristics consistent with thrombus .
one thrombus adhered to a circular mapping catheter ( figure 1 ) , the other thrombus was visualized adherent to a transseptal sheath ( figure 2 ) .
in both cases , thrombi were detected early after transseptal access and before radiofrequency energy delivery .
disruption of the endocardial surface by radiofrequency ablation thus can not be responsible for thrombus formation .
the thrombus attached to the transseptal sheath was detected before administration of heparin , whereas the thrombus which was attached to the circular mapping catheter was detected after heparin administration . in the latter case ,
act measured immediately after thrombus detection was 289 s. these two patients did not differ regarding baseline parameters from the whole group . in both cases ,
additional heparin was given to achieve an act > 300 s. sheaths and catheters were retracted to the right atrium while strong suction was applied to the sheath 's sidearm , carefully flushed , and another transseptal puncture performed .
no thrombi could be detected in the aspirate , neither were any clinical signs suggestive of central , peripheral , or pulmonary embolism noted .
based on the frequent observation of thrombi , we changed the anticoagulatory regimen to administration of heparin before transseptal access and a subsequent bolus after transseptal access to reach a target dose of 100 u / kg . in all following procedures ,
no intracardiac thrombi could be observed . due to the fact that the first act was measured 15 min
after transseptal access rather than heparin administration , it was significantly shorter in group i than in group ii ( 244.3 40.7 versus 301.7 68.7 s , p = .002 ) .
mean acts during the procedure did not differ between groups ( 297.9 40.9 versus 319.4 43.7 , p = .11 ) .
we observed one cardiac tamponade with the need for pericardiocentesis , which occurred during ablation on the ridge between the left atrial appendage and the left superior pulmonary vein .
one periprocedural stroke occurred in one group ii patient ( 1/47 ; 2.1% ) in whom we did not observe an intracardiac thrombus formation during ice monitoring .
the patient complained about difficulties speaking without any further neurologic deficits . while initial examination by the neurologist and cerebral imaging by computed tomography immediately after the procedure did not show signs of cerebral ischemia , repeat imaging on the next day revealed an ischemic area in the cerebellum .
he was then anticoagulated , and the repeat tee before the procedure did not show an intracardial thrombus .
we observed intracardiac thrombi in 2 group i patients ( 2/13 ; 15.4% ) versus none in group ii patients ( 0% ; p = .04 ) and one cva in one group ii patient ( 1/47 ; 2.1% ) versus none in group i patient ( 0% , p = ns ) , resulting in a total event rate of 15.4% in group i versus 2.1% in group ii ( p = .11 ) ( figure 3 ) .
the use of effective anticoagulation is out of question during left atrial ablation procedures in order to minimize the risk of thrombus formation and embolism . despite the use of anticoagulation
, the risk of periprocedural stroke reported ranges from 0% to 7% [ 5 , 9 , 10 ] , with most of these occurring within 24 h of the ablation procedure .
predictors of periprocedural stroke seem to be a chads - score 2 and the history of a cerebrovascular accident .
subclinical embolism as detected by magnetic resonance tomography in clinically asymptomatic patients has been reported in 11% of patients after left atrial ablation , with risk factors being left ventricular hypertrophy , left ventricular dilatation , and the presence of coronary artery disease .
it is common practice to start administration of heparin after transseptal access has been obtained safely , in order not to aggravate a possible pericardial effusion related to transseptal puncture .
this approach is endorsed by a german consensus document on peri- and postinterventional stroke during cardiac catheter procedures .
however , the short dwell time of foreign material within the slow flow area of the left atrium may be sufficient for thrombus formation .
of note , measurements of acts in our study show that whenever the operator decides to administer heparin after transseptal access , the patient is exposed to an increased risk of left atrial thrombus due to suboptimal act levels ( 244 versus 301 , p = .002 ) for at least 15 minutes .
the presented data show an incidence of 15.4% of thrombi adherent to sheaths and catheters within the left atrium almost immediately after left atrial access when heparin was given after transseptal puncture , as compared to an incidence of 0% when heparin was given before transseptal access .
the most likely mechanism for thrombus formation is the presence of foreign material within the left atrium , since both thrombi ( a ) were adherent to sheaths and catheters , respectively , and ( b ) were detected early before energy delivery at a point when no endothelial disruption induced by ablation had occurred .
clinical characteristics previously described as risk factors for periprocedural stroke or subclinical embolism did not differ between these groups .
ren et al . found an incidence of left atrial thrombi as detected by ice in 10.3% of patients , in all of whom heparin was given after transseptal access .
our observed incidence is also comparable to another study , which found left atrial thrombi in 9.1% of cases , when heparin was given after the second transseptal puncture , as compared to 0% when heparin was given before transseptal access . in this retrospective analysis ,
possible clinical risk factors of embolism as described above and the intensity of anticoagulation were not evaluated . however , our data and the data provided by ren et al . and bruce et al . strongly suggest that ( a ) ice is useful in thrombus detection and ( b ) thrombus formation must be expected in 1015% of cases when heparin is given after transseptal access . in these studies , as well as in our population ,
removal of sheaths to the right atrium while applying suction and careful flushing was safe and did not provoke clinical evident embolism . similarly to other authors , we believe that immediate withdrawal of the thrombus by retraction of sheaths and catheters , respectively , is the best strategy once a thrombus has been detected , since further manipulation of catheters carries the risk of thrombus dislodgement and embolism .
the only clinical evident embolism in our series of patients developed in a group ii patient in whom no intracardiac thrombus was detected prior to embolism .
the overall combined event rate of thrombus detection and clinical evident embolism therefore was not significantly different ; however , this retrospective study might be underpowered to detect such a difference .
still , it might be that heparin administration , even if started early , might not be the optimal strategy for periprocedural anticoagulation .
it remains unclear whether the observation of an intracardiac thrombus is predictive of embolism , however , it seems to be intuitive to avoid such a thrombus formation .
an association of left atrial thrombus and embolism was also not be found in other studies [ 13 , 14 ] , which may be related to ( a ) the small number of la thrombi overall detected , ( b ) the early detection by ice and subsequent removal , or ( c ) subclinical embolism . of note
. continued oral anticoagulation with a therapeutic inr at the time of ablation is another strategy for peri - interventional management of anticoagulation .
low event rates of embolism as well as bleeding have been described in experienced centers , and this evidence supports the strategy of maintaining therapeutic levels of oral anticoagulation during ablation procedures [ 1517 ] .
current evidence also shows that cardiac tamponade is not more frequent , more severe , or more difficult to manage when occurring during continued oral anticoagulation [ 17 , 18 ] .
intracardiac thrombus formation is frequent when heparin is administered after transseptal access and not associated with previously described risk factors for clinical and subclinical embolism .
early administration of heparin ( i.e. , before transseptal access ) diminishes this risk but did not affect the rate of cvas in this study .
further prospective investigation should address the issue whether early peri - interventional anticoagulation reduces the risk of embolism and stroke .
the study results must be appraised according to the retrospective and nonrandomized study design and the low event rate . additionally , despite consequent observation of the ice images , as a two - dimensional imaging modality
therefore , we can not exclude that thrombi developed in an unmonitored part of the left atrium and thus would have been overlooked . | objective . despite the use of anticoagulation during left atrial ( la ) ablation procedures , ischemic cerebrovascular accidents ( cvas ) are recognized as a serious complication .
heparin is usually given after safe transseptal access has been obtained , resulting in a short unprotected dwell time of catheters within the la , which may account for cvas .
we investigated the frequency of cvas and la thrombus formation as detected by intracardiac ultrasound ( ice ) depending on the timing of heparin administration .
methods and results .
sixty la ablation procedures with the use of ice were performed in 55 patients .
patients were grouped by heparin administration after ( group i , n = 13 ) and before ( group ii , n = 47 ) transseptal access .
group i patients were younger ( 56.6 13.7 versus 65.9 9.9 years , p = .01 ) ; other clinical and echocardiographic characteristics did not differ between groups .
early thrombus formation was observed in 2 ( 15.4% ) of group i patients as compared to 0% of group ii patients ( p = .04 ) .
one cva ( 2.1% ) occurred in one group ii patient without prior thrombus detection , and none occurred in group i patients ( p = ns ) .
conclusion .
early administration of heparin reduces the risk of early intracardiac thrombus formation during la ablation procedures .
this did not result in reduced rate of cvas . |
precise preoperative planning is a vital principle of success in orthopaedic surgery . arguably , no field in medicine is as dependent for its success on accurate planning and implementation of alignment correction and implant placement .
traditionally , preoperative planning has been performed on standard radiographs with various techniques , including the use of clear plastic templates [ 79 , 11 ] .
recently , digital templating was proposed as a method to electronically overlay templates from a digital library on clinical radiographs for arthroplasties .
the advocates of this technique cite the wide variety of available templates , the speed and precision of the technique , and elimination of hard - copy printouts of radiographs with their associated cost .
the disadvantages of digital templating are the dependence on the digital library , cost of the software , and limitations in software design for each application .
the purposes of this study are to describe ( 1 ) the techniques used in deformity analysis and preoperative surgical planning using standard radiographs for joint arthroplasty and corrective osteotomies of the extremities , ( 2 ) the use of ct scans to analyze rotational deformities in the presence and absence of joint prostheses and in planning corrective rotational osteotomies or revision joint replacement , and ( 3 ) the techniques for analyzing angular deformities of the spine . for all these applications , the specific use of a widely available image analysis software is discussed .
for this study i proposed using commercially available software ( adobe photoshop 6.0 ; adobe systems inc , san jose , ca ) for a wide variety of orthopaedic surgical applications .
however , any software program that allows calibration of measurements for lines and angles can be used .
they can be used with a step - by - step technique for orthopaedic applications with lower cost and increased flexibility than commercial orthopaedic software ( tables 13 ; figs . 1 , 2).table 1general techniques for adobe photoshoptechniques / steps1 .
multiple layers can be created by going to the layer menu at the top and selecting new and then layerb .
1 , large white arrow ) on the toolbarc . draw the path for the lines by pressing the left mouse button and dragging the mouse to the end of the line and then releasing the left mouse buttond .
finalize the line on the new layer by placing the cursor over the line , pressing the right mouse button and selecting fill path .
the line will now be placed definitively on the new layer established as shown in 1a2 .
1 , small black arrows)b . to measure an angle abc , press the left mouse button and drag from points a to b c. at this point , a linear measurement is given at the top of the screend .
keep the cursor over point b and press the alt button ; the ruler is then converted to an angle icon on the imagee . press the left mouse button again and drag the mouse to point cf . at this point ,
an angular measurement is given for the angle abc in the " info " window ( under " window " menu at top of screen)3 .
placement of texta . creating a new layer and then selecting the text tool ( fig . 1 , letter t ) b. press the left click button and drag the mouse to create a text box ; text can be typed into the text box c. if text is not visible , be sure the font is large enough to match the size of the image and change the color to improve contrast table 2technique for radiographic templating using adobe photoshopprocedures / steps1 . image and template software entrya .
convert images to black and white by selecting the image toolbar followed by mode ; select grayscalec . save template image files in separate folders as jpeg ( .jpg ) files ( select file toolbar followed by save as and select a file name with a format as jpeg)d .
enter desired radiographs , ct images , or mr images into software by directly downloading them or by scanning theme . in cases of standard radiographs
, some form of calibration template must be placed on the skin at the level of the jointf . in cases of ct or mri ,
menu on the photoshop menu window at the top of the screen containing the items : file , edit , image , layer , etch .
collect and save all images used for that specific case on the computer desktop or in a separate folderj .
the template image is inverted to make it more visible by selecting the image menu , followed by
from the photoshop toolbar ( fig . 1 ) , select the top left marquee tool ( black arrow ) in the shape of a rectangle d. on the template image , the marquee tool is used to outline the template along with a 10-cm segment of the calibration ruler found on most implant templatese .
select the preoperative radiograph image and press ctrl v to paste the desired portion of the template image onto the preoperative radiograph imageh . at this point , under the layers palette
2 ) , one will see a new layer ( that of the template image)i .
toggle the visibility of this layer by pressing the visibility toggle , which appears as an eye on the layers palette ( fig . 2 , black arrow)j .
change the opacity of the new template image by selecting the template layer on the layers palette ( fig . 2 ) and using the opacity tool on the upper right of the layers palette ( fig .
2 , large black arrowhead ) ; slide it to 50% ; at this point , the template image becomes partially transparentk .
change the brightness / contrast of the new template image by selecting the template layer as described previously and selecting the image toolbar followed by adjust ; select brightness / contrast3 . transforming the template imagea .
next , the size of the selected portion of the template image is adjusted to the radiographb
the template layer c. once this has been selected , a rectangle encloses the entire template image layerd .
the rectangle can be resized either vertically or horizontally by passing the cursor over any of its sides , pressing the left mouse button , and dragging the mousee .
the rectangle can be rotated by passing the cursor over its corners , pressing the left mouse button , and dragging the mousef .
ensure the magnification change seen at the top of the page is equal for both the height and width to avoid template image distortiong .
select the layer of the template image in the layers palette ( fig . 2 , template image not shown)b . use the move tool in the top right of the photoshop toolbar ( fig .
1 , large black arrowhead)c . left click on the template image and drag it to the desired position with the mouse5 . calibrating the template imagea .
rotate the template image , resize , and reposition it as directed in 3a e so that its ruler portion superimposes the calibration of the preoperative radiograph ; for example , the 100-mm ruler should be exactly matched to the 100-mm radiographic marker on the skinb . once the sizing has been selected , the template image can again be rotated and repositioned ( but not resized ) and placed in the desired position over the bonec . the same technique
can be performed for multiple templates ( ie , femoral and acetabular for tha , femoral and tibial for tka , calibrated rulers , etc)d .
while working on a new template , make the previous template layers invisible by toggling the layer visibility icon on the layers palette ( fig . 2 ,
black arrow)table 3technique for rotational analysis of ct / mr images using adobe photoshopstepsa .
copy and paste desired cuts as separate layers into new image document ( fig .
make all images invisible by toggling the layer visibility icon on the layers palette ( fig . 2 , black arrow)c .
alter the opacity of the image listed higher on the layer list to 50% ( fig . 2 , black arrowhead)d .
draw lines along the axes that are to be measured ( eg , femoral neck axis , transmalleolar axis , etc ) as directed in general techniques ( table 1)f .
1the toolbar from the software package is shown with common tools needed for digital templating : line tool ( white arrow ) ; measure tool ( small black arrows ) ; text tool ( letter t ) ; marquee tool ( black arrow ) ; and move tool ( large black arrowhead).fig .
2the software layers palette is shown with visibility toggle ( black arrow ) and layer opacity adjustment ( large black arrowhead ) .
general techniques for adobe photoshop technique for radiographic templating using adobe photoshop technique for rotational analysis of ct / mr images using adobe photoshop the toolbar from the software package is shown with common tools needed for digital templating : line tool ( white arrow ) ; measure tool ( small black arrows ) ; text tool ( letter t ) ; marquee tool ( black arrow ) ; and move tool ( large black arrowhead ) .
the software layers palette is shown with visibility toggle ( black arrow ) and layer opacity adjustment ( large black arrowhead ) .
the alignment objective in tka is to restore a projected anteroposterior weightbearing axis of the lower extremity to pass through the center of the knee .
traditionally , in the coronal plane , the goal is to perform the distal femoral cut exactly perpendicular to a line from the femoral head to the apex of the femoral notch distally .
the anatomic axis is defined as the line of best fit in the femoral diaphysis that passes through the center of the distal femur .
the position of this axis is obtained intraoperatively by placement of an intramedullary rod in the femur starting at a point just anterior to the origin of the posterior cruciate ligament on the inferior trochlea . on the femur , the angle between the mechanical axis and the anatomic axis ( fig .
this angle is measured in the software package and usually is between 4 and 7 ( fig . 3b , black arrow ) .
this angle is equivalent to the valgus angle set on the distal femoral cutting guide , thus achieving a distal femoral cut perpendicular to the mechanical axis of the femur . on the tibia ,
the goal is to cut the tibial surface exactly perpendicular to the line connecting the midpoint of the medial and lateral tibial spines and the center of the ankle .
a perpendicular line is drawn to this mechanical axis line in the software package , which then can be translated proximally and distally depending on the desired degree of bone resection . by placing the distal femoral and proximal tibial cut lines on their respective bones , the profile and thickness of each cut can be predicted preoperatively ( fig .
3b ) and both bone cuts will be perpendicular to their respective mechanical axes in the coronal plane . the same strategy can be applied in the sagittal plane with the measurement of the native proximal tibial slope and distal femoral flexion / extension as well as the analysis of any post - traumatic or congenital deformities involving the femur or tibia.fig .
a preoperative radiograph shows the mechanical axis of the femur connecting the central distal femur and femoral head and the anatomic axis of the femur ( the line running from the distal femur center up the femoral shaft ) .
the tibial mechanical axis is shown as the line connecting the midpoint between the medial and lateral tibial eminence and the center of the ankle .
( b ) a higher - magnification view of lower extremity alignment is shown at the level of the knee .
the black arrow represents the angle between the mechanical and anatomic axes of the femur , which can be used to set the distal femoral valgus cut angle used intraoperatively during tka .
( c ) the radiographic templates are imported into the software and are pasted onto the preoperative radiograph ( these templates do not contain a calibration ruler ) .
postoperative ( d ) anteroposterior and ( e ) lateral radiographs after the tka are shown .
( a ) a preoperative radiograph shows the mechanical axis of the femur connecting the central distal femur and femoral head and the anatomic axis of the femur ( the line running from the distal femur center up the femoral shaft ) .
the tibial mechanical axis is shown as the line connecting the midpoint between the medial and lateral tibial eminence and the center of the ankle .
( b ) a higher - magnification view of lower extremity alignment is shown at the level of the knee .
the black arrow represents the angle between the mechanical and anatomic axes of the femur , which can be used to set the distal femoral valgus cut angle used intraoperatively during tka .
( c ) the radiographic templates are imported into the software and are pasted onto the preoperative radiograph ( these templates do not contain a calibration ruler ) .
postoperative ( d ) anteroposterior and ( e ) lateral radiographs after the tka are shown .
a second objective in planning tka using digital templating is to select the size of the implants preoperatively .
this can be performed by scanning each of the templates into a separate image file and calibrating the templates to the calibration markers placed on the radiograph ( table 2 ) .
the preoperative plan can be performed by overlaying the template images digitally on the radiographs ( fig .
this technique allows a close approximation of the ultimate implant sizes preoperatively ( fig .
the goal of tha is to replace the painful hip . for long - term success , many believe that the restoration of hip biomechanics may lead to improved outcomes . with appropriate preoperative planning , the hip center of rotation and the offset between the proximal femur and the acetabulum can be improved .
the offset is defined as the lateral displacement of the femur relative to the pelvis .
the surgeon can adjust these factors based on the depth of reaming and subsequent cup placement , vertical placement of the femoral implant , type of implant used ( standard versus high offset ) , use of offset polyethylene liners , and variations in femoral head sizes .
digital templating allows the surgeon to precisely preplan these factors to restore hip biomechanics and to minimize leg length inequality ( fig .
( a ) a preoperative radiograph with acetabular and femoral components is shown with the implants in the desired positions and with the templates resized to match the image calibration .
( a ) a preoperative radiograph with acetabular and femoral components is shown with the implants in the desired positions and with the templates resized to match the image calibration .
resurfacing tha has gained worldwide interest in recent years as a method to address younger patients with advanced hip arthritis while preserving bone for future surgical procedures .
varus positioning of the femoral component in the coronal plane is one risk factor for such failure resulting from the tensile stresses applied to the lateral neck bone .
this can be the result of an excessively small femoral component diameter or a femoral preparation that is excessively in the valgus position in which the cylindrical reamer penetrates the cortical bone of the lateral neck .
on the lateral projection , the femoral head sometimes can be translated posteriorly on the neck forcing the starting point for the guide pin into a more anterior position on the head to remain central in the neck .
additionally , excessive anteversion , retroversion , anterior translation , or posterior translation of the femoral component could lead to notching or impingement against the acetabular component .
my preferred technique is to minimize the size of the femoral component while avoiding notching and thus to decrease bone resection on the acetabular side ( fig . 5 ) .
based on these observations , it is clear there is little room for error in placement of the femoral component in either varus or valgus directions .
some guidance systems use a pin in the lateral femoral cortex to determine the trajectory of the guide pin , measuring the vertical distance from the top of the greater trochanter to the entry site on the femoral shaft ( 65 mm ; fig . 5b , white arrows ) .
others reference the intertrochanteric crest and measure the distance between the projection of the guide pin and the lesser trochanter ( 45 mm ; fig . 5b , black arrow )
. both of these measurements can be preplanned by placing calibrated rulers in the desired position in the software.fig .
( a ) a preoperative radiograph shows the size template ( 10 cm ) on skin .
( b ) a preoperative radiograph with the calibrated template is shown in the desired position on the femoral component to avoid notching and to achieve the desired stem shaft angle ( acetabular sizing requires different positioning of the template ) .
calibrated rulers have been imported to determine the distance for guide pin placement along the lateral femoral cortex or along the intertrochanteric crest ( based on preferred guidance technique ) .
( a ) a preoperative radiograph shows the size template ( 10 cm ) on skin .
( b ) a preoperative radiograph with the calibrated template is shown in the desired position on the femoral component to avoid notching and to achieve the desired stem shaft angle ( acetabular sizing requires different positioning of the template ) .
calibrated rulers have been imported to determine the distance for guide pin placement along the lateral femoral cortex or along the intertrochanteric crest ( based on preferred guidance technique ) .
the indications for osteotomy of the proximal tibia include localized osteoarthritis and deformities of the lower extremity from congenital or traumatic etiologies .
i have used the technique of digital templating to preplan a proximal tibial osteotomy for a posttraumatic valgus deformity ( fig .
6 ) and to perform a virtual correction in the coronal plane with the software . using this technique
, the size and shape of an opening wedge and closing wedge can be predicted and planned before surgery .
additionally , plate templates can be used in the same way as for joint arthroplasty to determine optimal plate length and the potential need for plate bending .
the virtually osteotomized tibia can then be freely transformed and rotated until the desired correction is achieved on the image ( fig .
the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position .
in addition to the coronal plane planning , the same strategies can be used in correcting congenital or posttraumatic deformities in the sagittal plane and in avoiding an iatrogenic sagittal deformity during correction of a pure coronal plane deformity.fig .
6a d digital preoperative planning of opening wedge varus high tibial osteotomy in a posttraumatic deformity is shown . in this case , a lateral opening wedge osteotomy is selected .
( a ) osteotomy level selection : the level of the initial transverse osteotomy is drawn on the long , standing radiograph .
next , the selection tool is used to select a rectangle to include the entire tibia , ankle , and foot ( not shown ) .
( b ) osteotomy fragment reorientation : this image selection is copied and pasted into a new layer in the software .
the virtually osteotomized tibia then can be rotated using the free transform function ( white box ) .
this view allows assessment of desired correction by drawing a line from the center of the femoral head to the center of the ankle ( not shown ) .
the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position and the lateral cortical opening can be measured on the preoperative plan .
( d ) postoperative radiograph : a postoperative radiograph was obtained after stabilization with a locking plate ( tomofix ; synthes , paoli , pa ) and after placement of a tricalcium phosphate wedge in the osteotomy site .
digital preoperative planning of opening wedge varus high tibial osteotomy in a posttraumatic deformity is shown . in this case , a lateral opening wedge osteotomy is selected .
( a ) osteotomy level selection : the level of the initial transverse osteotomy is drawn on the long , standing radiograph .
next , the selection tool is used to select a rectangle to include the entire tibia , ankle , and foot ( not shown ) .
( b ) osteotomy fragment reorientation : this image selection is copied and pasted into a new layer in the software .
the virtually osteotomized tibia then can be rotated using the free transform function ( white box ) .
this view allows assessment of desired correction by drawing a line from the center of the femoral head to the center of the ankle ( not shown ) .
the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position and the lateral cortical opening can be measured on the preoperative plan .
( d ) postoperative radiograph : a postoperative radiograph was obtained after stabilization with a locking plate ( tomofix ; synthes , paoli , pa ) and after placement of a tricalcium phosphate wedge in the osteotomy site .
rotational malalignment of the lower extremity is an important source of disability and pain . in many cases
physical examination methods such as the quadriceps angle ( q angle ) , j sign , femoral anteversion , and tibial torsion are examiner - dependent and do not provide quantitative information on the degree and location of deformities ( ie , femoral or tibial ) .
thus , axial ct scans of the lower extremity provide a valuable role in measurement of these deformities . by superimposition of these images using the software package and by changing the opacity and visible layers
, these relationships can be measured precisely and the optimal corrective procedures can be selected ( table 3 ) . the technique requires a ct scan of the pelvis , which includes the femoral heads down to the lesser trochanters , the knee from the distal femur to the level of the tibial tubercle , and the ankle to include both malleoli . to determine the femoral neck axis ,
i have used the method described by teitge and torga - spak ( fig .
7a ) by superimposing the axial image with the femoral head center on a second axial image at the base of the neck with its center .
using the layering functions of the software , a line is drawn connecting these two points ( tables 1 , 2 ) . a second line is drawn along the posterior femoral condyles ( fig .
the angle between the femoral neck axis and the posterior femoral condylar line is measured and defined as the femoral anteversion .
this can be performed by superimposition of the layers of the distal femur and those of the femoral neck and head ( fig .
an identical technique can be used to determine external tibial torsion ( fig . 7d , white arrow ) using a coronal line bisecting the tibial plateau .
measurements of these values are important in the determination of congenital and posttraumatic deformities and in planning corrective osteotomies.fig .
( a ) superimposed axial images of the femoral head centers and bases of femoral necks are shown .
( b ) an axial ct scan of the distal femur is shown with posterior condylar lines .
the angle between the posterior condylar lines and femoral neck axis is defined as the femoral anteversion .
( d ) tibial torsion is measured by bisecting the proximal tibiae ( white arrow ) and defining the transmalleolar axis ( black arrowhead ) .
( a ) superimposed axial images of the femoral head centers and bases of femoral necks are shown .
( b ) an axial ct scan of the distal femur is shown with posterior condylar lines .
the angle between the posterior condylar lines and femoral neck axis is defined as the femoral anteversion .
( d ) tibial torsion is measured by bisecting the proximal tibiae ( white arrow ) and defining the transmalleolar axis ( black arrowhead ) .
berger et al . used rotational analysis for assessment of patellar subluxation and dislocation in tka .
they compared 30 patients with patellofemoral complications after tka with 20 patients with successful outcomes .
they developed a method to measure the component rotation of the femoral and tibial components independently by superimposing the axial ct images .
femoral component rotation was measured relative to the transepicondylar axis and tibial component rotation was measured relative to the tibial tubercle ( fig .
they reported a combined internal rotation of 1 to 4 was associated with lateral tracking and patellar tilt .
larger degrees of combined internal rotation ( 717 ) were associated with early patellar dislocation or late patellar failure . in the case of tka ,
the ct images need only include the knee down to the level of the tibial tubercle unless there is an issue regarding proximal femoral or distal tibial deformity . using this technique , the relationship between the transepicondylar axis ( fig . 8a , black arrow ) and
8c ) can be evaluated to determine whether tibial malrotation is an underlying factor leading to patellar subluxation or dislocation ( fig .
8a drotational malalignment in tka can be determined according to the method of berger et al . .
( a ) the transepicondylar axis ( black arrow ) and posterior condylar axis of the prosthesis ( white arrow ) are marked on this axial ct section of the distal femur .
( b ) an axial ct scan of the proximal tibia is shown at the level of the polyethylene liner with the center point of the liner shown ( white dot ) .
( c ) an axial ct of the proximal tibia is shown at the level of the tibial tubercle .
is calculated as the angle between a line bisecting the insert sagittally from anterior to posterior running through the center point and a line bisecting the tibial tubercle and running through the center point .
rotational malalignment in tka can be determined according to the method of berger et al . .
( a ) the transepicondylar axis ( black arrow ) and posterior condylar axis of the prosthesis ( white arrow ) are marked on this axial ct section of the distal femur .
( b ) an axial ct scan of the proximal tibia is shown at the level of the polyethylene liner with the center point of the liner shown ( white dot ) .
( c ) an axial ct of the proximal tibia is shown at the level of the tibial tubercle .
is calculated as the angle between a line bisecting the insert sagittally from anterior to posterior running through the center point and a line bisecting the tibial tubercle and running through the center point .
numerous methods have been used to measure kyphotic , scoliotic , and pelvic deformities in the axial skeleton .
they can include measurements of the cobb angle in scoliosis , kyphotic angles after vertebral fractures , and pelvic obliquity .
one such example would be measurement of the kyphotic angle in guiding operative versus nonoperative treatment of burst fractures of the spine ( fig . 9 ) .
in the method described by knight et al . , the angle subtended by a line overlying the inferior end plate of the vertebra above the fracture and the line overlying the inferior end plate of the fractured vertebra is measured using the techniques described here ( fig .
9a c the kyphotic angle for a thoracic burst fracture is calculated according to the method of knight et al . .
( a ) a lateral radiograph of the thoracic spine is shown from a patient who has sustained a burst fracture .
( c ) the kyphotic angle is measured according to the method of knight et al . .
the kyphotic angle for a thoracic burst fracture is calculated according to the method of knight et al . .
( a ) a lateral radiograph of the thoracic spine is shown from a patient who has sustained a burst fracture .
( c ) the kyphotic angle is measured according to the method of knight et al . .
the purpose of this article is to provide technical guidelines for use of a widely digital imaging software program to achieve three objectives : the use of deformity analysis and preoperative surgical planning of standard radiographs in joint arthroplasty and corrective osteotomies , the use of ct scans to analyze rotational deformities , and the analysis of angular deformities of the spine . using these techniques , preoperative planning
can be performed from anywhere and at any time as long as the images can be scanned or downloaded directly from the radiographic archive into the software program
. the critical requirements of any software program used for this application include the ability to select and resize certain segments of any image , the use of multiple layers , and the ability to alter the opacity of various layers .
the same approaches can be used for essentially any orthopaedic procedure requiring linear or angular correction and/or implantation of size - specific implants .
the techniques are limited to two - dimensional imaging and each view needs to be templated independently .
this limitation makes management of complex three - dimensional deformities difficult , if not impossible .
another limitation of this technique is that the accuracy and precision have not been validated .
the techniques require a baseline investment of time from each operator with a variable learning curve .
this can lead to insufficient interobserver and intraobserver reliability . finally and most importantly , when used in preoperative planning , the techniques presented here provide a tool to place the implants and perform osteotomies based on a preoperative goal
. however , the ideal position of implants and extremity alignment in arthroplasty and corrective osteotomies are points of ongoing debate .
detailed a method to optimize images of radiographs obtained with a digital camera using photoshop software .
however , the emphasis of their study was not on preoperative planning and/or the use of the overlay template technique discussed in this article .
the segmentation of mr images has been a difficult challenge often requiring either semiautomated or fully manual segmentation of images to separate bone from the surrounding soft tissues .
park et al . described the use of special functions of photoshop software to perform semiautomatic segmentation of mr images .
they then used these segmented images to make three - dimensional reconstructions of various anatomic structures , including the skin , bones , digestive tract , respiratory tract , urinary tract , cardiovascular system , and nervous system .
they were able to fulfill their objective of substantially accelerating the segmentation process over manual segmentation .
in yet another medical application of the same type of software , carvalho et al . described a digital subtraction radiography technique to evaluate chronic periapical dental lesions .
however , by using the digital subtraction technique , the authors were able to determine if the lesions were healing or expanding and also to quantify the size of the osseous lesions at each time point .
the techniques for image rotation , sizing , and the use of layers are similar to those discussed in the current article . despite the limitations , the techniques presented here recapitulate the same techniques that have been used for decades using planning tools such as tracing paper , plastic templates , and printed radiographs .
the ultimate objective of this study was to show that a universally available digital imaging software package can be used for a wide variety of orthopaedic applications , including analysis of deformities , size - specific templating of implant - related procedures , and planning of corrective surgical procedures .
with progression of the digital age and gradual elimination of hard - copy radiographs , digital deformity analysis and surgical planning may soon become the standard technique . | analysis of deformity and subsequent correction are the basis for many orthopaedic surgical procedures . in advanced cases of joint degeneration , arthroplasty may be the only available treatment option . until recently
, these analyses and preoperative surgical plans have been performed using standard radiographs , tracing paper , and/or plastic overlays .
numerous customized , commercially available , computer - based preoperative planning software programs have been introduced .
the purposes of this study were to describe ( 1 ) the techniques used in deformity analysis and preoperative surgical planning using standard radiographs for joint arthroplasty and corrective osteotomies of the extremities , ( 2 ) the use of computed tomography ( ct ) scans to analyze rotational deformities in the presence and absence of joint prostheses and in planning corrective rotational osteotomies or revision joint replacement , and ( 3 ) the techniques for analyzing angular deformities of the spine .
all these applications were performed with a widely available image analysis software . |
metal
ions are both ubiquitous to and crucial in biology . in classical
simulations , they are typically described as simple van der waals
spheres , making it difficult to provide reliable force field descriptions
for them .
an alternative is given by nonbonded dummy models , in which
the central metal atom is surrounded by dummy particles that each
carry a partial charge . while such dummy models already exist for
other metal ions , none is available yet for cu2 + because
of the challenge to reproduce the jahn teller distortion .
this
challenge is addressed in the current study , where , for the first
time , a dummy model including a jahn teller effect is developed
for cu2 + .
we successfully validate its usefulness by studying
metal binding in two biological systems : the amyloid- peptide
and the mixed - metal enzyme superoxide dismutase .
we believe that our
parameters will be of significant value for the computational study
of cu2 + -dependent biological systems using classical models . | |
chronic myeloid leukemia ( cml ) is a clonal myeloproliferative disorder of hematopoietic stem cells characterized by the reciprocal translocation t(9;22 ) ( q34;q11 ) , the philadelphia chromosome ( ph ) .
the resulting breakpoint cluster region - abelson 1 bcr - abl1 oncogene has a deregulated tyrosine kinase activity , which produces a constitutive proliferative signal responsible for the transformed phenotype of cml cells .
the age - adjusted incidence rate is 1.6 cases per 100,000 individuals per year and the median age at diagnosis is 65 years , with a slight male predominance .
proliferative retinopathy is a rare form of presentation of cml and few cases have been reported .
in january 2011 a 48-year - old caucasian male presented to the eye casualty department , hospital de santo antnio - centro hospitalar do porto , porto , with a 1 1 day history of acute visual loss and floaters in his left eye ( le ) .
the patient also complained of a 1 week history of blurred vision in the right eye ( re ) .
he had been diagnosed with hypertension and dyslipidemia in 2009 and he was medicated with candesartan / hydrochlorothiazide 16mg/12.5 mg once per day ( qd ) and simvastatin 20 mg qd .
both his parents had hypertension and dyslipidemia , without major ocular complications ; besides this the remaining family history was unremarkable .
in the last appointment ( march 2010 ) his best corrected visual acuity ( bcva ) was 20/20 with a myopia correction of 1d in both eyes ( be ) . when asked specifically about other symptoms , he stated he did note some fatigue over the last 3 months but it was not severe enough to seek medical attention as it disappeared with rest .
he denied any history of radiation treatment , injury , weight loss , fever , rash , bone pain , abdominal discomfort , left upper quadrant pain or night sweats . on presentation , his bcva was 20/50 in the re and 20/200 in the le with no improvement with pinhole .
on slit - lamp examination , the anterior segment had no abnormalities with an intraocular pressure of 14mmhg ( goldmann applanation tonometry ) in be .
no relative afferent pupillary defect was observed ; ocular movements were preserved in all fields of gaze .
dilated fundus examination showed the presence of multiple vascular abnormalities in the posterior pole and in all four quadrants of the peripheral retina in be , including , dilated and tortuous veins , widely scattered dot - blot and flame - shaped retinal hemorrhages , microaneurysms , multiple sea fan peripheral retinal neovascularisations with arteriovenous anastomosis , and vitreous hemorrhage more evident in the le [ figure 1 ] .
fluorescein angiography showed some degree of blockage due to the vitreous hemorrhage and the retinal hemorrhages in be .
fluorescein angiography also showed widely scattered microaneurysms , marked areas of peripheral retinal non - perfusion due to capillary dropout , arteriovenous anastomosis , and peripheral retinal neovascularisations with a sea fan configuration but without evidence of vasculitis [ figures 2 and 3 ] .
a diagnosis of bilateral proliferative retinopathy was made and an initial systemic evaluation was performed .
several laboratory test results were within normal limits , including : 0 the fasting glucose and hemoglobin a1c ; lipid profile ; reactive protein c activity ; erythrocyte sedimentation rate ; homocysteine level ; serum protein s , protein c , and antithrombin iii .
factor ii mutation , factor v leiden mutation , anti - cardiolipin antibodies , and lupus anticoagulant antibodies were negative .
complete blood count and peripheral blood smear evaluation revealed hyperleukocytosis ( 24810 cells / mm ; reference value , 4.5 - 11.010 cells / mm ) , thrombocytosis ( 68410/mm ; reference value , 150 - 45010/mm ) , normochromic normocytic anemia ( 10.8g / dl ; reference value , 13.0 - 18.0g / dl ) , hyperuricemia and elevated serum levels of lactate dehydrogenase ; the white blood cell differential count indicated an increased number of circulating mature and immature granulocytes , with the presence of blasts ( 1% ) and a basophilia of 5% .
color fundus photographs of posterior pole of the right eye ( a and b ) and left eye ( le ) ( c - d ) demonstrate dilated and tortuous veins , dot - blot and flame - shaped retinal hemorrhages and microaneurysms in both eyes , and also mild vitreous hemorrhage in the le ( a - f ) : fluorescein angiography of the right eye shows peripheral retinal non - perfusion with leaking peripheral neovascularisation ( a - f ) : fluorescein angiography of the left eye shows some degree of blockage due to the vitreous hemorrhage ; it also demonstrates multiple arteriovenous anastomosis with marked areas of capillary dropout and extensive leaking peripheral neovascularisations based on the ocular findings and hematological abnormalities , the patient was referred to a hematologist for further management .
the physical examination revealed a palpable splenomegaly 10 cm below the left costal margin and mild signs of anemia .
the bone marrow was hypercellular with an elevated myeloid - to - erythroid ratio and increased number of megakaryocytes but without fibrosis ; the blast percentage was 4% .
the morphologic findings combined with the cytogenetic analysis showing the reciprocal chromosomal translocation t(9;22 ) were diagnostic of cml .
the patient was started on hydroxyurea ( 2 g twice a day ) , allopurinol ( 300 mg qd ) and imatinib mesylate ( 400 mg qd ) which brought about a reduction in his white cell and platelets counts over a period of 5 weeks .
the patient achieved complete cytogenetic remission 6 months after initiation of therapy , and after 9 months of therapy he had undetectable levels of bcr - abl .
bilateral scatter panretinal photocoagulation ( prp ) of the peripheral avascular retina was performed in several sessions .
a vitrectomy with peripheral proliferative membrane peeling and endolaser was also performed in the le as the persistent vitreous hemorrhage did not allow the laser photocoagulation to be performed .
the patient has been stable for the last 10 months and his bcva is 20/25 in be at the most recent visit .
the natural history of cml in the absence of treatment is characterized by a triphasic course comprising a chronic phase ( cp - cml ) , followed by an accelerated phase and an invariably progression to a final fatal blastic phase in a variable period of time .
the prompt recognition of his ocular fundus findings and early referral to oncologists for adequate management may have been a major factor in determining his long - term outcome because patients with cp - cml at the time of diagnosis have the best long - term prognosis with treatment .
however , the treatment strategies of this type of leukemia have undergone revolutionary developments in the last decade mainly due to a detailed knowledge of its molecular pathogenesis .
the bcr - abl1 fusion protein has been the target for drug design and treatment of this disorder , which have enabled the development of effective tyrosine kinase inhibitors ( tkis ) that have the potential to eradicate the ph chromosome - positive clone . at the time of diagnosis our patient was started on imatinib mesylate at the initial recommended dose of 400 mg qd , given orally , and to date , with good results as it has been capable of inducing complete hematologic , cytogenetic , and major molecular remission in a tolerable manner .
achieving these responses early during the course of therapy may have been one of the most important factors in determining his long - term prognosis .
this drug , a first generation tki approved by the united states food and drug administration in 2001 , has become the initial treatment of choice for most patients with cml because it results in a marked increase in the long - term survival with preservation of an acceptable quality of life .
the patient reported here will be maintained on this therapy for the long - term , unless he develops resistance or intolerance to imatinib , or until other evidence regarding the duration of imatinib therapy for those in cp - cml is consistently available .
this drug has a rapid onset and short duration of action and so it permitted the control of the hyperviscosity syndrome without marked or prolonged myelosuppression .
as the white cell count was dramatically increased to 248,000cells/l hydroxyurea was started in a high dose .
the patient also had hyperuricemia prior to chemotherapy which reflected the high cell turnover rate and probably would have been exacerbated by the use of the antineoplastic agent hydroxyurea .
therefore , at the time of receiving cytotoxic therapy , the patient was started on oral allopurinol ( a xanthine oxidase inhibitor , which inhibits uric acid production ) as well as adequate hydration to maintain high urine output .
the ocular fundus findings observed are not pathognomonic of cml as they may be present in various local and systemic diseases involving the eye . according to mahneke et al .
the stage of retinopathy at the time of diagnosis may not affect the overall prognosis of these patients , even though it may adversely compromise visual outcome if not timely managed as recently reported by mandava et al .
this was one of our main concerns when the therapeutic approach of the bilateral proliferative retinopathy was planned .
vitreoretinal surgery with proliferative membrane peeling and endolaser was performed due to persistent vitreous hemorrhage in the le along with bilateral prp of the avascular zones .
this ocular treatment was delineated according to the principles , which have proven beneficial for other proliferative retinopathies of various etiologies .
the therapy has been performed by mandava et al . in a case of bilateral advanced proliferative retinopathy due to cml .
our patient is being carefully monitored for recurrent ocular disease because this may occur without any systemic evidence of relapse of cml as previously reported by nobacht et al .
it is important to try to elucidate the various physiopathological mechanisms that may be involved in the ocular manifestations of cml as they may reflect the effects of this disease throughout the body . in the present case ,
various mechanisms may have independently contributed to the final via of reduced blood flow , vascular stagnation , retinal capillary dropout , ischemia and neovascularisation .
the exact pathophysiology remains obscure but different mechanisms have been proposed in other diseases that may , or not , play a role in cml , including : 0 anemia ; leukostasis ; hyperviscosity syndrome ; leukoembolization ; endothelial lesion and localized thrombosis secondary to toxic products released by the leukemic cells ; angiogenic factors released by the ischemic retina ; increased serum levels of angiogenic growth factors , including increased levels of vascular endothelial growth factor , fibroblast growth factor 2 , hepatocyte growth factor and matrix metalloproteinases .
further studies are necessary to evaluate the possible pathogenic role of these various mechanisms as this may result in potential clinical implications , for instance in the evaluation and prognosis of these patients and in the development of new therapeutic targets .
treatment of the underlying causes of the ocular findings could actually result in their improvement or even resolution . in summary
, ophthalmologists should consider a thorough systemic evaluation for leukemia in patients with bilateral proliferative retinopathy as this may be the first sign of cml as previously reported by other authors .
our case illustrates that prompt treatment can result in resolution of the ocular findings and allow for restoration of visual function .
finally a timely diagnosis , an adequate supportive care and the initiation of imatinib mesylate have been essential for securing a favorable prognosis in this patient . | the authors report a rare case of a 48-year - old male with chronic myeloid leukemia ( cml ) who initially presented with a bilateral proliferative retinopathy .
the patient complained of recent visual loss and floaters in both eyes ( be ) .
ophthalmologic evaluation revealed a best corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/200 in the left eye ( le ) .
fundoscopy showed the presence of bilateral peripheral capillary dropout with multiple retinal sea fan neovascularisations , which were confirmed on fluorescein angiography .
full blood count revealed hyperleukocytosis , thrombocytosis , anemia , and hyperuricemia .
bone marrow aspiration and biopsy showed the reciprocal chromosomal translocation t ( 9;22 ) , diagnostic of cml .
the patient was started on hydroxyurea , allopurinol and imatinib mesylate .
he received bilateral panretinal laser photocoagulation and a vitrectomy was performed in the le .
the patient has been in complete hematologic , cytogenetic , and major molecular remission while on imatinib and his bcva is 20/25 in be . |
multiple injections of insulin is the main form of treatment for type 1 diabetes mellitus ( t1 dm ) . however , despite the combination treatment of ultrarapid and longacting insulin , it is not always easy to achieve an ideal blood glucose diurnal profile .
the diabetes control and complications trial ( dcct ) has confirmed that reductions in blood glucose level in t1 dm can reduce the risk of microvascular complications and cardiovascular events .
however , at least in type 2 diabetes , not only total glucose exposure , but also glucose fluctuations seem to be associated with the onset of cardiovascular disease . accordingly , strict blood glucose control with consideration of glucose swings is recommended for the therapeutic aim of suppressing the onset or progression of macrovascular complications in both type 1 and 2 diabetes .
glucosidase inhibitor ( gi ) , a drug that inhibits disaccharidehydrolyzing enzymes in the small intestine , suppresses postprandial elevation of blood glucose by delaying the digestion and absorption of sugars , and thus flattens out the postprandial fluctuations in plasma glucose . for type 2 diabetes , gi
is used in combination therapy with other oral hypoglycemic agents of different mechanisms of action or insulin . in patients with t1 dm ,
the use of gi , which inhibits the postprandial rise in blood glucose without involving endogenous insulin secretion , together with insulin therapy , is considered highly beneficial . unlike other types of gi ( acarbose , voglibose )
it has been reported that miglitol , compared with similar drugs , strongly suppresses spikes in plasma glucose , particularly soon after meals ( 0.51 h postprandially ) . at the same time , because the time to peak postprandial glucose level is delayed by the administration of miglitol , the incidence of hypoglycemia in between meals decreases .
these findings show that miglitol reduces the range of fluctuations in plasma glucose throughout the day , and combined treatment with miglitol is thought to be effective in patients with t1 dm with intensive insulin treatment and that it also prevents the occurrence of hypoglycemia . in the present study , we investigated the efficacy and safety of 12week miglitol therapy in combination with insulin therapy in patients with t1 dm .
the study subjects were patients with t1 dm who visited abe clinic , asahi internal medicine department clinic , fukuoka city medical association hospital , matsuba clinic , matsunami general hospital , misaki internal medicine clinic , naka memorial clinic or jinnouchi hospital from august 2004 to september 2005 , and had been diagnosed with t1 dm by diabetes specialists in past medical treatment .
the inclusion criteria were : ( i ) plasma glucose at 1 or 2 h after meal 180 mg / dl ; ( ii ) hba1c6.5% ; ( iii ) patients treated with multiple selfinjections of insulin ; and ( iv ) age 20 years .
the study consisted of a 4 to 10week observation period and a 12week treatment period . during the treatment period ,
all subjects were instructed to take a 50 mg miglitol tablet immediately before meals three times a day . throughout the observation and treatment periods ,
the dosage of insulin was adjusted to achieve blood glucose control as judged by the attending physician ; however , changes in the type of insulin or drugs were prohibited .
a meal tolerance test was carried out at the start of treatment and after 12 weeks of treatment in order to measure postprandial plasma glucose .
plasma glucose was measured before breakfast and at 0.5 , 1 and 2 h after the start of breakfast .
the content of breakfast served before and after the treatment in each patient was the same .
all undesirable events ( symptoms and signs ; abnormal fluctuations in laboratory values ) that appeared after the start of the administration of miglitol were collected as adverse events .
adverse events , other than those for which any relation to the study drug could be ruled out , were taken as adverse drug reactions .
general blood tests , blood chemistry tests , and urinalysis including hba1c and 1,5anhydroglucitol ( 1,5ag ) were carried out at the start of treatment and at 4 , 8 and 12 weeks of treatment . in order to check for symptoms of hypoglycemia ,
each subject was provided with a hypoglycemia notebook in which they recorded their condition at the time of the appearance of hypoglycemic symptoms during the treatment period .
the present study was carried out in accordance with the declaration of helsinki and with approval from the institutional review board of the medical institution .
statistical analysis of both efficacy and safety was carried out for data from all the subjects who proceeded to the treatment phase .
the basic statistics for efficacy analysis were calculated at each timepoint and a paired onesample ttest for the baseline was carried out .
data at 12 weeks of treatment or at the time of withdrawal were used as data for the end of treatment . for safety analysis ,
the study subjects were patients with t1 dm who visited abe clinic , asahi internal medicine department clinic , fukuoka city medical association hospital , matsuba clinic , matsunami general hospital , misaki internal medicine clinic , naka memorial clinic or jinnouchi hospital from august 2004 to september 2005 , and had been diagnosed with t1 dm by diabetes specialists in past medical treatment .
the inclusion criteria were : ( i ) plasma glucose at 1 or 2 h after meal 180 mg / dl ; ( ii ) hba1c6.5% ; ( iii ) patients treated with multiple selfinjections of insulin ; and ( iv ) age 20 years .
the study consisted of a 4 to 10week observation period and a 12week treatment period . during the treatment period ,
all subjects were instructed to take a 50 mg miglitol tablet immediately before meals three times a day . throughout the observation and treatment periods ,
the dosage of insulin was adjusted to achieve blood glucose control as judged by the attending physician ; however , changes in the type of insulin or drugs were prohibited .
a meal tolerance test was carried out at the start of treatment and after 12 weeks of treatment in order to measure postprandial plasma glucose .
plasma glucose was measured before breakfast and at 0.5 , 1 and 2 h after the start of breakfast .
the content of breakfast served before and after the treatment in each patient was the same .
all undesirable events ( symptoms and signs ; abnormal fluctuations in laboratory values ) that appeared after the start of the administration of miglitol were collected as adverse events .
adverse events , other than those for which any relation to the study drug could be ruled out , were taken as adverse drug reactions .
general blood tests , blood chemistry tests , and urinalysis including hba1c and 1,5anhydroglucitol ( 1,5ag ) were carried out at the start of treatment and at 4 , 8 and 12 weeks of treatment . in order to check for symptoms of hypoglycemia ,
each subject was provided with a hypoglycemia notebook in which they recorded their condition at the time of the appearance of hypoglycemic symptoms during the treatment period .
the present study was carried out in accordance with the declaration of helsinki and with approval from the institutional review board of the medical institution .
statistical analysis of both efficacy and safety was carried out for data from all the subjects who proceeded to the treatment phase .
the basic statistics for efficacy analysis were calculated at each timepoint and a paired onesample ttest for the baseline was carried out .
data at 12 weeks of treatment or at the time of withdrawal were used as data for the end of treatment . for safety analysis ,
the investigational drug was given to 43 patients ( 22 men , 21 women ; mean age 43.7 13.2 years , sd , table 1 ) with body mass index of 22.9 2.8 kg / m and disease duration of 13.0 7.2 years .
the details of insulin therapy and the number of patients who received each therapy are as follows : oncedaily injections of longacting insulin with bolus doses of rapidacting insulin given three times daily at mealtime ( n = 18 ) ; oncedaily injections of nph insulin with bolus doses of rapidacting insulin given three times daily at mealtimes ( n = 7 ) ; twicedaily injections of nph insulin with bolus doses of rapidacting insulin given three times daily at mealtimes ( n = 5 ) ; oncedaily injections of longacting insulin with bolus doses of regular insulin given three times daily at mealtime ( n = 4 ) ; oncedaily injections of nph insulin with bolus doses of regular insulin given three times daily at mealtimes ( n = 2 ) ; three times daily injections of premixed insulin ( n = 4 ) ; and twicedaily injections of premixed insulin ( n = 3 ) .
data are number of patients ( categorized data ) or mean sd ( quantitative data ) .
figure 1 shows the serial changes in plasma glucose levels after a meal , before and after treatment .
levels at the start of treatment were 217.6 82.1 mg / dl ( n = 43 ) before breakfast , 261.4 83.8 mg / dl ( n = 43 ) at 0.5 h , 274.6 91.8 mg / dl (
n = 43 ) at 1 h and 250.7 102.0 mg / dl ( n = 43 ) at 2 h after the meal .
at the end of treatment they were 208.7 88.0 mg / dl ( n = 41 ) , 199.5 85.7 mg / dl ( n = 39 ) , 199.4 91.3 mg / dl ( n = 39 ) and 212.0 95.8 mg / dl ( n = 39 ) , respectively , and the differences at 0.5 , 1 and 2 h after the meal were significant ( onesample ttest ; p < 0.001 , p < 0.001 and p = 0.01 , respectively ) .
the treatment with miglitol was discontinued in four subjects and the meal tolerance test after treatment was not carried out with them .
thus , the postprandial blood glucose data from these four cases are not included . in two of these four discontinued cases ,
data are mean sd . * p < 0.01 , * * p < 0.001 compared with before treatment .
hba1c level decreased slightly from 7.68 0.89% before treatment to 7.56 0.87% at 4 weeks , 7.57 0.92% at 8 weeks and 7.59 0.97% at 12 weeks of treatment , and the decrease was significant at 4 weeks of treatment ( onesample ttest , p = 0.023 ) .
* p < 0.05 compared with before treatment .
figure 3 shows the serial changes in 1,5ag levels during the 12week treatment .
1,5ag rose from 2.87 2.06 g / ml at the start of treatment to 4.94 3.55 g / ml at 4 weeks of treatment , 5.16 4.16 g / ml at 8 weeks and 4.87 4.01
g / ml at 12 weeks of treatment , and the increases at 4 , 8 and 12 weeks of treatment were significant ( onesample ttest , all p < 0.001 ) .
the incidence of adverse events was 97.7% , and the major symptoms and signs were hypoglycemia and digestive symptoms .
hypoglycemia was observed in 38 of the 43 ( 88.4% ) patients , and the frequency of hypoglycemia over the course of 30 days was 4.11 3.99 times / person/30 days .
one patient with loss of consciousness that required treatment with glucagon was judged to have developed moderate hypoglycemia , whereas the others were considered to have developed mild hypoglycemia and improved without treatment when patients took glucose , supplements or meals .
the major digestive symptoms were flatulence ( 20.9% [ 9/43 ] ) , abdominal distention ( 14.0% [ 6/43 ] ) and diarrhea ( 11.6% [ 5/43 ) ] ) , most of which occurred in the early phase of treatment .
bodyweight was 60.9 10.0 kg at the start of treatment , 61.3 10.1 kg at 4 weeks , 61.0 10.5 kg at 8 weeks and 60.9 10.8 kg at 12 weeks of treatment .
although there was a significant increase at 4 weeks compared with the start of treatment ( p = 0.015 ) , no such increase was noted at 12 weeks of treatment .
the dosage of insulin at the end of treatment , including subjects for whom administration of the test drug was discontinued , was decreased in 17 subjects , increased in 2 and was unchanged in the remaining 24 subjects .
the total amount of insulin used per day was 41.6 17.1 u / day before treatment and 39.8 17.4 u / day at the end of the treatment , with a change of 1.9 3.0 u / day ( onesample ttest , p < 0.001 ) .
the subjects were categorized into two groups : the 17 in whom the dosage of insulin was decreased and the other 26 .
the background data are shown in table 2 , and the hba1c and plasma glucose levels in table 3 . in the insulin reduction group ,
the change in insulin dosage was 4.9 2.8 u / day . in the insulin dose increase or no change group ,
the change in insulin dosage was + 0.1 2.8 u / day .
although no marked differences in background data were observed between the two groups , the age was somewhat higher and the hba1c values somewhat lower for the subjects for whom insulin was reduced . in both groups , a similar inhibition of postprandial elevation of plasma glucose level as a result of administration of miglitol was observed , and favorable control over plasma glucose levels was shown after the reduction in dosage of insulin . with regard to bodyweight change , a modest decrease in bodyweight was observed in the insulin reduction group , whereas an increase in bodyweight was observed in the insulin dose increase or no change group , at the endpoint .
the change in bodyweight was significantly different between the two groups ( insulin reduction group , 0.5 1.2 kg vs insulin dose increase or no change group , + 0.6 1.2 kg ; p = 0.006 ) .
data are number of patients ( categorized data ) or mean sd ( quantitative data ) .
in the present study , 43 t1 dm patients receiving intensive insulin therapy were given miglitol in combination with the usual therapy for a period of 12 weeks .
although no significant reduction in hba1c was noted under the combination treatment , postprandial elevation of plasma glucose was suppressed by the administration of miglitol .
in addition , the administration of miglitol reduced the required insulin dose . in terms of adverse events , no serious hypoglycemia or gastrointestinal symptoms
were observed , and the drug was well tolerated . regarding the incidence of hypoglycemia , only one moderately severe case requiring the injection of glucagon
the dcct study showed that strict control of plasma glucose levels is important in diabetes patients in order to prevent the development and progression of vascular disorders .
however , the results of ukpds 35 showed that the correlation between the development of macroangiopathy and hba1c level is not as strong as that existing in the case of microvascular disease .
in contrast , the results of the decode / decoda study and the funagata diabetes study clearly showed postprandial hyperglycemia to be an independent risk factor in cardiovascular events , which suggests that strict control of plasma glucose levels , including not only hba1c but also postprandial hyperglycemia , is important from the viewpoint of preventing the progression of macroangiopathy . in recent years
, it has been reported that repeated sharp fluctuations in plasma glucose can induce apoptosis of vascular endothelial cells , increase oxidative stress and promote the appearance of adhesion molecules .
the mechanism whereby repeated postprandial hyperglycemia triggers vascular disorders is being elucidated and results have shown that endothelial disorders caused by postprandial hyperglycemia can be improved by correcting the hyperglycemia with drug therapy .
the dcct / edic study , in which t1 dm patients were enrolled , also confirmed that the incidence of cardiovascular events can be reduced by strictly controlling fluctuations in plasma glucose through intensive insulin therapy .
based on these reports , we postulate that the inhibition of postprandial hyperglycemia and the flattening out of postprandial fluctuations in plasma glucose by the miglitol
insulin combination treatment in t1 dm patients could prevent the occurrence and progression of macroangiopathy in such patients .
one of the objectives of the combined use of oral diabetes drugs in diabetic patients treated with insulin is to reduce the required dosage of insulin and lower the risk of hypoglycemia .
weight gain can also become a problem in patients receiving insulin therapy and the ability to reduce the risk of weight gain by lowering the dosage of insulin is thought to be another advantage . in 17/43 subjects in the present study ,
the combined use of miglitol made it possible to reduce the daily dosage of insulin . in order to assess the advantages and disadvantages of reducing insulin dosage
, we used the data obtained from this study to carry out comparisons of baseline values and plasma glucose control status between subjects in whom insulin dosage was reduced and those in whom it was not .
comparison of the baseline values showed that the age was somewhat higher and the hba1c values somewhat lower for the subjects for whom insulin was reduced .
this result might suggest that insulin dose tends to be reduced for older patients with better glycemic control receiving miglitol treatment .
in contrast , both the fasting and postprandial plasma glucose levels were about the same as for the other subjects .
the data obtained at the end of treatment with miglitol showed that postprandial elevation of plasma glucose level was similarly inhibited in both the subjects in whom insulin dosage was reduced and those in whom it was not , and no deterioration in hba1c values as a result of reduced insulin dosage was observed .
these results suggest that good plasma glucose control is maintained even after reducing the dosage of insulin . furthermore , bodyweight tended to decrease in the subjects receiving the reduced dosage of insulin , suggesting that plasma glucose can be controlled without triggering weight gain by adopting a combination treatment with miglitol that allows for reduction of the insulin dosage .
recently , we found that miglitol increases plasma glp1 level after meals . considering that glp1 suppresses appetite and glucagon secretion
, the patients with a high response of glp1 secretion by miglitol should show the decreased bodyweight by the reduced appetite and required insulin dose as the result of the reduction of glucagon secretion .
accordingly , the difference observed by the analysis in the present study between insulin dose reduction group and insulin dose increase or no change group might be derived at least in part from the response of glp1 .
several parameters , including glp1 concentration , are to be measured to examine the details of this hypotheses in future research .
the incidence of hypoglycemia in the present study was 88.4% , at a frequency of 4.11 times / person/30 days .
there are numerous reports on the prevalence of hypoglycemia among patients with t1 dm , as in japanese ( 3.93 times / person/30 days ) and overseas ( 3.46 times / person/30 days ) studies of insulin glulisine and the overseas study of insulin aspart ( 3.7 times / person/30 days ) , and the incidence of hypoglycemia in the present study is not greatly different from these reference reports .
this study was carried out as an open study , in which a control group was not set .
additionally , the adjustment of insulin dose in response to the blood glucose control was carried out by each doctor without a shared rule of insulin dose adjustment .
therefore , the discussion based on the comparison between control and treated groups , and the detailed estimation of the influence on insulin adjustment are not available in the present study . considering that all the participating doctors are diabetes specialists in japan , the data in the present study represents the effect of miglitol under the current control of diabetes specialists in japan . in the present study ,
important clinical data were gathered on the efficacy and safety of the coadministration of miglitol in patients with t1 dm on insulin therapy . | abstractaims / introduction : patients with type 1 diabetes mellitus often show a precipitous postprandial rise in blood glucose that can not be controlled , even by intensive insulin therapy .
the combined use of an glucosidase inhibitor with insulin seems to be highly beneficial in such cases.materials and methods : we investigated the efficacy and safety of miglitol , an glucosidase inhibitor , for 12 weeks in 43 type 1 diabetes patients on intensive insulin therapy.results : coadministration of miglitol resulted in only a modest and temporal decrease in hba1c level .
however , it resulted in a significant reduction of plasma glucose level after breakfast ( 250.7 102.0 mg / dl at 2 h after breakfast before treatment ; 212.0 95.8 mg / dl at 2 h after breakfast after treatment for 12 weeks , p = 0.01 ) and a significant reduction of insulin dosage ( 41.6 17.1 u / day before treatment ; 39.8 17.4 u / day 12 weeks after treatment , p < 0.001 ) . during the study period , 88.4% ( 38/43 ) of subjects experienced hypoglycemia , but all events were mild except for one case , which was considered to be moderate .
no unexpected adverse events were observed during the study period.conclusions : coadministration of miglitol in type 1 diabetes patients on intensive insulin therapy resulted in an improvement of postprandial hyperglycemia with the reduction of insulin dosage .
considering the importance of postprandial hyperglycemia in the onset of cardiovascular disease , the combination therapy of miglitol and insulin could be advantageous in type 1 diabetes mellitus patients . |
reactive oxygen species ( ros ) such as superoxide ( o2 ) , hydrogen peroxide ( h2o2 ) , and the hydroxyl radical ( oh ) are produced in all aerobic organisms within the cell and are normally in balance with antioxidant molecules .
oxidative stress occurs when this critical balance is disrupted because of depletion of antioxidants or excess accumulation of ros ( figure 1 ) .
ros were originally considered to be exclusively detrimental to cells , but it is now recognized that redox regulation involving ros is key to the modulation of critical cellular functions ( figure 2 ) .
regardless of how or where they are generated , an increase in intracellular oxidants results in two very important effects : damage to various cell components and activation of specific signaling pathways , both of which influence numerous cellular processes leading to proper cell functions or to cell death . mounting evidence links oxidants and oxidative stress to a variety of human diseases and aging , as well as to senescence , impaired photosynthesis , and necrosis in plants . on the other hand ,
there are clear examples of how ros are put to constructive uses , the most powerful being the crucial role that o2 plays against invading microbes , by serving as a broad - spectrum antibiotic . plants also mount a broad - range response to invading pathogens by a rapid and transient production of ros via the ' oxidative burst ' .
this article considers a number of recent genome - wide analyses of the response to ros in bacteria , yeasts and arabidopsis in the light of previous knowledge about oxidative stress responses .
when oxidative stress occurs , cells function to counteract the resulting oxidant effects and to restore the redox balance .
all organisms have adaptive responses to oxidative stress , with antioxidant defense enzymes being induced by changes in the levels of h2o2 or o2 , leading to the activation or silencing of genes encoding defensive enzymes , transcription factors and structural proteins .
ros have also been proposed to function as second messengers independent of oxidative stress and to signal such cellular fates as cell proliferation , necrosis , and apoptosis . although various observations have led to the suggestion that cells have the means to sense ros and to induce specific responses , the underlying mechanisms are still not fully understood .
regulators of oxidative stress responses are currently best characterized in bacteria , but progress is now also being made towards understanding such regulators in higher eukaryotes . the transcriptional network that responds to ros in eukaryotes
seventeen years ago , it was shown by two - dimensional gel electrophoresis that the expression of approximately 30 proteins was induced in bacteria by h 2o2 . of these 30 proteins ,
12 were maximally induced within 10 minutes and the other 18 between 10 and 30 minutes .
subsequent work led to the discovery of the oxyr regulatory protein , which was shown to regulate the expression of 9 of the 12 rapidly induced proteins .
the tetrameric oxyr protein is a member of the lysr family of transcription activators and exists in two forms , reduced and oxidized ; the latter is the only form able to activate transcription .
similarly , the superoxide response or soxrs regulatory proteins were found to regulate expression of o2-responsive proteins in bacteria .
regulation of the soxrs regulon occurs by conversion of soxr protein to an active form , which enhances soxs transcription .
in addition to soxr and oxyr , several other transcriptional regulators modulate the expression of antioxidant genes in bacteria , indicative of the complexity and connectivity of overlapping regulatory networks .
there are no apparent homologs of oxyr , soxr , or soxs in eukaryotes , but a number of other transcription factors have been found to play a role in regulating eukaryotic antioxidant genes . in yeast ,
transcriptional regulators of antioxidant genes include ace1 , mac1 , yap1 , yap2 , hap1 , and hap2/3/4 . in higher eukaryotes ,
oxidative stress responses are more complex and are modulated by several different regulators . in mammalian systems ,
nuclear factor b ( nfb ) and activator protein-1 ( ap-1 ) are involved in regulating the oxidative stress response .
the antioxidant responsive element ( are ) is present in the promoter region of genes encoding mammalian glutathione s - transferase ( gst ) , metallothionein - i and manganese superoxide dismutase ( mnsod ) , and is responsible for the induction of these genes in response to oxidants .
the promoters of antioxidant genes in higher plants also contain the are and the nfb and ap-1 binding sites .
the role of these factors is not unique to activation of antioxidant genes , however : nfb , in particular , is known to play central roles in regulating cellular responses to stresses other than oxidants , as well as regulating normal growth and metabolism .
there is substantial evidence to suggest that a variety of biotic and abiotic stresses induce h2o2 , which serves as a common factor in regulating various signaling pathways .
similar stresses also activate mitogen - activated protein ( map ) kinases , with kinetics that either precede or parallel h2o2 production , suggesting that map kinases may be among the many converging points in the defense - signaling network .
in addition , exogenous application of several plant hormones and toxins has been shown to induce synthesis of o2 and h2o2 , leading to differential induction of some antioxidant genes and isozymes .
thus , from a utilitarian viewpoint , the identification of all genes and proteins regulated by h2o2 is an important step toward treatments that might confer tolerance to multiple , but interrelated , stresses .
in addition to induction or repression of antioxidant defense genes , ros are known to similarly affect expression of a variety of other genes involved in different signaling pathways in microbes , yeast , plants , and animals .
efforts to identify ros - responsive genes on a global scale were limited until the advent of microarray - based gene - expression analysis : dna microarrays are now being used to comprehensively examine gene expression networks during oxidative stress .
reports on the stress responses of escherichia coli , yeast , and higher plants have now provided significant progress in surveying gene expression in response to h2o2 .
the transcriptional profile of e. coli cells exposed to h2o2 was examined with a dna microarray composed of 4,169 e. coli open reading frames .
gene expression was measured in isogenic wild - type and oxyr - deletion mutants ( oxyr ) to confirm that the h2o2-response regulator oxyr activates most of the h2o2-inducible genes .
there was a very rapid and strong induction of a set of oxyr - regulated genes in the wild - type but not in the oxyr strain , providing an internal validation of the experiment and confirmation of the induction of the oxidative stress genes identified 17 years ago by other means .
in addition , several new h2o2-inducible genes were identified : some are members of the oxyr regulon and some are induced by an oxyr - independent mechanism .
several genes that are known to be repressed by oxyr were found to be significantly expressed in the oxyr mutant .
overall , the mrna of 140 genes in the wild - type and 167 genes in the oxyr strain were significantly induced after h2o2 treatment .
it was also found that the superoxide response transcription factor gene soxs was induced by h2o2 , indicating an overlap with other regulatory pathways .
also highly induced by h2o2 in both wild - type and oxyr cells were two genes known to be members of the soxrs regulon , fpr and soda ( encoding nadh - ferredoxin oxidoreductase and manganese - superoxide dismutase , respectively ) .
the microarray data revealed an overlap between the oxidative stress and heat - shock and ' sos ' dna - damage responses .
thus , the results from e. coli microarrays clearly indicate that the activities of transcription factors in addition to oxyr and soxrs are likely to be modulated by oxidative stress .
in addition to bacteria , the transcriptional profile in response to oxidative stress has also been characterized in eukaryotes . in one broad - ranging study ,
the expression profile was examined in saccharomyces cerevisiae cells exposed to h2o2 , in addition to other stresses , and the global set of genes induced or repressed by each environmental signal was identified . the results indicated that about two - thirds of the genome is involved in the response to environmental changes and the response to oxidative stress involves about one - third of the yeast genome , with the maximal effects on gene expression occurring slightly later relative to other stresses examined during similar time courses .
most of the transcriptome returns to pre - stress levels within 2 hours of exposure to h2o2 .
genes that are repressed for approximately 1 hour after exposure to h2o2 are only transiently repressed in other stress time courses .
thus , genes encoding the translation apparatus and its regulators are remarkably coordinated in the responses to each environmental change , although the dynamics of each response are different .
the expression programs following h2o2 or o2 treatment were essentially identical , despite the fact that different ros are involved .
there was strong induction of genes known to be involved in detoxification of both h2o2 and o2 , such as catalase , superoxide dismutase , and glutathione peroxidase , as well as genes involved in oxidative and reductive reactions ( for example , thioredoxin , glutathione reductase , and glutaredoxin ) .
the genes most strongly induced in response to h2o2 and o2 were dependent on the transcription factor yap1 for their induction .
genes that are moderately induced by ros and other signals are regulated by different transcription factors , depending on the conditions , and their response may be governed by different upstream signaling pathways .
recently , it has also been demonstrated that h2o2 activates the sty1 ( stress - activated map kinase ) pathway in schizosaccharomyces pombe in a dose - dependent manner , via two sensing mechanisms . at low h2o2 levels ,
sty1 is regulated by a two - component signaling pathway that feeds into either of the two - wak1 or win1 - stress - activated map kinase kinase kinases upstream of sty1 .
in contrast , at high h2o2 levels , sty1 activation is controlled mainly by an independent two - component mechanism , requiring the function of both wak1 and win1 .
in addition , the individual bzip transcription factors pap1 and atf1 were found to function within a limited range of h2o2 concentrations : pap1 activates target genes at low h2o2 concentration , whereas atf1 controls transcriptional responses to high h2o2 , with some minor overlap . some apparent cross - talk among sty1 , atf1 , and
thus , s. pombe deploys a combination of stress - responsive regulatory proteins to gauge and trigger the appropriate transcriptional response to increasing h2o2 concentrations .
this yeast mounts two separate responses to oxidative stress : an adaptive response to low - level h2o2 exposure that protects it from subsequent exposures to higher h2o2 levels , and an acute response that allows the cell to survive a sudden , potentially lethal dose of h2o2 .
the oxidative - stress response has also recently been characterized in an organism more complex than yeast and bacteria .
a recent large - scale cdna microarray analysis of the arabidopsis transcriptome during oxidative stress identified 175 non - redundant expressed sequence tags ( ests ) from a sample of 11,000 that are regulated by h2o2 .
of these , 62 are repressed and 113 are induced ; and rna blots showed that some of the h2o2-regulated genes are also modulated by other signals known to involve oxidative stress . furthermore , a substantial number of these genes have predicted functions in defense responses , cell signaling , transcription , and cell rescue ( from environmental insults and developmental arrest ) , underscoring the pleiotropic effects of h2o2 in the response of plants to stress .
overall , the microarray used was estimated to represent only about 30% of the arabidopsis genome , depending on redundancy , and 1% to 2% of the genes represented in the array are affected by h2o2-imposed oxidative stress , which is comparable to the situation in yeast . of the 175 genes identified as h2o2-responsive ,
most have no obvious direct role in oxidative stress but may be linked to oxidative stress indirectly , as a consequence of other biotic and abiotic stresses , explaining their sensitivity to h2o2 . among the genes induced by h2o2
were genes encoding transcription factors , suggesting that they may mediate downstream h2o2 responses consistent with genomic studies in other species .
also , expression of the map kinases in arabidopsis is induced by oxidative stress , as in other organisms , which in turn can mediate the induction of oxidative stress - responsive genes .
during the past ten years , the traditional view of ros as mere indiscriminate reactive byproducts of cellular metabolism has undergone a metamorphosis .
this change is primarily as a result of the discovery that ros , and particularly h 2o2 , may act as signal - transducing molecules , and that activation of intracellular transcription factors such as oxyr , soxrs , nfb , and ap-1 occur via interaction with ros , leading to gene transcription ( figure 3 ) .
more recently , genome sequencing and expression profiling using dna or oligonucleotide microarrays , and related technologies , have been used effectively in the study of global gene - expression patterns in response to different growth and environmental conditions to which organisms are exposed .
subsequent hierarchical clustering methods allow for the allocation of genes , co - regulated temporally or in response to a given signal , into specific expression groups , or regulons .
the numbers of genes that can be detected by these methods in response to h2o2 or any given environmental or developmental signal far exceed the limited number that could have been detected only a few years ago . given that transcription of genes into mrna is governed by transcription factors ,
which bind to cis - regulatory regions of the dna in the vicinity of their target genes , the question arises as to whether large co - regulated groups of genes share cis - regulatory elements that bind to common transcription factors .
the data available with respect to oxidative stress seem to suggest this is indeed the case .
cis - acting elements within the promoters of ros - activated genes are being defined as well as their cognate trans - acting factors .
a comparative analysis of promoter sequences of genes with similar expression profiles should provide a basis for unraveling common regulatory sequences and overlapping gene - expression networks modulating ros - responsive genes .
the antioxidant enzymes catalase and superoxide dismutase play key roles in modulating the levels of endogenous h2o2 and o2 , which at specific concentrations act , in turn , to modulate the expression of other ros - responsive genes . despite some limitations , mentioned in the papers cited [ 16 , 19,20,21 ] , it has become clear through the use of microarrays that there are far more genes responding to ros than previously thought .
we currently have a large and growing number of sequenced genomes and emerging technologies presenting us with enormous opportunities to advance biological science and our knowledge of how genomes perceive signals to respond to variable environments . knowing the sequences of tens of thousands of ros - responsive genes , however , only reminds us that we still do not know the many proteins they encode , nor the biochemical or biological function of the great majority of such proteins .
how such proteins interact with ros to drive the various physiological processes in aerobic organisms remains a great puzzle .
for the future , the fundamental challenge will be to integrate the information now being obtained on gene - expression patterns with structural and functional parameters and interactions of the various proteins encoded by ros - responsive regulons , and to view the cell in which they function holistically .
research from the author 's laboratory has been supported by grants from the us epa , nsf , usda , and nih .
oxidative stress results from imbalance between the levels of reactive oxygen species ( ros ) and antioxidants ( aox ) . under normal circumstances , cells are able to balance the production of oxidants and antioxidants ( such as catalase and superoxide dismutase ) , resulting in redox equilibrium .
oxidative stress occurs when cells are subjected to excess levels of ros , or as a result of depletion in antioxidant defenses .
they have many roles as signaling molecules in addition to their scavenging protective functions in biological systems . the major signaling pathways activated in response to oxidative stress .
ros can originate from metabolic activity or from external environmental signals and are modulated by antioxidants to nontoxic levels at which point they serve as signaling molecules .
ros can activate gene transcription in two ways : either via transcription factors ( such as , nfb , ap-1 and are - binding proteins , are - bp ) that can interact directly with specific dna motifs , including are , on promoters of target genes ; or via activation of map kinase cascades , which in turn activate transcription factors that trigger target gene transcription .
the degree to which a given pathway is activated is dependent on the nature and duration of the stress , as well as on cell type and developmental stage . | oxidative stress arises from an imbalance between generation and elimination of reactive oxygen species , often leading to cell death .
genomic tools are expanding our understanding of the antioxidant defenses aerobes have evolved and the recently discovered role(s ) of reactive oxygen species in signaling . |
according to the centers for disease control and prevention , 9% of us adults meet the criteria for current depression while 3.4% meet the criteria for major depression .
as depression affects such a large portion of the us population , research investigating the risks it imposes is increasing .
depression has been linked to poorer control of risk factors for cardiovascular disease ( cvd ) , including blood pressure , hemoglobin a1c , and body mass index [ 24 ] .
cvd is one of the leading complications and underlying causes of death among persons with diabetes [ 5 , 6 ] .
therefore , increased cvd risks associated with depression pose a particular threat to the diabetic population .
diabetics not only have poorer control of these risk factors as compared to nondiabetics , but are also nearly three times as likely to be depressed .
depression poses a major risk to this vulnerable group . the question of whether the presence of depression is associated with increased blood pressure , hba1c , and body mass index ( bmi ) among people with diabetes is largely unexamined , but studies that have analyzed these relationships are described later . of the nondiabetes
specific studies that examined the relationship between blood pressure and depression , some found a decrease in blood pressure among those who are depressed [ 8 , 9 ]
. however , nabi and colleagues showed that age - related risks of hypertension increase more within the depressed group than the nondepressed group , arguing that the risk of hypertension increases with multiple depressive episodes over time .
few studies have examined whether depression is associated with hba1c levels and those that did have mixed results .
some studies found an elevated hba1c among diabetic patients with depression compared with their nondepressed counterparts .
one study of diabetic patients found that hba1c is significantly correlated with depression for type 1 patients but not type 2 . a few studies examined depression treatment and diabetic outcomes with mixed results , possibly due to the variation in side effects of antidepressant medications [ 4 , 1316 ] .
although the treatments positively affect mood , some could negatively affect cvd risk factors , thus negating the positive effects of mood improvement on these same factors .
however , one study found that those who remained in depression remission 6 months following the completion of psychotherapy treatments showed significant improvements in hba1c .
katon and colleagues showed that patients who were classified as having major or minor depression were significantly more likely to be obese than those without depression .
as depression affects a high percentage of persons with diabetes , it is important to understand whether the presence of depression , presumably due to undertreatment , is associated with poorer control of these intermediate cardiovascular risk factors . using data from the translating research into action for diabetes ( triad ) study
, this report examines whether there is an association between depressive symptoms and cardiovascular risk factors among diabetics .
first , the triad dataset is a large , geographically diverse dataset of diabetic patients .
as the personal and medical health costs associated with poorer control over cvd risk factors are greater than their nondiabetic counterparts , it is important to analyze this group in particular for depression - related trends .
therefore , the study provides an estimate of patient health information for those who are already connected to care but who may be lacking proper depression diagnoses and treatment . in the triad dataset
, only 44% of those who were identified as depressed by the phq-8 screener were taking antidepressant medications .
we hypothesized that presence of depressive symptoms will be associated with poorer control of blood pressure , hba1c , and body mass index .
this study uses cross - sectional data for a diabetic sample to compare the outcomes of interest ( blood pressure , hba1c , and body mass index ) among those with and without depression . these analyses
triad is a multicenter prospective longitudinal study of persons with type 1 or type 2 diabetes in managed care settings .
eligible patients were community dwelling , not pregnant , had diabetes for more than 1 year , spoke english or spanish , were continuously enrolled in their health insurance plan for 18 months or more , used at least 1 diabetes - related medical service , and were able to provide informed consent .
the institutional review boards at each participating site approved the study and all participants provided informed consent .
this report specifically analyzes data from the 2003 wave ( wave 2 ) of triad in which depression was measured in the patient sample with diabetes .
these analyses of wave 2 include data from five of the six centers contained in the original baseline survey .
this sample includes those who were managed - care patients , 18 years of age and older , had diabetes , and were willing to participate in the wave 2 survey .
less than 5% of the sample had type 1 diabetes , defined as age < 30 years and use of insulin but no oral hypoglycemic agents .
the data were collected from patients using a mailed survey or computer - assisted telephone interviews and medical record reviews .
triad originally enrolled a random sample of 11,927 adults , with 8,334 completing the initial survey and medical record review . while 6,760 patients completed the wave 2 survey ,
medical chart data were unavailable for 1,928 patients ( 28.5% ) , leaving 4,832 patients completing surveys with available medical charts .
the sample size was further reduced by 1,279 patients due to missing data on at least one chart variable in our analyses , leaving 3,553 patients with complete chart data .
the sample size was again reduced by 1,212 due to missing data on at least one survey variable in our analyses , leaving an analytic sample size of 2,341 with complete survey and chart data .
there were three key outcome measures : blood pressure , hba1c , and body mass index ( bmi ) .
blood pressure and hba1c were obtained from the last recorded result of the patient medical record .
these three variables were selected because of their relevance to cardiovascular health in diabetics as well as the completeness of the data within the triad dataset .
depression was measured using the patient health questionnaire ( phq-8 ) , in which anyone with a score of 10 or greater was classified as depressed and anyone with a score of 9 or less was classified as nondepressed .
the charlson comorbidity index was a covariate and was calculated from comorbidity data from the patients ' medical records .
other variables used as covariates in the regression analyses included sex , age , race and ethnicity , income , education , insulin treatment , duration with diabetes , the charlson index , and whether the patient was married or living together with a partner , which came from the patient self - reported survey .
all of the independent variables were categorical except for duration with diabetes and the charlson index and were represented using a series of indicators for each category , with one omitted reference category .
these variables were categorized in order to discern any possible nonlinear trends and to ensure that there were enough participants in each category for the analysis . using linear regression to adjust for patient demographic and health variables as well as fixed research site effects , we compared the adjusted means between the depressed and nondepressed diabetic patients .
results are presented as adjusted means between the groups with and without depression with associated p - values .
we examined sensitivity analyses looking at the associations of depression on the outcome variables after inclusion of smoking , physical activity , and antidepressant use .
descriptive statistics on outcomes and covariates in the depressed and nondepressed groups , and unadjusted tests of differences between depression groups on each of these variables , are shown in table 1 .
hba1c and bmi scores ( but not blood pressure ) were found to differ significantly between depression groups before regression adjustment .
all covariates except duration with diabetes were found to differ significantly between the depressed and not depressed groups . it should be noted that 56% of those categorized as depressed had not taken antidepressant medication and might be considered undertreated in the absence of data on other treatments such as psychotherapy .
differences between patients with and without depression in the three outcomes changed only minimally after adjusting for the above covariates .
table 2 displays the results of the linear regressions , testing the adjusted associations of depression with the three outcomes .
the regression coefficients in table 2 show the amount of change in each outcome per single - unit change in each independent variable . for categorical independent variables ,
the coefficients indicate the value of the difference in the means of each outcome between the categories specified in the row of table 2 and their reference category .
our results showed that blood pressure is more strongly associated with age and race than with any other independent variables .
most older age categories differed significantly from the reference category , while african americans had higher systolic blood pressure levels than whites , and latinos had lower systolic blood pressure levels than whites .
this is consistent with previous study results [ 21 , 22 ] . unlike blood pressure
diabetic patients with depression had hba1c levels that were an average of 0.3 points higher than their nondepressed counterparts .
similar to blood pressure , age and race were also strongly associated with hba1c , which decreased with age and was higher on average in minority groups .
depression had a significant association with bmi , with depressed patients having an average bmi that was 1.55 units higher than that of nondepressed diabetic patients .
gender , age , race , and education also showed significant associations with bmi .
the results showed only a small change in magnitude for blood pressure and no change for hba1c .
there was a moderate change in magnitude on the association of depression on bmi , but the sign and significance of depression did not change .
as these results did not change our conclusions , we only show the original analyses .
to summarize , among the diabetic patients in the triad cohort , presence of depression is associated with poorer control of hba1c and bmi .
however , persons with depression did not , on average , have poorer blood pressure control than those without depression .
in addition to adding evidence to the literature by analyzing a large and both geographically and ethnically diverse sample receiving care from multiple health plans and providers , our findings also show that despite having access to care , depressed diabetic patients still had a more difficult time managing these risk factors . although the cross - sectional study design can not establish causation with regard to the association of depression and control of cardiovascular risk factors , among persons with diabetes , those with depression may be less likely to maintain a healthy lifestyle of weight management and medication adherence .
in addition , these patterns of depression and poor control have been found to be cyclical .
found that depression and obesity are cyclically linked , and depression can lead to obesity while obesity can also lead to depression .
also , sacco and bykowski argue that a perceived lack of control over one 's health , due to both poor adherence and adverse medical outcomes , can also be a cause of depression .
this creates a cyclical pattern that may potentially be broken by appropriate screening , diagnosis , and treatment of depression .
first , our measure for depression was the phq-8 questionnaire , which is a screening test .
although the score is correlated with depression , it does not conclusively confirm the diagnosis .
second , our data do not allow for us to control patients who are receiving nonpharmacologic treatment for depression .
fewer than half of the depressed patients in our sample were treated with antidepressants , but others might have received other treatment modalities such as psychotherapy .
third , although the overall triad study was longitudinal , this particular analysis was conducted using a single wave of data . due to the cross - sectional nature of our analyses
, we can not ascertain whether depression causes a worsening of these cardiovascular risk factors over time , and the impact of previous and/or multiple episodes of depression can not be captured .
fourth , we were unable to control other potentially important unmeasured factors such as family history of depression or other environmental factors .
finally , as our sample was limited to persons with diabetes with managed - care insurance , our findings may not generalize to the uninsured or those with other forms of coverage .
the above results , along with other discussed complications from untreated depression , highlight the importance of screening all persons with diabetes for depression .
although others have shown that sustained improvement in depression from successful treatment can lead to an improvement in hba1c and bmi , in the immediate term , treatment for depression is associated with an increase in hba1c and blood pressure .
however , these results may be due to issues related to selection if patients with more severe depression and associated poorer cvd risk factor control are more likely to receive treatment for depression .
gois et al . found that sertraline and interpersonal psychotherapy both caused depression remission while also decreasing hba1c levels .
echeverry et al . also observed decreases in hba1c and blood pressure when depressed diabetic patients were treated with sertraline .
these findings point toward the need for further research on the impact of various treatments of depression among persons with diabetes if we are to achieve the critically important goals of improving symptoms of depression and decreasing the risk of cardiovascular disease among this vulnerable group with multiple chronic conditions . | diabetic patients are nearly three times as likely to have depression as their nondiabetic counterparts .
patients with diabetes are already at risk for poor cardiovascular health . using cross - sectional data from the translating research into action for diabetes ( triad ) study ,
the authors tested the association of depression with cardiovascular risk factors in diabetic patients .
depression was measured using the patient health questionnaire ( phq8 ) .
patients who scored greater than 9 on the phq8 were classified as depressed and were compared with those who were not depressed ( n = 2,341 ) .
depressed patients did not have significantly different blood pressure levels than those who were not depressed .
however , those who were depressed had higher hba1c levels than those who were not depressed ( p < 0.01 ) and higher bmis than those who were not depressed ( p < 0.01 ) .
these results indicate that depressed diabetic patients are at greater risk of having poor control of cardiovascular risk factors and suggest that depression screening should be a standard practice among this patient group . |
pth modulates osteoblast activity and osteoclast resorption , increases renal tubular reabsorption of calcium , and stimulates conversion of 25-hydroxyvitamin d to 1,25-dihydroxyvitamin d by 1-hydroxylase in the kidney .
in addition , pth acts on bone cells to increase expression of fibroblast growth factor-23 ( fgf-23 ) .
higher pth concentrations are associated with an increased mortality risk among general older populations [ 2 , 3 ] . in the presence of hypovitaminosis
d , absence of secondary hyperparathyroidism is characterized by lower rates of bone turnover and reduced mortality when compared with counterparts who manifest a physiological pth elevation in response to vitamin d deficiency .
pth stimulates interleukin-6 ( il-6 ) production by osteoblasts and liver cells [ 6 , 7 ] . in turn
, il-6 may modulate acute - phase protein synthesis in the liver [ 8 , 9 ] .
it has been proposed to administrate vitamin d in the elderly to reduce serum levels of il-6 and c - reactive protein ( crp ) and , possibly , to decrease the risk of thromboembolic vascular events . in this context ,
patients with hyperparathyroidism will , theoretically , have higher levels of il-6 , crp , or tumor necrosis factor- ( tnf- ) . however , previous studies have had conflicting results .
some showed elevated levels of these inflammatory markers in hyperparathyroidism [ 1114 ] , whereas others found that levels of crp , il-6 , and leukocytes were similar in patients and controls [ 15 , 16 ] .
studies have showed decreased , increased [ 1618 ] , or unchanged [ 13 , 19 ] levels of inflammatory markers after parathyroidectomy .
such association studies may be underpowered to detect meaningful differences due to small sample sizes .
serum concentrations of 25-hydroxyvitamin d and pth were found to independently associate with blood pressure and hypertension in the national health and nutrition examination survey ( nhanes ) in 2003 through 2006 .
in addition , other parameters such as il-6 and tnf- were not available in the nhanes database .
recently , we have validated the significance of another inflammatory marker , neutrophil - to - lymphocyte ratio , in differentiated thyroid cancer . in the present study
, we hypothesized that pth levels might be associated with inflammatory status in the general us population . to test this hypothesis
, we examined the cross - sectional associations between demographic , lifestyle , and serum factors and intact pth using the large - scale data set released by the us nhanes 2003 - 2004 and 2005 - 2006 .
nhanes provides nationally representative cross - sectional data for the health status of the us civilian noninstitutionalized population . in this analysis
, we used data from the 2003 - 2004 and 2005 - 2006 nhanes that obtained serum pth levels .
the design and operation are available on the centers for disease control and prevention website [ 22 , 23 ] .
ethical approval for the study was obtained from the research ethics review board of national center for health statistics , and all participants gave written informed consent .
sociodemographic information such as age , gender , and race / ethnicity was recorded during the interview .
current smokers were defined as participants who were currently smoking and had smoked 100 cigarettes in their life .
blood pressure was measured at the mobile examination centers by physicians with mercury sphygmomanometers using a standard protocol . up
the complete blood count and leukocyte differential count were measured with the beckman coulter maxm analyzer .
red cell distribution width ( rdw ) was derived from the coefficient of variation of the red cell volume distribution histogram .
serum creatinine concentration was measured by kinetic jaffe assays with picrate ( beckman lx20 ) .
estimated glomerular filtration rate ( egfr ) was determined using the chronic kidney disease epidemiology collaboration ( ckd - epi ) equation .
serum total calcium was measured by an indirect ion - selective electrode method ( beckman lx20 ) . when serum albumin level was < 4.0 g / dl , serum calcium levels were corrected using the following formula : corrected calcium ( mg / dl ) = measured total calcium ( mg / dl ) + 0.8 [ 4.0 serum albumin ( g / dl ) ] .
serum pth was measured by electrochemiluminescence immunoassay on an elecsys 1010 autoanalyzer ( roche diagnostics ) .
participants with an elevated c - reactive protein concentration ( > 1 mg / dl ) and a decreased albumin concentration ( < 3.5 g / dl ) were assigned score 2 .
those with an elevated c - reactive protein concentration ( > 1 mg / dl ) were assigned score 1 , and patients with a c - reactive protein concentration of 1 mg / dl and any albumin concentration were assigned score 0 .
all statistical analyses were computed by using survey commands of stata ( stata corporation ) to incorporate sample weights and to adjust for clusters and strata of the complex sample design . where distributions appeared nonnormal , we used natural log transformations to normalize their right - skewed distributions ( white blood cell count , triglycerides , crp , and pth ) .
serum pth levels were stratified into clinically relevant categories ( 639 , 4059 , 6099 , and 100 ) .
potential confounding factors were chosen on the basis of previous studies or of their biologic plausibility .
sample weights , which account for the differential probabilities of selection , nonresponse , and noncoverage , were incorporated into the variance estimation process .
univariate modeling was performed to determine whether a directional trend existed between variables and three inflammatory markers ( crp , rdw , and plr ) .
we examined whether the observed associations persisted within the subgroups stratified by egfr and pth .
subsequently , we constructed full multivariable linear regression models to test associations between inflammatory markers and serum pth .
multivariate models were adjusted for age , sex , race / ethnicity , smoking status , glycohemoglobin , albumin , high - density lipoprotein , ( logged ) triglycerides , corrected total calcium , 25-hydroxyvitamin d , and egfr .
trends across pth categories were also assessed in linear regression models by using continuous ( logged ) pth values .
in addition , we performed logistic regression with a dichotomous outcome of increased mgps ( > 0 ) , adjusting simultaneously for the same covariates .
amongst the 8948 participants who formed our main analysis sample , the weighted mean age was 49.4 years .
the results indicate that with increasing pth levels , age , body mass index , systolic and diastolic pressure , glycohemoglobin , and serum levels of creatinine tended to increase , while egfr and serum levels of albumin , calcium , phosphorus , and vitamin d tended to decrease with increasing pth levels . in all , egfr level was a major determinant of pth levels .
three inflammatory markers ( crp , rdw , and plr ) were examined in our study .
the weighted mean ( 95% ci ) crp , rdw , and plr was 0.49 ( 0.470.51 ) mg / dl , 12.87% ( 12.84%12.91% ) , and 139.6 ( 137.7141.5 ) , respectively .
the unadjusted egfr - stratified levels of crp , rdw , and plr by pth categories are displayed in figure 1 .
crp and rdw values increased at higher pth categories independent of egfr tertile ( all p values < 0.01 ) .
the unadjusted plr tended to increase across pth categories , but the statistically significant difference was observed only among participants in the lowest tertile of egfr ( p = 0.004 ) .
results of the multivariate linear regression predicting levels of three inflammatory markers are presented in table 2 . after adjusting for age and sex ( model 1 ) , the linear relationships of pth with crp and rdw were statistically significant ( p < 0.001 ) .
these relationships persisted after further adjusting for covariates including race / ethnicity , smoking status , glycohemoglobin levels ( model 2 ) , and other potential confounders ( model 3 ) .
after further adjusting for pth determinants including calcium , vitamin d , and egfr ( model 4 ) , crp and rdw remained significantly associated with pth categories ( p < 0.001 ) .
moreover , when ( logged ) pth was entered as a continuous variable in linear regression models , it was independently associated with crp and rdw ( both p < 0.001 ) .
plr was marginally associated with pth after multivariate adjustment for covariables , assessed either by categories of or by continuous pth ( p = 0.190 and p = 0.095 , resp . ) .
additionally , systemic inflammation was examined as a dichotomous variable ( mgps > 0 ) . excluding subjects with missing data of crp ( n = 2 ) and albumin ( n = 34 ) , 1083 ( 12% ) participants had increased mgps .
we found significant correlations between mgps and other inflammatory markers , namely , crp , rdw , and plr ( all p < 0.001 ) .
participants with increased mgps had higher pth concentrations ( weighted mean 51.9 and 45.5 , resp . ; p < 0.001 ) .
as shown in figure 2 , the multivariate odds ratios for increased mgps according to increasing pth categories were 1 , 1.05 , 1.29 , and 1.27 ( odds ratio for trend 1.11 ) .
the trend remained significant when ( logged ) pth levels were included in the logistic regression as a continuous variable ( p values for model 1 to model 4 were < 0.001 , < 0.001 , < 0.001 , and 0.013 , resp . ) .
the odds ratio was 1.39 , 1.42 , 1.46 , and 1.31 from model 1 to model 4 , respectively .
this is consistent with the observation that serum albumin levels were inversely associated with pth concentrations .
recently , we have shown that in dialysis patients with secondary hyperparathyroidism , the symptom burden had a negative impact on patients ' quality of life , and parathyroidectomy significantly improved symptoms and quality of life .
epidemiological studies have consistently reported higher total and cardiovascular mortality associated with pth levels [ 2 , 3 , 28 , 29 ] .
in addition , associations with cancer mortality and noncancer , noncardiovascular mortality have been reported .
pleiotropic effects of pth are also reflected by numerous nonskeletal , nontraditional manifestations of primary hyperparathyroidism .
furthermore , in obese adolescents , pth levels were closely correlated with crp levels and triglycerides : high - density lipoprotein ratio .
the pth level , but not the vitamin d level , is an independent predictor of metabolic syndrome .
however , a paradoxical increase in crp levels after parathyroidectomy was observed in some studies [ 16 , 18 ] .
therefore , the cause - and - effect relationship between pth and inflammation remains unclear .
interestingly , elevated levels of crp are associated with increased risk of several types of cancer . in this study
, we found a positive association between pth and crp , after adjustment for multiple potential confounders .
our results are in agreement with findings from epidemiological studies showing that pth correlates with cardiovascular and even cancer mortality .
rdw is a quantitative measure of variability in the size of circulating erythrocytes , with higher values reflecting greater heterogeneity in cell sizes ( i.e. , anisocytosis ) .
rdw is an easy , inexpensive , routinely reported parameter as a part of the complete blood count test .
recent studies exhibit a strong correlation between rdw and crp [ 3739 ] . of note
, data from nhanes iii showed that rdw was associated with cardiovascular disease , cancer , and chronic lower respiratory tract disease .
rdw was shown to be a powerful predictor of mortality in community - dwelling older adults . as a surrogate marker for the inflammatory state
accompanied with systemic inflammation , release of various immunological mediators ( including il-6 ) increases circulating platelet counts as a result of megakaryocyte proliferation .
platelets release the thromboxanes and other mediators , and consequently , patients with higher platelets may have increased inflammation .
smith and colleagues found that plr is a prognostic marker in patients with periampullary cancer .
subsequent study showed that plr is an independent predictor of long - term mortality after non - st segment elevation myocardial infarction . in dialysis patients ,
plr was positively correlated with il-6 levels , and plr was superior to neutrophil - to - lymphocyte ratio in terms of inflammation prediction .
however , the association was no longer statistically significant in the fully adjusted model ( i.e. , model 4 ) .
the nonsignificant results may be due to direct effects of pth on platelets and lymphocytes [ 4547 ] .
alternatively , there may be a nonlinear relationship between pth levels and blood cell derangements induced by the acute phase reaction .
hepatic albumin biosynthesis is downregulated by inflammatory stimuli as part of a negative acute phase reaction .
previous studies suggest that mgps is superior to the original gps and has greater consistency and is of more use .
the observation was based on the results that a low albumin concentration alone was uncommon and was not significantly associated with cancer - specific survival in many cancers . in this study , increased mgps was analyzed as a dichotomous variable ( > 0 versus = 0 ) and taken as another marker of inflammation .
although mgps is usually used to predict outcome in cancer patients , our results indicate that serum pth levels were strongly associated with increased mgps .
overall , our consistent findings buttress the validity of the association found between pth levels and inflammation .
a major strength of our study is that it was based on nationwide , population - based sampling survey data .
first , our analyses point to an association between pth levels and inflammation but do not indicate a direction for the causal relationship because of the observational and cross - sectional study design .
for example , in the nhanes database , there were no data on the fgf-23/klotho axis .
higher fgf-23 levels were reported to be independently associated with higher levels of inflammatory markers in patients with chronic kidney disease . moreover , both systemic and local inflammation may decrease kidney klotho expression , and klotho downregulates inflammation .
our finding may be a reflection of the association between low klotho levels and inflammation .
third , no repeat measurements of pth or other inflammatory markers were available , and therefore , the data set was limited to measures collected at a single time point for each participant .
finally , caution should be taken in interpreting results in relation to populations other than us adults .
in conclusion , using the nhanes data from the us adult population , we found an association between higher serum concentrations of pth and several inflammatory markers ( crp , rdw , plr , and mgps ) .
studies have shown that vitamin d supplementation has a suppressive effect on pth levels , but it is still unclear whether the suppression has any impact on clinical outcomes . given that inflammation is involved in the pathophysiology of cardiovascular disease , cancer , aging , and other conditions , our results provide clues on which to base further investigations of the mechanistic aspects of the pth - inflammatory link . | background and aims .
high levels of parathyroid hormone ( pth ) appear to be associated with an increased mortality .
previous studies concerning the relationship of inflammatory markers with hyperparathyroidism have yielded inconsistent results .
this study investigated whether serum pth concentrations were independently associated with several inflammatory markers among the us adults .
materials and methods . using data from the national health and nutrition examination survey , we examined the relation between serum pth and c - reactive protein ( crp ) , red cell distribution width ( rdw ) , and platelet - to - lymphocyte ratio ( plr ) levels with weighted linear regression . additionally , we examined the relation with increased modified glasgow prognostic score ( mgps ) by using weighted logistic regression . results .
crp , rdw , and plr values increased with increasing serum pth concentration .
after extensively adjusting for covariates , crp and rdw increased linearly and across pth categories ( all p < 0.001 ) , while plr marginally increased ( p = 0.190 and p = 0.095 using pth as a categorical and continuous variable , resp . ) .
the odds ratio of increased mgps was 1.11 and 1.31 across pth categories and with increasing pth levels continuously .
conclusion .
these nationally representative data indicate that serum pth levels are independently associated with several inflammatory markers in the us population .
the casual relationship between pth levels and inflammation remains to be elucidated . |
many environmental pollutants have estrogenic activity in animals .
xenobiotic estrogens include many pesticides and industrial chemicals that biocumulate .
the impact of these common pollutants on the reproductive success of wildlife may be considerable , particularly in threatened or endangered species .
this research examined the use of plasma vitellogenin in males as a biomarker for estrogenic xenobiotics in reptiles and amphibians .
adult male turtles ( trachemys scripta ) and frogs ( xenopus laevis ) were given ip injections of estradiol-17 beta ( e2 ) , diethylstilbestrol ( des ) , or o , p'-ddt ( 1-chloro-2-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl)benzene ) daily for 7 days , and plasma was collected on day 14 . the estrogenic activity of each compound was determined by measuring the induction of plasma vitellogenin .
vitellogenin was identified by precipitation , electrophoresis , western blot , and enzyme - linked immunosorbant assay ( elisa ) . in both species ,
estradiol and des treatments induced the most vitellogenin , whereas ddt treatments induced smaller amounts of vitellogenin in a dose - dependent fashion .
these data indicate that induction of plasma vitellogenin in males may be a useful biomarker of xenobiotic estrogen activity in wild populations of reptiles and amphibians.imagesfigure 1.figure 3 . | |
its pathogenesis is related to human papilloma virus ( hpv ) types 6 and 11 .
the occurrence of these lesions is influenced by smoking , co - existent infections , dietary deficiencies and hormonal changes .
squamous papillomas are traditionally divided into two types : isolated - solitary and multiple - recurring .
the former is usually found in an adult 's oral cavity , while the latter is mostly found in a child 's laryngotracheobronchial complex .
these lesions commonly occur between the ages of 30 and 50 years , and sometimes can occur before the age of 10 years . oral squamous papilloma accounts for 8% of all oral tumors in children .
the common site predilection for the lesion is the tongue and soft palate , and may occur on any other surface of the oral cavity such as the uvula and vermilion of the lip .
hpv involvement in head and neck carcinogenesis was first proposed by syrjanen et al . in 1983 .
recurrence is uncommon , except for lesions in patients infected with human immunodeficiency virus ( hiv ) .
a 14-year - old male patient was reported with a painless growth on the palatal surface of the mouth since 8 months .
the patient noticed the growth around 8 months back , which started as a small growth , gradually increased in size over a period of 6 months and attained the present size of 3 3 cm .
there was no history of pain , parasthesia or numbness associated with the growth and no similar lesions were present elsewhere .
intraoral examination revealed the presence of a solitary , well - defined , oval - shaped exophytic growth on the right half of the hard palate measuring 3 3 cm in size between the first and second molar regions on the palatal side [ figure 1 ] .
it was non - tender , firm in consistency and arose from the underlying soft tissue .
the lesion was surgically excised without any post - operative complications [ figure 2 ] .
the excised lesion [ figure 3 ] was sent for histopathological evaluation , which confirmed the squamous papilloma [ figure 4 ] .
squamous papilloma on the right side of the maxilla at the palatal surface excised squamous papilloma lesion post - excision area of the palate histopathological picture showing presence of finger - like projections and confirm squamous papilloma
oral squamous papilloma is a generic term used for papillary and verrucous growths composed of benign epithelium and minor amounts of connective tissue .
squamous cell carcinomas are the most common malignancies in adults , but are exceptionally rare in pediatric patients , particularly those involving the oral mucosa .
papillomas generally measure 1 cm in range and appear as pink to white exophytic granular or cauliflower - like appearance .
ribeiro et al . reported a case of oral squamous cell carcinoma that is uncommon .
squamous papillomas are classified into two types : isolated - solitary and multiple - recurring .
isolated solitary is usually found in adults oral cavity while multiple recurring occurs commonly in children .
isolated lesions are exophytic and pedunculated growths , resembling cauliflower appearance as seen in our case .
histologically , these lesions present as many long , thin and finger - like projections extending above the mucosal surface . each finger - like projection is lined by stratified squamous epithelium and connective tissue centrally .
the upper epithelial layer shows pyknotic nuclei , often surrounded by edematous or optically clear zone , the so - called
the differential diagnosis of solitary oral squamous papilloma are verruciform xanthoma , papillary hyperplasia and condyloma acuminatum .
verruciform xanthoma has predilection for gingiva and alveolar ridge , while condyloma are larger than papilloma with broader base and appear pink to red .
blood investigations such as enzyme - linked immunosorbent assay and the polymerase chain reaction test can be performed to detect the presence of virus .
surgical removal of the lesion is the treatment of choice for oral squamous papilloms , either by surgical or electrocautery excision , cryosurgery , intralesional injections of interferon or laser ablation .
| oral squamous papillomas are benign proliferating lesions induced by human papilloma virus
. these lesions are painless and slowly growing masses . as an oral lesion ,
it raises concern because of its clinical appearance .
these lesions commonly occur between age 30 and 50 years , and sometimes can occur before the age of 10 years . oral squamous papilloma accounts for 8% of all oral tumors in children
.
common site predilection for the lesion is the tongue and soft palate , and may occur on any other surface of the oral cavity such as the uvula and vermilion of the lip . here
, we are presenting a case of squamous papilloma on the palate . |
the shoulder and chest muscles regulate normal motion in the shoulder joint by stabilizing
the scapula in the chest while the upper arms are raised1 . the serratus anterior muscle ( sa ) , the upper trapezius muscle ( ut ) ,
the lower trapezius muscle ( lt ) , the rhomboid muscle , and the levator scapula muscle form
force couples that effect this stabilization2 .
the sa , which is considered of primary importance in stabilizing
the scapula , acts as a lever when the vertical axis through the sternoclavicular joint
protracts and also causes abduction and upward rotation of the scapula , thereby keeping the
scapula flat against the ribs .
weakening of the serratus anterior muscle leads to
excessive activation of the upper trapezius muscle and reduced activation of the serratus
anterior muscle and lower trapezius muscle , reducing the dynamic stability of the scapula .
when the arms are abducted , incomplete dynamic stability moves the humerus through
contraction of the deltoid muscle and the supraspinatus muscle , triggering a clash between
the subacromion and the head of the humerus .
a weakened sa can be found in workers who have
impingement syndrome in the shoulder joint , baseball players who have instability in the
shoulder joint , and swimmers who experience pain in the shoulder joint .
reduction in the
activity of this muscle is closely related to abnormal motion of the shoulder joint3 , 4 . for those experiencing problems with movement of the scapula due to imbalance of muscle
force couples , isometric and upper extremity exercise therapy programs aimed at restoring
the normal position of
, upper extremity
exercise programs using closed kinetic chain exercises , such as push - up exercises , push - up
plus exercises ( pupes ) , and isometric ball exercises , are quite often applied .
when applying
therapy to stabilize the shoulder joint , it is more effective for muscle strengthening to be
performed on an unstable platform , which introduces dynamic movement , rather than performing
static and continuous movement on a stable surface .
the push - up exercise is a common
exercise performed in a prone position by raising and lowering the body using the arms . when
performing push - up exercises , it is more effective to use an unstable surface than a stable
surface in order to increase muscle activity and simulate the proprioception of the shoulder
joint .
the sling is a device that hangs from the ceiling and facilitates the performance of
stability exercises for weak muscles by stimulating proprioception due to the instability of
the platform on which the exercises are performed .
this system results in a dynamic exercise
method for strengthening various muscles and thereby increases the stability of the joints
and the proximal portion of the torso . in pupes ,
moseley et
al.5 reported that the pupe was one of
the most effective closed kinetic chain training methods for selectively strengthening the
sa and for stabilizing the shoulder joint .
most previous studies on pupes have been
conducted using both stable and unstable platforms .
few studies have measured the
electromyographic activity of shoulder stabilizer muscles according to changes in hand
position while using both surface conditions . therefore , the goal of this study was to investigate the electromyographic activity of the
shoulder stabilizer muscles according to changes in hand position while performing the pupe ,
a closed kinetic chain exercise , using a sling device as an unstable platform and a push - up
bar as a stable platform .
this study included 20 healthy young male students in their 20s attending y university in
chungbuk province , korea .
subjects who did not
have musculoskeletal or neurological diseases in the upper or lower extremities , did not
have a lesion in the spine or the upper extremities , and did not have a history of surgery
were selected and randomly divided into groups .
the subjects were divided into an unstable
surface group ( usg ) ( n=10 ) and a stable surface group ( ssg ) ( n=10 ) . the average age of the
subjects in the usg was 23.7 1.21 years , the average height was 175.16 4.42 cm , and the
average weight was 73.01 8.67 kg .
the average age of subjects in the ssg was 23.3
1.45 years , the average height was 174.27 3.29 cm , and the average weight was 74.41
7.49 kg .
the subjects were informed about the purpose and method of this study prior to the
experiments , and voluntarily provided consent to participate . to provide an unstable surface while performing pupe , a sling device ( terapimaster plus ,
nordisk terapi as , norway ) was used , and a push - up bar ( fe 0168
, korea sports , seoul , korea )
was used to provide a stable surface . both the sling device and the push - up bar were set at
a height of 10 cm from the ground . the subjects who performed pupes using the sling device assumed a quadruped starting
posture , keeping both the hands and knees shoulder - width apart , so that the body weight was
supported by the hands and knees .
when the hands gripped the sling device , they were at a
height of 10 cm and perpendicular to the ground .
while flexing the shoulder joint , the
femoral / knee joint flexion was at 90 degrees in the quadruped posture , and the head , spine ,
and pelvis were positioned in a straight line , in a neutral state , with the eyes gazing at a
point on the ground .
the elbow joint was fully extended , and the ankle joint was maintained
in a plantar flexion posture . in order to keep the cervical vertebrae in a neutral position ,
the cervical and thoracic vertebrae were set in a straight line .
hand positions were alternated to the neutral position
( np ) , in which the upper extremities remained in the neutral position ; the internal rotation
position ( irp ) , in which the upper extremities were internally rotated to 90 degrees ; and
the external rotation position ( erp ) , in which the upper extremities were externally rotated
to 90 degrees . if the standardized starting posture , movement , and finish posture were not
maintained , data were not recorded .
the experimenter instructed the subjects to assume the
correct posture and measured each posture twice .
then , the
subjects were allowed to exercise for 10 minutes to sufficiently familiarize themselves with
conversion from the quadruped posture to the pupe posture . at this time
, the subjects were
told that the thoracic vertebrae should not be lifted excessively as a compensatory
mechanism .
the subjects using the push - up bar
performed pupes under the same conditions as the subjects using the sling device .
an mp150 ( biopac systems , santa barbara , ca , usa ) with a 2-cm diameter ag - ag / cl electrode
was used to measure changes in the electromyographic activity of the muscles related to
stabilization of the scapula . for manual muscle testing , maximum voluntary isometric
contractions ( mvic ) were measured to quantify the action potential of all the muscles .
since
the subjects were all right - hand dominant , surface electrodes were attached to the upper
extremities on the right side at the region of the back halfway between the spinous process
of c7 and the acromion for measurement of the ut ; the region near the inner 1/4 position
between the thoracic vertebrae and the inferior angle of the scapula for measurement of the
lt ; the region in front of the latissimus dorsi muscle at the no . 5 and no .
6 ribs for
measurement of the sa ; and at a region 2 cm from the inner side of the anterior axillary
border .
the emg signals were processed as full - waves once they were collected at a sampling signal
acquisition rate of 1,000 hz . the data were summarized using the acqknowledge version 3.81
( biopac systems , santa barbara , ca , usa ) software , performing band - pass filtering at
30500 hz and processing the signal by filtering at 60 hz to remove noise . the raw data
measured for standardization were converted to root mean squares ( rms ) .
the measurement
values from the first and last seconds were excluded ; thus , only the values measured during
the three middle seconds were included in the analysis .
each rms value was divided by the
maximum value of isometric contraction ( mvic ) , and then the results were obtained by
performing normalization of a range between 0% and 100% to the % mvic values . for statistical processing ,
a repeated measures one - way anova was performed in order to
determine the electromyographic activity of the muscles within the group according to the
hand position changes .
an independent samples t - test was also performed to establish the
significance of the electromyographic activity of the muscles between the groups according
to the changes in hand position .
usg , unstable surface group ; ssg , stable surface group ; ut , upper trapezius ; lt , lower
trapezius ; sa , serratus anterior ; pm , pectoralis major ; np , neutral position ; irp , 90
internal rotation position ; erp , 90 external rotation position .
meansd ,
* repeated one - way anova . independent sample t - test .
p<0.05 the results showed significant differences in electromyographic activity when comparing
changes in hand position within each group ( usg and ssg ) ( p<0.05 ) .
there were significant
differences in the electromyographic activity of the sa in the erp among the changes in hand
position in the between - group comparison ( p<0.05 ) ( table 1 ) .
in this study , we examined the electromyographic activity of the ut , lt , sa , and pm
according to np , irp , and erp hand positions , when performing the pupe , a closed kinetic
chain exercise , using a sling device to provide an unstable surface and a push - up bar to
provide a stable surface .
oh et al.6 reported that , as it is a
dynamic exercise method , a sling exercise on an unstable surface , as opposed to one on a
stable surface , strengthened various muscles by improving the stability of proximal joint
parts .
accordingly , the present study investigated the pupe with changing hand positions
under two different conditions : on an unstable surface using a sling device , and on a stable
surface using a push - up bar . in a study of 30 healthy adults ,
ludewig et al.7 stated that electromyographic activity of the sa improved and that
of the ut was reduced while subjects were performing push - up exercises , claiming this type
of exercise is best for strengthening the sa .
choi8 reported that in patients experiencing chronic shoulder pain for
more than three months , push - up exercises for seven weeks showed no difference in
electromyographic activity of the anterior deltoid muscle and lt but that the
electromyographic activity of the ut was significantly reduced and that of the sa was
significantly increased . in this study ,
the electromyographic activity of the sa was higher
when exercises were performed on an unstable surface than on a stable surface using the erp
hand position , whereas no differences were found in the ut and lt .
this means that the
changes in hand position did not have a significant influence on the electromyographic
activity on the ut and lt .
de oliveira et al.9 reported that the sa
showed no difference between push - up exercises for upper extremities performed on a stable
surface and those performed on an unstable surface using a large rubber ball . according to
park and you10 , pupes were more effective
than push - up exercises in activating the sa .
in particular , it was more effective to
exercise on an unstable surface than on a stable one . however , lehman et al.11 reported that changes in height during
pupes resulted in significant differences in the electromyographic activity of the sa ,
irrespective of the stability of the surface .
yoon et al.12 showed that the
electromyographic activity of the sa was highest in the erp and lowest in the np on a stable
surface .
lee et al.13 showed that
exercises performed in the np showed higher electromyographic activity than those performed
in the irp when comparing only those two hand positions .
the present study results showed
higher electromyographic activity of the sa on an unstable surface than on a stable surface
when using the erp .
this is because the biceps brachii and triceps could support upward and
downward movements of the shoulder joint on the unstable surface better than on the stable
surface and the ut , lt , and pm could support the anterior and posterior movements , thereby
inhibiting shoulder movement and further maintaining stability . among the hand positions ,
the erp influenced stability by more than the irp and np , increasing the electromyographic
activity of the sa .
first , because the study included only a small
number of normal adult male subjects , the results can not be generalized .
second , only the
right ( dominant side ) upper extremity muscles were measured , so the changes on the other
side are unknown .
third , the sling device , used as the unstable surface , was hung from the
ceiling with a rope ; it was hard to continue holding the sling with the hands under static
conditions .
fourth , the sling handle was made of thin cloth , which slipped slightly and was
hard to control .
future studies are needed to investigate the effects of changes in the
positions of the hands and feet on the shoulder stabilizer muscles during the pupe on both
stable and unstable surfaces . | [ purpose ] the purpose of this study was to determine the effect of hand position changes
on electromyographic activity of shoulder stabilizers during push - up plus exercise ( pupe )
performed on both stable and unstable surfaces .
[ subjects ] this study was performed on a
cohort of 20 normal adults divided into an unstable surface group ( usg ) ( n=10 ) and a
stable surface group ( ssg ) ( n=10 ) . [ methods ] a sling device was used to provide an
unstable surface , and a push - up bar was used to provide a stable surface .
pupes were
performed with hands in various positions : the neutral position ( np ) , the internal
rotation position ( irp ) , or the external rotation position ( erp ) .
electromyography was
used to determine and analyze the electromyographic activity of the upper trapezius muscle
( ut ) , the lower trapezius muscle ( lt ) , the serratus anterior muscle ( sa ) , and the
pectoralis major muscle ( pm ) .
[ results ] comparison of the results within the usg and ssg
showed significant differences depending on the hand position used during the exercise .
comparison between the usg and ssg showed that the erp hand posture resulted in
significant differences in electromyographic activity of the sa in the usg . [ conclusion ]
the electromyographic activity of the sa indicated that performing pupes using the erp on
an unstable surface provided more effective intervention for shoulder stabilization than
erp on a stable surface . |
in the past two decades , dental implants have become increasingly popular as a procedure to restore missing teeth .
a number of patient and procedure related parameters determine the success of the implant treatment .
diabetes mellitus ( dm ) is the most common systemic disease which is generally considered as a relative and not an absolute contraindication for implant therapy.1 among men and women over 55 years of age , where the rates of edentulism are higher , about 18.4 percent of individuals have some form of diabetes affecting the whole body.2 since tooth loss is greater in diabetic than in non - diabetic individuals , the validity of dental implant treatment options for these patients has to be realized.3 the metabolism of phosphorus and calcium is essential for bone mineralization & remodeling and is affected by hyperglycemia , which alters the response of the parathyroid hormone .
diabetes mellitus also inhibits osteoblastic differentiation.4 the success of replacing lost teeth by dental implants in diabetic patients was evaluated in a study in 1999 by balshi and wolfinger.2 the study found a success rate of 94.3% with 227 brnemark implants for a follow up period of two years , and concluded that the success rates of dental implants are higher in controlled diabetics.2 in 2003 peled et al .
evaluated the clinical outcome of dental implants in a group of controlled diabetics over a five year period and they reported an overall success rate of 97.3%.5 further a study done in 2003 by al jabbari et al .
, showed that diabetic geriatric patients with acceptable glucose control demonstrated a 92.7% success rate , 1 year after first stage surgery.6 the above literature indicates that diabetic patients are eligible candidates for implant therapy provided their plasma glucose level is under metabolic control .
the aim of the study was to evaluate the efficacy of implant as tooth replacement in diabetic patients with acceptable metabolic control with early non - functional loading protocol evaluated at periodic time intervals for three months .
survival of the implants is evaluated by radiographic evaluation to measure the crestal bone heights , clinical assessment by bleeding on probing and microbiological colonization around the implants .
the study group consisted of five patients ( three females and two males ) of age ranging from 35 - 65 years attending the outpatient clinic of department of prosthodontics , faculty of dental sciences , sri ramachandra university , chennai , india.the patients were diagnosed as diabetic and were on a modified diet or receiving oral medication , insulin or combination therapies and with good metabolic control as evaluated by laboratory investigation .
efforts were made to meet the plasma glucose levels recommended by the american diabetes association ( fasting plasma glucose - of 140 ml / dl and 2-hour post prandial glucose of 200 ml / dl).5 all cases were indicated for single tooth maxillary central incisor replacement ( fig .
1 ) with adjacent teeth intact or restored with functionally and aesthetically acceptable restorations.the exclusion criteria were patients with a history of smoking habit , alcohol or drug abuse , health conditions not permitting surgical procedures , history of bone grafting at the implant site , patients who had previously failing implants in the selected implant locations and patients who had evidence of oral pathology such as tumour , chronic bone disease or previous irradiation .
the study group consisted of five patients ( three females and two males ) of age ranging from 35 - 65 years attending the outpatient clinic of department of prosthodontics , faculty of dental sciences , sri ramachandra university , chennai , india .
the patients were diagnosed as diabetic and were on a modified diet or receiving oral medication , insulin or combination therapies and with good metabolic control as evaluated by laboratory investigation .
efforts were made to meet the plasma glucose levels recommended by the american diabetes association ( fasting plasma glucose - of 140 ml / dl and 2-hour post prandial glucose of 200 ml / dl).5 all cases were indicated for single tooth maxillary central incisor replacement ( fig .
the exclusion criteria were patients with a history of smoking habit , alcohol or drug abuse , health conditions not permitting surgical procedures , history of bone grafting at the implant site , patients who had previously failing implants in the selected implant locations and patients who had evidence of oral pathology such as tumour , chronic bone disease or previous irradiation .
the patients signed an informed consent and were explained about all the procedures that would be done during the study .
the approval for this clinical study was obtained from the institutional review board of sri ramachandra university . under local , anesthesia ,
a crestal incision was made and a full thickness muco - periosteal flap was raised .
single stage screw tapered , self threaded implants ( uniti implants , equinox medical technologies , zeist , holland ) with a diameter of 3.7 mm and length 13 mm were placed with an insertion torque of 45 ncm ( fig .
patients were recalled after an interval of 4 days from the day of surgery , for evaluation of the implant and for cementation of the provisional restoration .
the provisional restoration was fabricated using heat cure acrylic resin and cemented with non - eugenol zinc oxide cement ( 3 m espe ) .
three parameters were selected for each patient to evaluate the survival of implants ; radiographic evaluation , microbiological assessment and clinical assessment .
the examinations were done at periodic intervals on the 4 , 20 , 40 , 60 and 90 day after the surgical procedure .
radiographic examination was carried out with radiovisuography ( rvg ) taken with rinn x ray holders ( rinn corp . com . ,
the distance between the 2 references points was measured digitally , using sopro digital imaging software .
microbiological assessment was done to identify micro - organisms located subgingivally along the implant site .
the micro flora was examined for their predominance of aerobic organisms and represented by means of colony forming units ( cfu ) .
all patients were instructed to brush twice daily and rinse the mouth after every meal . on the fourth day , the provisional restoration was removed and the abutment was isolated with sterile gauze at the palatal and buccal sites .
specimens were collected using sterile swab which were inserted into the depth of peri - implant site and kept for 15 - 20 seconds ( fig .
colonies were identified by gram stain , growth was reported for , cocci , bacilli , gram positive ( staphlylocoocus aureus , streptococcus species ) and gram negative ( pseudomonas species ) pathogens .
the number of colonies of each sub cultured isolate was related to the total colony count ( cfu ) to obtain the relative proportion .
this sampling procedure was also repeated at regular intervals of 4 , 20 , 40 , 60 day and at the end of three months .
since the restoration was cemented using a temporary cement , it could be removed easily without causing damage to the soft tissues around the implant .
bleeding on probing ( bop ) is a widely used clinical indicator to detect the presence of inflammation . by clinical observation of bleeding on probing using calibrated manual periodontal probe ,
three parameters were selected for each patient to evaluate the survival of implants ; radiographic evaluation , microbiological assessment and clinical assessment .
the examinations were done at periodic intervals on the 4 , 20 , 40 , 60 and 90 day after the surgical procedure .
radiographic examination was carried out with radiovisuography ( rvg ) taken with rinn x ray holders ( rinn corp
the distance between the 2 references points was measured digitally , using sopro digital imaging software .
microbiological assessment was done to identify micro - organisms located subgingivally along the implant site .
the micro flora was examined for their predominance of aerobic organisms and represented by means of colony forming units ( cfu ) .
all patients were instructed to brush twice daily and rinse the mouth after every meal . on the fourth day , the provisional restoration was removed and the abutment was isolated with sterile gauze at the palatal and buccal sites .
specimens were collected using sterile swab which were inserted into the depth of peri - implant site and kept for 15 - 20 seconds ( fig .
colonies were identified by gram stain , growth was reported for , cocci , bacilli , gram positive ( staphlylocoocus aureus , streptococcus species ) and gram negative ( pseudomonas species ) pathogens .
the number of colonies of each sub cultured isolate was related to the total colony count ( cfu ) to obtain the relative proportion .
this sampling procedure was also repeated at regular intervals of 4 , 20 , 40 , 60 day and at the end of three months .
since the restoration was cemented using a temporary cement , it could be removed easily without causing damage to the soft tissues around the implant .
bleeding on probing ( bop ) is a widely used clinical indicator to detect the presence of inflammation . by clinical observation of bleeding on probing using calibrated manual periodontal probe ,
the microbial colonization , bleeding on probing and crestal bone loss data was transferred onto a spreadsheet programme .
the data were analyzed using statistical packages ( spss for windows ) for statistical analysis .
initially the mean , the standard deviations and ranges were calculated for the quantitative data .
paired t - test was done to calculate the difference in the microbial colonization , bleeding on probing and crestal bone loss data .
the differences in the crestal bone measurements were investigated using paired t - tests ( table 2 ) .
p value ( probability value ) between the 4 day and the end of three months was less than 0.05 to indicate statistical significance .
the mombelli et al.7 scores indicate that there was a definite amount of inflammation present in all patients after implant placement , especially till the 20 day . by the end of three months
there was significant reduction in bleeding on probing , indicating healthy tissues surrounding the implant .
results of microbiological examination indicate that colonization was significantly reduced by the end of three months and was similar to that of contra lateral tooth .
differences between quantitative variables were tested with wilcoxon ranked sign test ( dependent data , i.e. within - group comparisons ) , ( table 4 ) .
p value ( probability value ) between the 4 day and the end of three months was less than 0.05 to indicate statistical significance .
the microbial colonization , bleeding on probing and crestal bone loss data was transferred onto a spreadsheet programme .
the data were analyzed using statistical packages ( spss for windows ) for statistical analysis .
initially the mean , the standard deviations and ranges were calculated for the quantitative data .
paired t - test was done to calculate the difference in the microbial colonization , bleeding on probing and crestal bone loss data .
the differences in the crestal bone measurements were investigated using paired t - tests ( table 2 ) .
p value ( probability value ) between the 4 day and the end of three months was less than 0.05 to indicate statistical significance .
the mombelli et al.7 scores indicate that there was a definite amount of inflammation present in all patients after implant placement , especially till the 20 day . by the end of three months
there was significant reduction in bleeding on probing , indicating healthy tissues surrounding the implant .
results of microbiological examination indicate that colonization was significantly reduced by the end of three months and was similar to that of contra lateral tooth .
differences between quantitative variables were tested with wilcoxon ranked sign test ( dependent data , i.e. within - group comparisons ) , ( table 4 ) .
p value ( probability value ) between the 4 day and the end of three months was less than 0.05 to indicate statistical significance .
it is estimated that 15% to 25% of the elderly population suffer from either insulin - dependent or noninsulin - dependent diabetes mellitus.8 the influence of diabetes on bone and bone forming cells imposed many speculations about the use of dental implants in diabetics and osteoblast function was shown to improve by glycaemic control.9 the present study evaluated the efficacy of implant as tooth replacement in diabetic patients with acceptable metabolic control with early non - functional loading protocol by radiographic evaluation , clinical assessment and microbiological colonization around the implants . loading protocol in diabetic patients is an important factor in the success of dental implant treatment .
balshi et al . in 2007 , identified that an immediate loading protocol in a diabetic patient can lead to successful osseointergration despite the effects that the disease has on the bone remodelling process.10 in a two year follow up study done by azza ezz ell - arab in 2000 assessed the durability of single tooth replacement implant in controlled diabetics versus non - diabetics.11 radiographic evidence of bone intergration was detected in both healthy and diabetic individuals .
it is essential to detect early signs of diabetes in the clinical peri - implant evaluation and plan the therapeutic intervention accordingly .
absence of bleeding on probing ( bop ) is widely considered as an useful clinical indicator for the absence of inflammation.12 in the present study , there was no association found between presence of microorganisms and amount of bone loss . between 4 day ( base line ) and at the end of 90 day , statistically different counts were found at the implant surfaces . by the end of 90 day
thus , significantly lower bacterial counts of putative pathogens were found at the end of three months .
the findings of the present study were similar to that reported by salvi et al .
in 2007.12 in the present study , all implants were well intergrated in bone for the duration of the study as evaluated by clinical and radiographic criteria . however , future studies are needed to establish definitive guidelines with objective criteria such as duration , type of diabetes and glycosylated hemoglobin levels .
better study designs , preferably longitudinal randomized clinical trials , are needed to evaluate various determining factors and prove such a statement .
the results of this retrospective study show that a growing number of diabetic patients can enjoy the benefits of dental implants provided their diabetes is under control
. a high success rate of dental implants is achievable in diabetic patients whose disease is under control . | purposethe study was conducted to evaluate the efficacy of implant supported tooth replacement in diabetic patients.materials and methodsthe study involved placement of implants ( uniti implants , equinox medical technologies , zeist , holland , diameter of 3.7 mm and length 13 mm ) in five diabetic patients ( three females and two males ) of age ranging from 35 - 65 years with acceptable metabolic control of plasma glucose .
all patients included in the study were indicated for single tooth maxillary central incisor replacement , with the adjacent teeth intact .
the survival of the restored implants was assessed for a period of three months by measurement of crestal bone heights , bleeding on probing and micro flora predominance .
paired t - test was done to find out the difference in the microbial colonization , bleeding on probing and crestal bone loss .
p values of less than 0.05 were taken to indicate statistical significance.resultsresults indicated that there was a significant reduction in bleeding on probing and colonization at the end of three months and the bone loss was not statistically significant.conclusionthe study explores the hypothesis that patients with diabetes are appropriate candidates for implants and justifies the continued evaluation of the impact of diabetes on implant success and complications . |
twenty - eight rhesus monkeys , imported in 2006 from guangxi grandforest scientific primate company , dayiling ping nan county , china were used in this study .
this colony consisted of 23 females weighting 3.70.67 kg , aged 4y 06 m and 5 males weighting 4.00.69 kg , aged 4y 14 m ( table 1 ) . in china farm , these monkeys have been raised in the outdoor housing facility with shelter and were provided vegetables ( e.g. , sweet potato ) , biscuits , and bread which were made in their facility . after being imported from china , a quarantine process of 1 month was concluded with the subjects in good general condition .
the monkeys were maintained in single - housed cages and had daily access to food ( ps diet , oriental yeast co. ltd , japan , fresh fruit , and vegetables ) and unlimited access to water .
their room was maintained at 244 and a relative humidity of 5010% , with an artificial light - dark cycle of 12:12 ( 7:00 am onset ) and with 13 - 18 air changes per hour .
all animals used in this study were cared for in strict accordance with the national institutes of health " guide for the care and use of laboratory animals . "
the monkeys were captured and chemically restrained with a 5 mg / kg dose of ketamine hcl prior to hair specimen collection .
hair is a good biomonitoring tool for heavy metal assessment , because it integrates the accumulation and concentration of heavy metals over time .
so , hair specimens were taken , with the investigator using latex gloves and surgical scissors , by clipping hair as close to the skin as possible from the flank and femoral regions of each animal .
all hair specimens ( 1 g ) were stored in sealed plastic bags until they were processed for analysis .
a microwave digestion system ( mls 1200 mega , milestone co. , italy ) and an inductively coupled plasma mass spectrometer ( elan 6000 , perkin - elmer co. , usa ) were used in this study .
hair samples of 0.5 g were put in a vessel and mixed with 65% hno3 8 ml and 30% h2o2 2 ml and digested by pressurization and heating for 20 minutes in the microwave digestion system . after cooling to room temperature , the samples were analyzed by icpms .
twenty - eight rhesus monkeys , imported in 2006 from guangxi grandforest scientific primate company , dayiling ping nan county , china were used in this study .
this colony consisted of 23 females weighting 3.70.67 kg , aged 4y 06 m and 5 males weighting 4.00.69 kg , aged 4y 14 m ( table 1 ) . in china farm , these monkeys have been raised in the outdoor housing facility with shelter and were provided vegetables ( e.g. , sweet potato ) , biscuits , and bread which were made in their facility . after being imported from china , a quarantine process of 1 month was concluded with the subjects in good general condition .
the monkeys were maintained in single - housed cages and had daily access to food ( ps diet , oriental yeast co. ltd , japan , fresh fruit , and vegetables ) and unlimited access to water .
their room was maintained at 244 and a relative humidity of 5010% , with an artificial light - dark cycle of 12:12 ( 7:00 am onset ) and with 13 - 18 air changes per hour .
all animals used in this study were cared for in strict accordance with the national institutes of health " guide for the care and use of laboratory animals . "
the monkeys were captured and chemically restrained with a 5 mg / kg dose of ketamine hcl prior to hair specimen collection .
hair is a good biomonitoring tool for heavy metal assessment , because it integrates the accumulation and concentration of heavy metals over time .
so , hair specimens were taken , with the investigator using latex gloves and surgical scissors , by clipping hair as close to the skin as possible from the flank and femoral regions of each animal .
all hair specimens ( 1 g ) were stored in sealed plastic bags until they were processed for analysis .
a microwave digestion system ( mls 1200 mega , milestone co. , italy ) and an inductively coupled plasma mass spectrometer ( elan 6000 , perkin - elmer co. , usa ) were used in this study .
hair samples of 0.5 g were put in a vessel and mixed with 65% hno3 8 ml and 30% h2o2 2 ml and digested by pressurization and heating for 20 minutes in the microwave digestion system . after cooling to room temperature , the samples were analyzed by icpms .
differences between sexes were calculated using the student t - test . any difference with p<0.05
the mean values and standard errors for heavy metal concentrations of arsenic ( as ) , beryllium ( be ) , cadmium ( cd ) , chromium ( cr ) , iron ( fe ) , lead ( pb ) , mercury ( hg ) , and selenium ( se ) in the hair of monkeys are presented in table 2 .
the mean values obtained in ppm were as follows : as ( 0.654 ) , be ( 0.005 ) , cd ( 0.034 ) , cr ( 11.349 ) , fe ( 87.106 ) , pb ( 0.656 ) , hg ( 0.916 ) , and se ( 3.200 ) .
there were statistically significant differences in be , cr , and as concentrations between males and females .
be concentrations were significantly higher in females ( 0.0060.003 ppm ) compared to males ( 0.0030.001 ppm ) ( p<0.01 ) .
cr concentrations in hair were also significantly higher in females ( 11.8884.460 ppm ) compared to males ( 8.7571.729 ppm ) ( p<0.05 ) . as concentrations in hair were also significantly higher in females ( 0.6900.353 ppm ) compared to males ( 0.4880.113 ppm ) ( p<0.05 ) .
our monkeys were imported from a breeding center that includes outdoor housing facilities in southwest china .
thus , it was suspected that they have sufficient opportunities for natural exposure to air , soil , and water pollution . in this study , we measured heavy metal concentrations in the hair of newly imported rhesus monkeys from southwest china .
mercury has gained worldwide attention due to its high toxicity , bio - accumulation , and difficulty to control , because it is readily released into the atmosphere from both natural and anthropogenic sources [ 14 - 18 ] .
exposure to methylmercury , a toxic form usually absorbed by ingestion , is absorbed more readily and excreted more slowly than other forms of mercury .
especially , china has been regarded as one of the largest anthropogenic sources of heavy metals such as mercury . in recent years
, more attention has been given to hg emissions in southwest china , which is known as the main habitat of most macaques .
mercury concentration ( ranging from 0.123 to 2.188 ppm ) in the hair of our monkeys was very higher than that of free - ranging rhesus monkeys in nepal ( ranging from 0.043 to 0.594 ppm ) .
the high levels of hg detected in our study suggest that the rhesus monkeys were exposed to contaminated atmosphere around the farm or contaminated vegetables and water .
generally , hg has been shown to damage the immune and nervous systems , and cause birth defects but our monkeys have not shown typical clinical signs related with hg .
common sources of lead include dust containing paint chips or lead released into the atmosphere from industrial or automotive emissions such as leaded gasoline .
acute exposure to lead is known to damage the nervous , renal , circulatory , hepatic , and reproductive systems .
these consequences are well recognized and often clinically obvious in humans and animals with acute exposure . however , the effects of lower levels of pb exposure are more subtle , but may be significant .
according to recent reports from nepal and singapore , the lead concentration in the hair of cynomolgus and rhesus monkeys were 2.51 and 6.0 ppm , respectively .
these levels are remarkable higher than that of our chinese rhesus monkeys . in the other words
, it may represent that the chinese rhesus monkeys was less exposed to the lead . in our results , the heavy metal concentrations detected in the hair of chinese rhesus monkeys were not enough levels which affect the monkeys clinically . although , we just report some heavy metal concentration in these monkeys , further studies about continuous monitoring , adverse effects , and comparison between blood and hair on heavy metal concentration are needed . | macaque monkeys are good sentinel to humans for environmental pollutions because their similarities in genetic and physiological characteristics .
so , their reference values about exposures to heavy metals are required for proper data interpretation . here ,
we report several heavy metals concentrations in the hair of rhesus monkeys which are widely used in biomedical research .
the hair of 28 imported rhesus monkeys from an animal farm in southwest china were examined for the presence of eight heavy metals ( arsenic , beryllium , cadmium , chromium , iron , lead , mercury , and selenium ) .
the analyzed data in parts per million ( ppm ) for hair concentrations of heavy metals in rhesus monkeys were as follow : as ( 0.6540.331 ) , be ( 0.0050.003 ) , cd ( 0.0340.022 ) , cr ( 11.3294.259 ) , fe ( 87.10630.114 ) , pb ( 0.6560.613 ) , hg ( 0.9160.619 ) , and se ( 3.2000.735 ) .
the concentrations of be , cr , and as showed significant higher in females than in males ( p<0.05 ) .
we present here the reference values of several heavy metals in healthy china - origin rhesus monkeys .
these data may provide valuable information for veterinarians and investigators using rhesus monkeys in experimental studies . |
colonoscopy is usually performed to identify or correct a problem in the colon and distal small bowel , and its clinical applications have increased enormously in recent years .
intestinal perforation and hemorrhage are well - known complications of colonoscopy , but the incidence of these problems is very low1 , 2 ) .
the first such case was reported in 1988 by houghton and aston3 ) , and there have been only a few reports since then .
fecaliths , lymphoid hyperplasia , foreign bodies and parasites can block the inner lumen , leading to increased intraluminal pressure , impaired blood flow and inflammation .
a 48-year - old woman underwent colonoscopy for colorectal disease screening at a health care center .
she also had a computed tomographic ( ct ) scan of the abdomen performed before the colonoscopic examination , which revealed no abnormalities except for a 76 cm multilobulated left renal cyst ( figure 1 ) . before the procedure
there was no sign of inflammation around the cecum or the appendicular orifice ( figure 2 ) .
she felt well after the colonoscopic examination , and she was discharged promptly after passing bowel gas .
however , that evening , she felt a right lower abdominal pain that gradually intensified .
four days later , she visited our outpatient department with complaints of right lower abdominal pain .
her vital signs were stable with a body temperature of 37. the abdomen was slightly distended with right lower quadrant tenderness and rebound tenderness .
the initial white blood cell count was 7,300 cells / mm with 5,100 neutrophils / mm ; the other blood profiles ( hemoglobin , hematocrit and the platelet count ) and tests for liver function , electrolytes and urinalysis were within normal limits .
acute appendicitis was suspected , and so ultrasonography was performed for making an accurate diagnosis .
the results showed a swollen appendix with pericecal fluid accumulation and inflammation of the terminal ileum and cecum ( figure 3 ) .
major complications such as bleeding , colonic perforation and postpolypectomy syndrome are rare . other minor complications such as abdominal pain , nausea , vomiting , bowel spasm and mucosal tears in the lining of the colon can occur frequently1 , 2 ) .
this procedure can on rare occasions lead to splenic rupture , pneumomediastinum , pneumothorax , incarcerated hernia , ileus and diverticulitis .
more often , hemodynamic alterations that occur during colonoscopy may be the cause of cardiovascular and cerebrovascular sequelae1 , 2 ) .
houghton and aston3 ) firstly described appendicitis as a rare complication of colonoscopy , and only 12 cases of acute appendicitis following colonoscopy have currently been reported in the english medical literature ( table 1)3 - 9 ) .
vender , et al.5 ) reported on three cases that occurred at two institutions where a total of about 8000 colonoscopic examinations were performed .
therefore , the incidence of appendicitis following colonoscopy was about 0.038% . this is lower than the incidence of bleeding ( 0.21% ) , colonic perforation ( 0.1% ) or abdominal pain ( 0.09%)2 ) after colonoscopy . because abdominal pain after colonoscopy is generally caused by retained gas or colonic spasm secondary to colonoscopy2 ) , it is not easy to suspect acute appendicitis as the cause of this pain .
it is also important to distinguish appendicitis from post - polypectomy syndrome , which is a combination of pain , peritoneal irritation , leukocytosis and fever after colonoscopy7 , 8) . among the 12 reported cases ,
there were eight men and four women with a mean age of 53.5 years ( range : 24 - 69 ) .
this high mean age may be because colonoscopy is generally performed on patients during their fifth and sixth decades .
most patients commonly present with right lower abdominal or suprapubic pain , with or without fever and chilling .
one unusual case revealed only abdominal pain and distension with ileus10 ) . in nine patients , these symptoms developed between several hours and two days after colonoscopic examination , but the symptoms occurred after two days in the other three patients .
four of the ten patients underwent surgery four days after the development of symptoms , and three showed a perforated appendix , which suggests that a delayed diagnosis may increase the likelihood of perforation .
acute appendicitis is thought to be mainly caused by luminal obstruction , like the so called barium appendicitis after a barium enema , and this can occur by obstruction of the appendiceal orifice via the retention of barium11 , 12 ) .
the suggested mechanisms of appendicitis after colonoscopy are 1 ) pre - existing subclinical disease of the appendix5 ) , 2 ) elevation of the intraluminal pressure with insufflated air can obstruct the luminal orifice by lengthening the appendix , 3 ) elevated intraluminal pressure can force bowel contents , and especially fecaliths , into the appendix6 ) , 4 ) direct intubation of the colonoscope into the appendiceal lumen can induce inflammation5 ) and 5 ) mucosal injury around the appendiceal orifice can cause local edema and obstruction of the appendiceal lumen10 ) .
seven of the reported cases suggested the importance of the procedure 's duration because therapeutic procedures such as polypectomy and hemostasis are lengthy .
the second and third mechanisms described above may be the causes in our case . in conclusion , appendicitis is a very rare but important complication of colonoscopy and this can be easily confused with other complications of the procedure .
prompt recognition of this complication can lead to early , effective treatment , and delayed diagnosis can lead to serious results .
therefore , clinicians should always keep the possibility of this complication in mind as the cause of abdominal pain after performing colonoscopic examination . | a 48-year - old woman who was without any abnormal past medical history underwent colonoscopy as a screening procedure for colorectal disease .
the procedure was uneventful and there was no sign of inflammation around the appendicular orifice or the luminal surface of the cecum .
the patient did not complain of pain or significant discomfort throughout the procedure .
she then developed pain in the right lower quadrant of the abdomen that evening and this persisted for four days .
she visited the outpatient department and underwent abdominal ultrasonography , which showed a swollen appendix with a collection of pericecal fluid .
surgical exploration and appendectomy were performed ; the final diagnosis was acute suppurative appendicitis .
colonoscopists should be aware of this rare complication and consider it when making the differential diagnosis of post - colonoscopy abdominal pain . |
hydatidosis or human cystic echinococcosis ( ce ) is a cosmopolitan zoonotic disease that can be found in people and livestock infected with the larval stage of the tapeworm parasite echinococcus granulosus .
dogs are the primary definitive hosts for this parasite , with livestock and humans acting as intermediate hosts .
the outcome of infection is cyst development in the liver , lungs , or other organ systems . depending on different factors such as cyst type , size , location , and presence / absence of complications , the options for treatment of ce are as follows : surgery , pair ( puncture , aspiration , injection of protoscolicidal agent , and reaspiration ) , antiparasitic drug treatment , or watch and wait .
the evidence supporting any of the four treatment modalities , from carefully designed clinical studies , is insufficient , and choosing treatment options for patients remains controversial . for inoperable cases , chemotherapy with the benzimidazole - methylcarbamate ( bz ) compounds as albendazole ( abz ) and mebendazole remains the only alternative .
approximately a third of patients treated with bz drugs have been cured and 3050% of ce - patients have demonstrated significant regression of cyst size . however , 2040% of cases do not respond favourably .
moreover , adverse reactions against bz under long - term chemotherapy include elevation of transaminases , alopecia , gastrointestinal disturbances , and leucopenia .
praziquantel ( pzq ) , a heterocyclic pyrazinoisoquinoline derivative , has been proposed to be used alongside bz in ce - patients .
nevertheless , further studies are required to determine whether there are significant benefits from combination therapy with abz and pzq over monotherapy with abz . with regard to these difficulties ,
the development of a new therapeutic drug for human treatment of cystic echinococcosis is necessary .
a problem that has emerged with the use of synthetic anthelmintics is the development of parasitic resistance , which can threaten the success of treatment in humans [ 712 ] .
consequently , the search of new therapeutic alternatives such as the use of traditional medicinal plants has been increased .
essential oils are volatile , natural , and complex compounds characterized by a strong odour and formed by aromatic plants as secondary metabolites .
these substances may be constituted for about 20 to 60 components at quite different concentrations .
therefore , the biological effects of essential oils may be the result of a synergism of all molecules or reflect only those of the main molecules present at the highest levels according to gas chromatographical analysis .
on the other hand , there are studies where the principal component of essential oil was more active on its own than when present in essential oil which suggests that antagonism may have occurred between components of the oil . because of its hydrophobic character , the essential oils or their components present a great potential for pharmacological applications as antimicrobial agents [ 14 , 1720 ] .
the role of essential oils against parasitic helminths has been studied by several authors [ 13 , 2123 ] . however , there are few publications about the effect of these substances on e. granulosus .
the in vitro effect of the essential oils of rosmarinus officinalis ( rosemary ) , mentha pulegium , m. piperita , pistacia khinjuk ( pistachio ) , and trachyspermum ammi ( ajowan ) was demonstrated against protoscoleces of e. granulosus [ 2427 ] .
moreover , the in vitro effect of thymol was shown against protoscoleces , microcysts , and cysts of e. granulosus [ 28 , 29 ] .
apoptosis is a form of cell death which is triggered by external factors and ultimately leads to the cell 's self - destruction .
the induction of apoptosis was demonstrated for several essential oils [ 3133 ] . on the other hand ,
proposed that apoptosis may be a cellular mechanism underlying hydatid cyst infertility , which is observed in some herbivores and humans .
moreover , drug - induced apoptosis in e. granulosus protoscoleces has been reported [ 30 , 35 ] .
thymus vulgaris ( thyme ) and origanum vulgare ( oregano ) are shrubs distributed in areas of the mediterranean and asia , both members of family lamiaceae . in vitro ,
thyme and oregano essential oils have significant antibacterial , antifungal , and antiparasitic activities [ 17 , 3739 ] .
the biological activity of these substances has been related to their phenolic compounds content such as thymol and carvacrol , which represent between 40 and 50% of oils .
the aim of the present work was to determine the in vitro effect of t. vulgaris and o. vulgare essential oils against e. granulosus protoscoleces and cysts .
oregano and thyme leaves , completely formed , were collected separately in sierra de la ventana ( 389 s , 6148 w ) , buenos aires province , argentina , between december and february .
specimens were classified and stored in the herbarium of the faculty of exact and natural sciences , universidad nacional de mar del plata .
fresh plant material was dried prior to distillation ( 2027c and ~50% rh ) ; and dried leave oils were obtained by hydrodistillation using a clevenger - type apparatus for 2 h. an average of 100 g of leaves was used in each experiment , and several distillations were performed until the volume required to run all trials was reached .
the oils were dried over anhydrous sodium sulphate and stored in screw - capped dark glass vials at 58c until further testing .
thin layer chromatography ( tlc ) of the essential oils was performed on silica gel plates ( 0.2 mm kieselgel 60 f254 , merck ) .
both oils were applied to two tlc plates using an aliquot of 5 l ( using drummond microcapillaries ) and developed ( 93 : 7 toluene / ethyl acetate ) .
separated compounds were sprayed with sulphuric acid in ethanol , and later on with vanillin in ethanol , followed by heating at 110c .
essential oil composition was elucidated by gas chromatography ( gc ) and gas chromatography - mass spectrometry ( gc - ms ) , using a shimadzu gc-17a chromatograph equipped with a flame ionization detector ( fid ) .
separations were performed using a db-1 fused silica column ( 60 m 0.248 mm , film thickness 0.25 mm ) .
oven temperature was programmed from 60 to 240c at 3c / min and the final temperature was held for 10 min .
injector and detector temperatures were set at 230c and 250c , respectively , and carrier gas
the gc / ms analysis was performed on a perkin- elmer , qmass 910 gc operating at 70 ev , equipped with a db-5 fused silica column ( 30 m 0.25 mm , film thickness 1.0 mm ) .
the injector and detector temperatures were 250c ; oven temperature was programmed from 60c ( 5 min ) , 60 to 220c at 3c / min , and 220c ( 8 min ) and carrier gas he at a flow of 1 ml / min .
the identification of eo 's components was based on the comparison of their mass spectra with those reported in literature [ 43 , 44 ] .
quantitative data were obtained by integration of fid area percents without the use of collection factors .
density to 20c , triplicate of 1 ml of essential oil was weighed and the average of the values obtained was calculated . the refractive index was determined at 20c 0.05c with an abbe refractometer , in compliance with aoac official method 921.08 .
oils were subjected to ultraviolet - visible ( uv - vis ) spectroscopy at a concentration of 200 ppm to oregano and 12,5 ppm to thyme , using a shimadzu uv-2101pc scanning spectrophotometer .
the infrared spectroscopy ( ir ) of the samples was recorded as a thin liquid film on nacl windows with ftir mattson , model genesis ii spectrophotometer .
the spectra were accumulated from 8 scans measured with a resolution of 2 cm in the range of 5004500 cm .
hydatid cysts from liver and lungs of naturally infected cattle were obtained from an abattoir located in the southeast of the buenos aires province , argentina .
protoscoleces were removed from cysts under aseptic conditions and washed several times with phosphate - buffered saline ( pbs , ph 7.2 ) .
viable and free protoscoleces ( 2000 per leightont tube ) were cultured in 10 ml of medium 199 ( gibco ) , containing 60 g / ml penicillin , 100 g / ml streptomycin , 50 g / ml gentamicin , and 4 mg / ml glucose .
essential oils of t. vulgaris and o. vulgare were dissolved in medium 199 using propylene glycol ( pg ) as an emulsifier and were added to the medium resulting in thymol final concentrations of 10 g / ml ( according to thymol concentration on these essentials oils determined by cg ) .
thymol ( sigma ) was dissolved in dimethyl sulphoxide ( dmso ) at a drug concentration of 10 mg / ml and was added to the medium resulting in final concentrations of 10 g / ml .
protoscoleces incubated with culture medium alone and with culture medium containing pg or dmso were used as controls .
samples of protoscoleces ( approximately 90100 protoscoleces in 180 l of incubation medium ) from each treatment and the controls were taken every 5 to 6 days for viability assessment .
animal procedures and management protocols were carried out in accordance with the 2011 revised form of the guide for the care and use of laboratory animals published by the us national institutes of health .
samples of protoscoleces that had been incubated for 60 days in the presence of thymol and essential oils of t. vulgaris and o. vulgare were rinsed in medium 199 .
after sedimentation , they were resuspended in medium 199 supplemented with 60 g / ml penicillin , 100 g / ml streptomycin , and 50 g / ml gentamicin and the concentration was adjusted to 3000 protoscoleces / ml . of this protoscoleces suspension resulting from the in vitro treatments ( t. vulgaris , o. vulgare , thymol , and pg control ) , 0.5 ml was inoculated into each of four female cf1 mice ( body weight 25 5 g ) by intraperitoneal injection . at 5 months after infection
gamma - glutamyl - transpeptidase ( ggt ) was proposed as an ideal viability marker during in vitro pharmacological studies against e. granulosus protoscoleces .
enzyme assay was carried out in sterile tissue culture plate ( nunc , 96 wells ) .
protoscoleces ( 1500 per well ) were cultured under aseptic conditions in 0.2 ml of medium 199 per well supplemented with antibiotics and glucose . in vitro incubations were performed at 37c without changes of medium .
enzyme activity was determined from culture protoscolex supernatants following the procedure described by cumino et al . .
the activity was measured at 37c in a recording shimadzu model uv - vis spectrophotometer , the volume of the reaction mixture was 1 ml with 50 l of enzyme samples , and measurements were made after 30 min of incubation .
gamma - glutamyl - transpeptidase ( ggt ) was determined following the szasz method , whereas the rate of increase in absorbance is due to release of p - nitroaniline .
the substrate solution is an aqueous buffered solution containing 2.9 mm l - gamma - glutamylp - nitroanilide , 100 mm glycylglycine , and 5 mm mgcl2 .
a commercially available kit ( apop tag plus in situ apoptosis detection kit s7101 ; chemicon international a serologicals company ; usa and canada ) was used to detect the 3-oh ends of the dna strands according to manufacturer 's instruction .
briefly , protoscoleces from different treated groups were fixed in karnovsky 's solution ( paraformaldehyde 2% ( w / v ) , glutaraldehyde 2.5% ( v / v ) , and 0.05 m cacodylate ph 7.2 ) at 4c for 48 h and then embedded in paraffin .
tissue sections ( 5 mm ) were deparaffinized and pretreated with proteinase - k solution ( 20 g / ml ) at room temperature for 15 min .
the endogenous peroxidase activity was quenched using 3% ( v / v ) hydrogen peroxide in phosphate - buffered saline ( pbs ) at room temperature .
sections were incubated in a mixture of terminal deoxynucleotidyl transferase and digoxigenin - labeled dideoxynucleotide in a humidified chamber at 37c for 1 hour .
after reacting with a stop buffer , the sections were incubated with an antidigoxigenin peroxidase conjugate for 30 minutes .
peroxidase activity was detected by exposing the sections to a solution containing 3.3-diaminobenzidine tetrahydrochloride ( dab ) .
negative controls were treated with distilled water in place of the terminal deoxynucleotidyl transferase enzyme .
nuclei of apoptotic cells were stained brown with tunel reagents ; normal nuclei had no brown staining but showeda blue color with hematoxylin .
female cf-1 mice ( body weight 25 g 5 ) were infected by intraperitoneal inoculation with 1,500 e. granulosus protoscoleces / animal , suspended in 0.5 ml of medium 199 ( gibco ) .
animals were housed in a temperature - controlled ( 22 1c ) , light - cycled ( 12-hour light / dark cycle ) room .
at 8 months after infection , mice with experimental secondary ce were euthanized and necropsy was carried out immediately thereafter . at necropsy ,
groups of 5 cysts were placed in leighton tubes containing 10 ml of medium 199 .
thymol and the essential oils of t. vulgaris and o. vulgare were added to the medium resulting in thymol final concentrations of 10 g / ml .
cultures were maintained at 37c without changes of medium during the entire drug incubation period .
samples of cysts from each of the dosing groups and the controls were taken and then fixed for electron microscopy .
the criteria for cysts viability assessment included the loss of turgidity , the collapse of cysts , and the ultrastructural observation of the germinal layer as described by elissondo et al . .
log - rank test was used to assess the survival differences of protoscoleces after exposure to thymol and essential oils of t. vulgaris and o. vulgare . for infectivity studies ,
differences in mean weight of cyst recovered from mice inoculated with treated and untreated protoscoleces were tested using the mann - whitney u nonparametric test ( wilcoxon rank sum test ) .
samples of protoscoleces and cysts cultured in vitro were processed for scanning and transmission electron microscopy ( sem and tem ) as described by elissondo et al .
the chemical composition of oregano and thyme essential oils obtained by hydrodistillation was determined by gc - ms .
aromatic compounds ( thymol and carvacrol ) dominated the composition . the main compounds and the rfs values determined by tlc of the essential oils extracted from the different plants studied
o. vulgare showed 5 bands , being the more intense the superior one to an rf of 0.60 corresponding to the mixture of thymol and carvacrol .
t. vulgaris showed two bands , being able to identify a pink band at rf 0.64 and another less intense blue band at a lower rf .
major compounds identified by tlc were consistent with data obtained from gc - es analysis .
no difference in the uv absorption curves of o. vulgare in relation to t. vulgaris essential oils was observed ; both showed a peak absorbance at 275 nm but in the case of thyme oil , this peak was more pronounced ( figure 2 ) . on infrared spectra
, it could be observed that both oils presented similar ir patterns ( figure 3 ) .
the survival of e. granulosus protoscoleces after exposure to t. vulgaris and o. vulgare essential oils and thymol is shown in figure 4 .
control protoscoleces cultured in medium 199 + pg or in medium 199 + dmso remained viable ( 92.2 2.4% and 85.9 2.1% , resp . ) after 60 days of incubation ( figure 4 ) .
essential oil of t. vulgaris provoked a protoscolicidal effect , significantly reducing ( p < 0.01 ) the viability of protoscoleces to 35.3 2.8% after 60 days of incubation ( figure 4 ) .
essential oil of o. vulgare significantly reduced ( p < 0.01 ) the viability of protoscoleces to 22.3 1.2% after 60 days ( figure 4 ) .
there was no significant difference ( p > 0.05 ) between the protoscolicidal effect of essential oils of t. vulgaris and o. vulgare .
viability was 0% at day 72 for both essential oils ( data not shown ) .
on the other hand , the loss of viability of the protoscoleces incubated with thymol became clearer after 12 days of postincubation ( p.i . ) , where the percentage value was 55.4 8.2% , reaching 0% after approximately 60 days ( figure 4 ) .
the results of the viability tests coincide with tissue damage observed at the structural and ultrastructural level .
control protoscoleces revealed no changes in structure and ultrastructure throughout the experimental period ( figures 5(a ) , 6(a ) , and 6(b ) ) .
after 3 days p.i . , the presence of numerous blebs in the tegument and contraction of soma region of essential oil treated protoscoleces was observed by inverted microscope ( figures 5(b ) and 5(c ) ) .
other alterations such as rostellar disorganization and loss of hooks could be seen after 6 days of incubation .
studies by sem revealed that ultrastructural damage was produced in drug - treated protoscoleces ( figure 6 ) .
after 6 days p.i . , alterations in the tegument and contraction of soma region were observed in essential oil treated protoscoleces .
moreover , rostellar disorganisation and shedding of microtriches of the rostellar region were detected ( figures 6(c ) and 6(e ) ) . at 36 days p.i . , loss of morphology was evidenced ( figures 6(d ) and 6(f ) ) .
control protoscoleces developed an average of 6 2.6 g of cysts , similar to the inoculation of freshly isolated protoscoleces .
this result showed that the infectivity of control protoscoleces was not affected during in vitro incubation for 60 days .
loss of infectivity in protoscoleces incubated with thymol and o. vulgare after 60 days was observed , since all of the protoscoleces failed to develop into cysts following their inoculation into mice .
protoscoleces incubated with t. vulgaris developed an average of 0.29 0.4 g of cysts .
however , the weight of cysts recorded in mice inoculated with t. vulgaris treated protoscoleces was significantly lower ( p < 0.05 ) than that obtained in the control group .
ggt activity in culture supernatants of protoscoleces treated with thymol and essential oils of t. vulgaris and o. vulgare is shown in figure 7 .
in contrast , the activity of ggt was detected in culture supernatants of drug treated protoscoleces at 5 days p.i .
interestingly , the viability of essential oil treated protoscoleces at similar incubation time was slightly decreased ( figure 4 ) .
figure 8 shows representative tunel images of protoscoleces treated with thymol and essential oils of t. vulgaris and o. vulgare .
the results of tunel assay clearly reveal presence of in situ dna fragmentation in the nuclei of treated protoscolex tissue .
, while in essential oil treated parasites it was observed after 16 h p.i . ( figures 8(b)8(d ) ) . in addition , protoscoleces with loss of the integrity of the tegument ( figures 8(b)8(d ) ) showed higher levels of apoptotic nuclei than protoscoleces with intact tegument ( figure 8(a ) ) .
all control cysts appeared turgid with no observable collapse of the germinal layer over the course of the in vitro experiment .
in contrast , a loss of turgidity was detected in cyst treated with t. vulgaris and o. vulgare essential oils and thymol ( table 3 ) .
control cultures exhibit no ultrastructural alterations in parasite tissue during the whole incubation period ( figure 9(a ) ) .
studies by sem revealed that the germinal layer of treated cysts lost the feature multicellular structure ( figures 9(b)9(d ) ) .
the present work describes for the first time the in vitro efficacy of t. vulgaris and o. vulgare essential oils against protoscoleces and cysts of e. granulosus .
essential oils of t. vulgaris and o. vulgare had a time - dependent effect provoking the loss of protoscolex viability . moreover , protoscoleces treated with o. vulgare and thymol lost their infectivity , since following inoculation of treated parasites in mice no cysts could be recovered after five months of postinfection .
the alterations included contraction of soma , blebs formation in tegument , rostellar disorganisation , and loss of hooks and microtriches .
these changes have been observed in e. granulosus protoscoleces following in vitro treatment with other essential oils or their components [ 24 , 26 , 28 , 29 ] .
moreover , the same alterations have also been reported by other authors working with synthetic drugs like bz [ 48 , 54 ] , praziquantel , ivermectin [ 51 , 56 ] , and nitasoxanide .
ggt activity in culture supernatants of protoscoleces treated with t. vulgaris and o. vulgare essential oils and thymol was also measured as a marker of tegumental damage .
ggt activity was readily detected into the culture supernatant of protoscoleces treated either with essential oils and thymol .
moreover , our observations are in accordance with those reported by cumino et al . .
the high sensitivity and accuracy of the enzymatic method was demonstrated in relation to ultrastructural studies and determination of viability by the methylene blue exclusion test .
the efficacy of t. vulgaris and o. vulgare essential oils and thymol was also demonstrated in vitro on e. granulosus murine cysts .
sem studies revealed that the germinal layer of cysts lost the characteristic multicellular structure , clearly showing disintegrated areas .
the ultrastructural changes were analogous to those reported by other authors [ 29 , 52 , 57 , 58 ] .
t. vulgaris essential oil showed thymol as a main compound in their composition while the predominant components in o. vulgare essential oil were thymol and carvacrol .
we suggest that the main components found in the essential oils assayed could be responsible for the markedly anthelmintic effect detected . despite the differences observed in their chemical composition ( see table 1 ) , the essential oils assayed had a similar anthelmintic activity .
data about the essential oil composition obtained with cg ms and tlc are in accordance with those reported by wagner et al . , bagamboula et al . , and gende .
physicochemical properties were analyzed with the aim of establishing the quality , purity , and chemical stability of the essential oil .
all physicochemical properties obtained ranged within the prospective values , if compared with the results by montes and retamar . in the present study , thymol had a slightly higher effect against e. granulosus metacestodes than did the essential oils assayed . however , there are studies where the biological activity of the essential oils was superior to thymol alone .
santoro et al . have shown that trypomastigotes are more sensitive to t. vulgaris essential oil in relation to purified thymol .
the discrepancies observed could be explained by the differences of their qualitative and quantitative composition .
the composition of essential oils can differ according to harvesting seasons , geographical sources , age , and vegetative cycle stage [ 6062 ] .
therefore , in order to obtain essential oils of constant composition , they have to be extracted under the same conditions from the same organ of the plant .
on the other hand , the inherent activity of the essential oils can be expected to relate to the proportions in which the components are present and to interactions between them .
thus , the observed anthelmintic activity of t. vulgaris and o. vulgare could be explained by the possible antagonic interactions between the components present in these essential oils .
antagonic effect between components was also described by liolios et al . working with other essential oils .
have suggested that the lower antibacterial of lippia sidoides essential oil when compared to thymol and carvacrol may be attributed to an antagonistic effect of the minor components on the activity of the essential oil .
the mechanism of action of essential oils and their components has not been studied in great detail .
since the essential oils are complex mixtures of several compounds , their antimicrobial activity is not attributable to one specific mechanism but there are several targets in the cell [ 66 , 67 ] . in eukaryotic cells , essential oils can provoke depolarisation of the mitochondrial membranes by decreasing the membrane potential [ 6870 ] .
essential oils change the fluidity of membranes , which become abnormally permeable resulting in leakage of radicals , cytochrome c , calcium ions , and proteins , as in the case of oxidative stress and bioenergetic failure .
permeabilization of outer and inner mitochondrial membranes leads to cell death by apoptosis and necrosis [ 71 , 72 ] .
apoptosis induced by thymol in cancer cells was associated with mitochondrial pathway [ 73 , 74 ] .
tunel assay revealed that thymol and essential oils of t. vulgaris and o. vulgare can induce apoptosis in the cells of protoscoleces .
as it was mentioned , drug - induced apoptosis in e. granulosus protoscoleces has also been reported by other authors [ 30 , 35 ] . in conclusion
, our data suggest that essential oils of t. vulgaris and o. vulgare have an anthelmintic effect against protoscoleces and cysts of e. granulosus .
however , the ggt activity measured in culture supernatants of protoscoleces treated with thymol or essential oil was similar .
further in vitro and in vivo bioassays will be required to fully evaluate the potential of these essential oils or some of their components as useful alternatives for the treatment of hydatid disease .
moreover , the evaluation of the in vitro effects of carvacrol alone and in combination with thymol is in progress and the results of this study could be a promising option for ce chemotherapy . | the aim of the present work was to determine the in vitro effect of t. vulgaris and o. vulgare essential oils against e. granulosus protoscoleces and cysts .
essential oils were added to the medium resulting in thymol final concentrations of 10 g / ml .
the essential oils had a time - dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation .
the results were confirmed at the ultrastructure level .
loss of infectivity in protoscoleces incubated with o. vulgare after 60 days was observed . on the other hand ,
the weight of cysts recorded in mice inoculated with t. vulgaris treated protoscoleces was significantly lower than that obtained in control group .
gamma - glutamyl - transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol .
t. vulgaris and o. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times .
the efficacy of t. vulgaris and o. vulgare essential oils was also demonstrated in vitro on e. granulosus murine cysts .
our data suggest that essential oils of t. vulgaris and o. vulgare have anthelmintic effect against protoscoleces and cysts of e. granulosus . |
this study used cross - sectional data from participants of the 20032004 and 20052006 nhanes study conducted in the u.s .
the three main aspects to the survey are an interview given by a trained interviewer in the participants home , a medical exam completed at a mobile examination center , and laboratory tests .
the national center for health statistics ethics review board approved the survey protocols , and informed consent was obtained for all subjects .
there were 14,584 people aged 6 years and older who participated in the accelerometer study in nhanes .
the 12,772 participants had at least 1 valid day of accelerometry data , which was defined as 10 or more hours of wear time ( 17 ) .
for the current study , we included participants 60 years of age and older ( n = 2,656 ) and who had 4 or more valid days of accelerometer wear time ( n = 2,401 ) . only participants scheduled for a morning blood draw had triglyceride and plasma glucose data because these samples were obtained after an overnight fast , bringing the final sample size to 1,367 ( 709 men and 658 women ) .
activity was assessed using the actigraph am-7164 accelerometer ( actigraph , fort walton beach , fl ) .
participants were instructed to wear the monitor for seven consecutive days and to remove the monitor before going to bed and during showers , bathing , swimming , or other water activities .
the monitors were returned by mail to the nhanes contractor where the data were downloaded , and the monitors were checked for calibration .
the uniaxial actigraph measures and records vertical acceleration as counts per minute , an arbitrary intensity indicator of the movements of each minute .
nonwear time was defined as intervals of at least 60 consecutive minutes of 0 counts with allowance for up to 2 min of counts between 0 and 100 .
active minutes were defined as 100 counts per minute and sedentary minutes as counts < 100 during wear time ( 18 ) .
a sedentary bout was defined as a period of time > 5 min when count values fell into the sedentary range with only 1 allowable minute outside the sedentary range .
a sedentary break was defined as an interruption in sedentary time when the count value exceeded 99 per minute . to identify specific sedentary activity parameters , five variables of interest
were included in this study : 1 ) duration of valid sedentary time ( hours ) ; 2 ) proportion of total valid wear time that was sedentary time ; 3 ) average duration of a sedentary bout ; 4 ) average intensity during sedentary time , which is used as a quantitative indicator of stillness during sedentary time and is defined by the average count value during sedentary time ; and 5 ) number of breaks in sedentary time .
metabolic syndrome was defined according to the atp iii guidelines as meeting three or more of the following criteria : 1 ) waist circumference 102 cm for men and 88 cm for women ; 2 ) serum triglyceride level of 150 mg / dl ; 3 ) hdl cholesterol level < 40 mg / dl for men and < 50 mg / dl for women ; 4 ) fasting glucose level 100 mg / dl or use of antidiabetic medications ( insulin or oral agents ) ; or 5 ) systolic blood pressure 85 mmhg and/or diastolic blood pressure 130 mmhg , or use of antihypertensive medications ( 2 ) .
up to four blood pressure reading were taken on participants . to determine blood pressure status ,
the last two blood pressure readings were averaged for participants that had three or four measurements .
if participants only had two blood pressure readings , the last reading was used in the analysis . if participants had only one blood pressure reading , that reading was used in the analysis .
measurements with a systolic reading of zero were considered missing . according to the guidelines , medications for elevated triglycerides and reduced hdl cholesterol ( fibrates and nicotinic acid )
people with one or two missing metabolic syndrome criteria who had three positive metabolic syndrome criteria were grouped in the metabolic syndrome group .
similarly , people with one or two missing metabolic syndrome criteria who had three negative metabolic syndrome criteria were grouped in the nonmetabolic syndrome group .
covariates included age , sex , self - reported race / ethnicity ( non - hispanic white , non - hispanic black , hispanic , other ) , education ( less than high school , high school or general equivalency diploma , more than high school ) , alcohol consumption ( never , former , current drinker , missing ) , smoking ( never , former , current ) , bmi ( kg / m ) , self - reported diabetes and heart disease , and physical activity ( moderate - to - vigorous physical activity [ minutes > 2,020 counts ] and average accelerometer count during active minutes ) .
data were analyzed using sas 9.2 software ( sas institute , cary , nc ) and sudaan 10.0 ( rti international , research triangle park , nc ) to account for the complex survey design and incorporate the sample weights .
sample weights were recalculated from the nhanes mobile examination center weights to reflect the nonresponse
reweighting was conducted within age ( 6069 years , 7079 years , and 80 + years ) , race / ethnicity ( non - hispanic whites , non - hispanic blacks , and mexican americans ) , and gender strata .
the t tests for continuous variables and tests for categorical variables were used to examine the differences in characteristics between people with and without metabolic syndrome .
analysis of variance was conducted to examine the association between sedentary variables and the metabolic syndrome components . adjusted means and standard errors adjusted for sex and age
logistic regression analyses were performed to examine the associations between the sedentary variables ( in quartiles ) and metabolic syndrome .
model 2 was additionally adjusted for education , race / ethnicity , alcohol consumption , smoking status , and bmi .
model 3 additionally adjusted for diabetes , heart disease , and physical activity by the average counts during active time was added to model 4 .
interactions between sex , age , race / ethnicity , and all sedentary variables were tested but were found to be nonsignificant at = 0.05 .
activity was assessed using the actigraph am-7164 accelerometer ( actigraph , fort walton beach , fl ) .
participants were instructed to wear the monitor for seven consecutive days and to remove the monitor before going to bed and during showers , bathing , swimming , or other water activities .
the monitors were returned by mail to the nhanes contractor where the data were downloaded , and the monitors were checked for calibration .
the uniaxial actigraph measures and records vertical acceleration as counts per minute , an arbitrary intensity indicator of the movements of each minute .
nonwear time was defined as intervals of at least 60 consecutive minutes of 0 counts with allowance for up to 2 min of counts between 0 and 100 .
active minutes were defined as 100 counts per minute and sedentary minutes as counts < 100 during wear time ( 18 ) .
a sedentary bout was defined as a period of time > 5 min when count values fell into the sedentary range with only 1 allowable minute outside the sedentary range .
a sedentary break was defined as an interruption in sedentary time when the count value exceeded 99 per minute . to identify specific sedentary activity parameters , five variables of interest
were included in this study : 1 ) duration of valid sedentary time ( hours ) ; 2 ) proportion of total valid wear time that was sedentary time ; 3 ) average duration of a sedentary bout ; 4 ) average intensity during sedentary time , which is used as a quantitative indicator of stillness during sedentary time and is defined by the average count value during sedentary time ; and 5 ) number of breaks in sedentary time .
metabolic syndrome was defined according to the atp iii guidelines as meeting three or more of the following criteria : 1 ) waist circumference 102 cm for men and 88 cm for women ; 2 ) serum triglyceride level of 150 mg / dl ; 3 ) hdl cholesterol level < 40 mg / dl for men and < 50 mg / dl for women ; 4 ) fasting glucose level 100 mg / dl or use of antidiabetic medications ( insulin or oral agents ) ; or 5 ) systolic blood pressure 85 mmhg and/or diastolic blood pressure 130 mmhg , or use of antihypertensive medications ( 2 ) .
up to four blood pressure reading were taken on participants . to determine blood pressure status ,
the last two blood pressure readings were averaged for participants that had three or four measurements .
if participants only had two blood pressure readings , the last reading was used in the analysis . if participants had only one blood pressure reading , that reading was used in the analysis .
measurements with a systolic reading of zero were considered missing . according to the guidelines , medications for elevated triglycerides and reduced hdl cholesterol ( fibrates and nicotinic acid )
people with one or two missing metabolic syndrome criteria who had three positive metabolic syndrome criteria were grouped in the metabolic syndrome group .
similarly , people with one or two missing metabolic syndrome criteria who had three negative metabolic syndrome criteria were grouped in the nonmetabolic syndrome group .
covariates included age , sex , self - reported race / ethnicity ( non - hispanic white , non - hispanic black , hispanic , other ) , education ( less than high school , high school or general equivalency diploma , more than high school ) , alcohol consumption ( never , former , current drinker , missing ) , smoking ( never , former , current ) , bmi ( kg / m ) , self - reported diabetes and heart disease , and physical activity ( moderate - to - vigorous physical activity [ minutes > 2,020 counts ] and average accelerometer count during active minutes ) .
activity was assessed using the actigraph am-7164 accelerometer ( actigraph , fort walton beach , fl ) .
participants were instructed to wear the monitor for seven consecutive days and to remove the monitor before going to bed and during showers , bathing , swimming , or other water activities .
the monitors were returned by mail to the nhanes contractor where the data were downloaded , and the monitors were checked for calibration .
the uniaxial actigraph measures and records vertical acceleration as counts per minute , an arbitrary intensity indicator of the movements of each minute .
nonwear time was defined as intervals of at least 60 consecutive minutes of 0 counts with allowance for up to 2 min of counts between 0 and 100 .
active minutes were defined as 100 counts per minute and sedentary minutes as counts < 100 during wear time ( 18 ) .
a sedentary bout was defined as a period of time > 5 min when count values fell into the sedentary range with only 1 allowable minute outside the sedentary range .
a sedentary break was defined as an interruption in sedentary time when the count value exceeded 99 per minute . to identify specific sedentary activity parameters , five variables of interest
were included in this study : 1 ) duration of valid sedentary time ( hours ) ; 2 ) proportion of total valid wear time that was sedentary time ; 3 ) average duration of a sedentary bout ; 4 ) average intensity during sedentary time , which is used as a quantitative indicator of stillness during sedentary time and is defined by the average count value during sedentary time ; and 5 ) number of breaks in sedentary time .
metabolic syndrome was defined according to the atp iii guidelines as meeting three or more of the following criteria : 1 ) waist circumference 102 cm for men and 88 cm for women ; 2 ) serum triglyceride level of 150 mg / dl ; 3 ) hdl cholesterol level < 40 mg / dl for men and < 50 mg / dl for women ; 4 ) fasting glucose level 100 mg / dl or use of antidiabetic medications ( insulin or oral agents ) ; or 5 ) systolic blood pressure 85 mmhg and/or diastolic blood pressure 130 mmhg , or use of antihypertensive medications ( 2 ) .
up to four blood pressure reading were taken on participants . to determine blood pressure status ,
the last two blood pressure readings were averaged for participants that had three or four measurements .
if participants only had two blood pressure readings , the last reading was used in the analysis . if participants had only one blood pressure reading , that reading was used in the analysis .
measurements with a systolic reading of zero were considered missing . according to the guidelines , medications for elevated triglycerides and reduced hdl cholesterol ( fibrates and nicotinic acid )
people with one or two missing metabolic syndrome criteria who had three positive metabolic syndrome criteria were grouped in the metabolic syndrome group .
similarly , people with one or two missing metabolic syndrome criteria who had three negative metabolic syndrome criteria were grouped in the nonmetabolic syndrome group .
covariates included age , sex , self - reported race / ethnicity ( non - hispanic white , non - hispanic black , hispanic , other ) , education ( less than high school , high school or general equivalency diploma , more than high school ) , alcohol consumption ( never , former , current drinker , missing ) , smoking ( never , former , current ) , bmi ( kg / m ) , self - reported diabetes and heart disease , and physical activity ( moderate - to - vigorous physical activity [ minutes > 2,020 counts ] and average accelerometer count during active minutes ) .
data were analyzed using sas 9.2 software ( sas institute , cary , nc ) and sudaan 10.0 ( rti international , research triangle park , nc ) to account for the complex survey design and incorporate the sample weights .
sample weights were recalculated from the nhanes mobile examination center weights to reflect the nonresponse
reweighting was conducted within age ( 6069 years , 7079 years , and 80 + years ) , race / ethnicity ( non - hispanic whites , non - hispanic blacks , and mexican americans ) , and gender strata .
the t tests for continuous variables and tests for categorical variables were used to examine the differences in characteristics between people with and without metabolic syndrome .
analysis of variance was conducted to examine the association between sedentary variables and the metabolic syndrome components . adjusted means and standard errors adjusted for sex and age
logistic regression analyses were performed to examine the associations between the sedentary variables ( in quartiles ) and metabolic syndrome .
model 2 was additionally adjusted for education , race / ethnicity , alcohol consumption , smoking status , and bmi .
model 3 additionally adjusted for diabetes , heart disease , and physical activity by the average counts during active time was added to model 4 .
interactions between sex , age , race / ethnicity , and all sedentary variables were tested but were found to be nonsignificant at = 0.05 .
out of the 1,367 people in the total analyzed sample , 665 ( 48.6% ) participants met the criteria for having metabolic syndrome .
as shown in table 1 , participants with metabolic syndrome were more often women ( p < 0.01 ) , had a lower educational level ( p < 0.01 ) , were more often nondrinkers ( p < 0.01 ) , had a higher bmi ( p < 0.001 ) , had a higher prevalence of diabetes and heart disease , spent less time in the moderate - to - vigorous activity range , and had a lower average accelerometry count during active minutes in the day ( p < 0.01 ) .
baseline characteristics of the study population * average age not representative of study population ; nhanes groups all people 85 years of age and older as 85-years - old .
on average , people spent 9.5 h ( 65% of wear time ) as sedentary .
the correlation between total sedentary time and percent sedentary time was high ( correlation coefficient 0.75 ) ; the correlation between total sedentary time with average length of a sedentary bout , intensity during sedentary time , and number of sedentary breaks was lower ( correlation coefficients 0.55 , 0.53 , and 0.09 , respectively ) .
table 2 shows the age- and sex - adjusted means of the five sedentary variables according to each metabolic syndrome component and metabolic syndrome .
participants with a large waist circumference , low hdl cholesterol , high triglycerides , and metabolic syndrome had a higher percentage of sedentary time out of total wear time , a longer average sedentary bout , a lower average intensity during sedentary time ( not statistically significant for hdl ) , and fewer breaks in sedentary time ( all p < 0.05 ) after adjusting for age and sex . adjusted means * ( se ) of five sedentary variables according to metabolic syndrome * adjusted for age and sex .
figure 1 shows the age- and sex - adjusted mean values of the five sedentary variables according to the number of metabolic syndrome criteria .
a significant positive trend with the number of metabolic syndrome criteria was found for the duration and percentage of sedentary time and the average length of a sedentary bout ( all p < 0.01 ) .
the intensity during sedentary time and the number of breaks in sedentary time had a significant negative trend with the number of metabolic syndrome criteria ( all p < 0.01 ) .
adjusted mean values * and standard errors of the five sedentary variables according to the number of metabolic syndrome criteria. * adjusted for age and sex .
model 1 , adjusted for age and sex , shows the greater likelihood of having metabolic syndrome in highest quartiles of sedentary time duration , percent sedentary time , and average length of a sedentary bout .
further , compared with the highest quartile , the lowest quartile of the intensity during sedentary time and the number of sedentary breaks was associated with an increased likelihood of metabolic syndrome .
model 2 was additionally adjusted for ethnicity , alcohol consumption , smoking , and bmi , and model 3 was additionally adjusted for diabetes and heart disease . in model 3 ,
a higher percentage of sedentary time out of total wear time ( quartile 2 : odds ratio [ or ] 1.52 [ 95% ci 1.042.21 ] ; quartile 3 : 1.59 [ 1.022.49 ] ; quartile 4 : 1.61 [ 1.052.48 ] ) and fewer sedentary breaks ( quartile 3 : 1.50 [ 1.022.21 ] ) were related to a significantly increased likelihood of metabolic syndrome .
percentage sedentary time and sedentary breaks remained statistically significant after additional adjustment for physical activity in model 4 .
a higher intensity during sedentary time was related to an increased likelihood of metabolic syndrome in model 2 ( quartile 4 : 1.66 [ 1.032.67 ] ) but was no longer statistically significant in models 3 and 4 ( 1.59 [ 0.962.62 ] ) , although the test for trend over the intensity categories remained statistically significant ( p = 0.05 ) . in additional analysis we adjusted for minutes of moderate - to - vigorous physical activity in model 4 instead of average counts per minute during active time , which did not change our results .
further , the relation between the intensity during sedentary time and sedentary breaks with metabolic syndrome did not change after adjustment for total sedentary time ( data not shown ) .
ors and 95% ci of metabolic syndrome according to quartiles of sedentary behavior * adjusted for age and sex .
adjusted for age , sex , ethnicity , education , alcohol consumption , smoking , and bmi .
adjusted for age , sex , ethnicity , education , alcohol consumption , smoking , bmi , diabetes , and heart disease .
adjusted for age , sex , ethnicity , education , alcohol consumption , smoking , bmi , diabetes , heart disease , and physical activity .
using nhanes data , the relationship between objectively measured sedentary time and metabolic syndrome was examined in a representative sample of older adults living in the u.s . compared with people without metabolic syndrome ,
people with metabolic syndrome spent more hours and a greater percentage of their wear time as sedentary .
further , people with metabolic syndrome had a longer average sedentary bout , a lower intensity during sedentary time , and fewer breaks in sedentary time . a higher percentage of total wear time spent as sedentary time was associated with a significantly greater likelihood of metabolic syndrome even after adjustment for physical activity . additionally , fewer sedentary breaks were associated with a higher likelihood of metabolic syndrome , and a significant trend was found for intensity during sedentary time where the likelihood of metabolic syndrome increased with a decrease in intensity .
previous research with self - reported data is consistent with the findings in this study on the association between time spent in sedentary behaviors and metabolic syndrome .
self - reported sedentary behavior , such as television viewing time , has been positively associated with metabolic risk factors and metabolic syndrome ( 8,9,19,20 ) .
several studies with objectively measured sedentary data have shown that the number of hours of sedentary time is related to metabolic risk factors ( 13,14,21 ) .
a study among australian adults found that sedentary time was positively associated with metabolic syndrome ( 14 ) .
other studies found that physical activity , but not sedentary time , predicts metabolic risk ( 21 ) and insulin resistance ( 22 ) . using a large nationally representative sample of older adults , our study was the first to explore different parameters of objectively measured sedentary behavior such as the average length of a sedentary bout ; the intensity during sedentary time , which is a quantitative indicator of stillness ; and sedentary breaks with metabolic risk in older adults . one previous study of adults ( mean age 53.4 years ) has been conducted on breaks in sedentary time and metabolic syndrome and corroborates our findings that fewer breaks in sedentary time were significantly associated with a larger waist , higher triglyceride levels , and higher 2-h glucose levels ( 13 ) .
the results of our study suggest that not only is total sedentary time an important health risk factor , but patterns ( i.e. , the way sedentary time is accumulated ) of sedentary behaviors might be important risk factors as well .
an important strength of our study is that sedentary behavior was measured objectively by accelerometry , but accelerometers are not sensitive to detect all activities such as biking , standing , and upper - body movement .
the cross - sectional design of this study limits inference about the direction of causality between the sedentary variables and metabolic risk .
current findings suggest that not only is it important to reduce sedentary time among older adults , but minimizing prolonged periods of uninterrupted sedentary time and increasing sporadic movements during sedentary time may also be protective . in practice , this may be achieved by taking brief activity breaks to disrupt prolonged periods of sitting or by increasing movements while sitting . these minimal changes may result in increases in daily energy expenditure ( 23 ) or could provide direct effects on metabolic regulation ( 24 ) . in summary
, the proportion of sedentary time was strongly related to metabolic risk , independent of physical activity .
older adults may be at a higher risk of developing metabolic syndrome based on this age - related increase of time spent in sedentary behaviors .
this was the first study to examine multiple sedentary parameters including sedentary intensity and sedentary breaks in relation to metabolic syndrome , and future studies should focus on these specific variables to corroborate our results .
current results , though cross - sectional in nature , suggest that potential behavioral interventions for the prevention of metabolic syndrome in older adults might include the promotion of physical activity as well as reduction in total sedentary time and disruption of prolonged sedentary bouts . | objectivethis study examined the association between objectively measured sedentary activity and metabolic syndrome among older adults.research design and methodsdata were from 1,367 men and women , aged 60 years who participated in the 20032006 national health and nutrition examination survey ( nhanes ) .
sedentary time during waking hours was measured by an accelerometer ( < 100 counts per minute ) .
a sedentary bout was defined as a period of time > 5 min .
a sedentary break was defined as an interruption in sedentary time ( 100 counts per minute ) .
metabolic syndrome was defined according to the adult treatment panel ( atp ) iii criteria.resultson average , people spent 9.5 h ( 65% of wear time ) as sedentary .
compared with people without metabolic syndrome , people with metabolic syndrome spent a greater percentage of time as sedentary ( 67.3 vs. 62.2% ) , had longer average sedentary bouts ( 17.7 vs. 16.7 min ) , had lower intensity during sedentary time ( 14.8 vs. 15.8 average counts per minute ) , and had fewer sedentary breaks ( 82.3 vs. 86.7 ) , adjusted for age and sex ( all p < 0.01 ) . a higher percentage of time sedentary and fewer sedentary breaks were associated with a significantly greater likelihood of metabolic syndrome after adjustment for age , sex , ethnicity , education , alcohol consumption , smoking , bmi , diabetes , heart disease , and physical activity .
the association between intensity during sedentary time and metabolic syndrome was borderline significant.conclusionsthe proportion of sedentary time was strongly related to metabolic risk , independent of physical activity .
current results suggest older people may benefit from reducing total sedentary time and avoiding prolonged periods of sedentary time by increasing the number of breaks during sedentary time . |
spinal intradural lipoma is a rare condition , accounting for < 1% of all spinal cord tumors.1 when encountered , most are found in the lumbosacral region with coinciding spinal dysraphism .
several reports in the literature have discussed spinal cord lipomas , especially in the pediatric population ; however , there are fewer reports of isolated cervical cord lipomas .
nondysraphic spinal cord lipomas usually present in the second or third decade of life , representing 55% of the cases .
only 12% of these tumors are localized to the cervical spine.2
3 we discuss a case of a nondysraphic cervical lipoma with an exophytic component in an adult .
symptoms were periodic but were getting progressively worse . the numbness was noted to be worse in the right hand .
physical examination showed the patient had a good range of motion in her neck , intact muscle strength in all groups , good muscle tone , normal reflexes , and no myelopathic signs .
radiographic work - up revealed a 2.54 1 cm hyperintense lesion on t1 with 40% cord compression .
the lesion spanned the entire length of the c6 vertebral body and 75% of the c7 vertebral body .
there was a heterogenic component to this lesion at the level of the c6c7 disk that may have indicated an infiltrative process ( figs . 1
2
3
4
5
6 ) .
a sagittal view , t1 with contrast , of the cervical spine .
an axial view , t1 with contrast , of the cervical spine .
a sagittal view , t1 without contrast , of the cervical spine .
there is a hyperintense , non contrast - enhancing mass on the dorsal aspect of the spinal cord causing cord compression .
an axial view , t1 without contrast , of the cervical spine .
there is a hyperintense , non contrast - enhancing mass on the dorsal aspect of the spinal cord causing cord compression .
the patient was taken to the operating room for a posterior cervical laminectomy and resection of the mass .
after a subperiosteal dissection , we used a high - speed drill to create troughs bilaterally .
the lamina was removed en bloc , hoping to secure the lamina after the tumor resection with laminoplasty trough plates .
a midline durotomy was performed , spanning both the cephalad and caudal poles of the tumor . on gross inspection of the tumor ,
the tumor was yellow and ellipsoid , involving both extramedullary and intramedullary components ( fig .
, we determined that a gross total resection of this tumor might not be possible due to the involvement of the spinal cord .
we began to resect layers of the tumor in a dorsal to ventral fashion . as we approached the spinal cord , it was deemed the remaining portion of the tumor was unresectable ( fig .
we used a small amount of progenix ( medtronic spinal and biologics , memphis , tennessee , united states ) in the lateral gutters to aid in bony regrowth .
the pathology specimen was reviewed by a neuropathologist , and the diagnosis was a lipoma .
the dorsal aspect of the tumor after the laminectomy and midline durotomy are performed .
these intraoperative pictures show the tumor .
the tumor after part of the exophytic portion was removed .
. left : cranial ; right : caudal ; down : left ; up : right .
it is well reported in the literature that spinal cord lipomas compromise < 1% of all spinal cord tumors.1 they are located dorsally and may have an exophytic component .
hydromyelia is present in 2% of the cases but was not present in this case .
dysraphism may include vertebral body anomalies , widening of the spinal canal and neural foramina , or anomalies of the posterior elements of the spine.4 uncertainty exists in the literature whether these lipomas are true neoplasms or hamartomas .
there are reports of nondysraphic lipoma recurring after resection due to hyperplasia , which supports it being a true neoplasm .
dysraphic lipomas are believed to be hamartomas.5
spinal cord lipomas are more frequently encountered in the pediatric population , possibly due to their embryological origin . depending on the location and involvement of neural structures , lipomas can present with a wide variety of symptoms .
mild neurologic dysfunction , such as sensory deficits , can be an indolent - presenting symptom for years .
other symptoms include growth retardation , dysphagia , respiratory difficulty , gait problems , and paresis ( the most common).6
7
8
lipomas consist of mature adipocytes with no atypical cellular features .
striated muscle , epithelial derivatives , bone , and neural tissue may also be found within lipomas . in our case ,
neural structures were involved on gross inspection , and adipocytes were the only cells present on microscopic inspection .
it is also believed that patients with a large body fat content are predisposed to developing lipomas . in our case , we did not recommend weight loss as a conservative treatment option because the patient was of normal weight.9 grossly , lipomas have historically been found to easily separate at the tips of the lesion ; the intraparenchymal margins are adherent.10 in our case , all margins of the tumor were adherent and embedded within the spinal cord .
although other pathologies can have similar characteristics on mri , a fat suppression sequence can help delineate a lipoma from blood or calcifications.10
11
there are several theories on the developmental mechanism of spinal cord lipomas .
most theories contribute the development of dysraphic spinal cord lipomas to an adipocyte migration problem during embryological development.12 one hypothesis is that if the neuroectoderm does not close properly , the mesoderm that migrates between the cutaneous and neural ectoderm can extend to the dorsal surface of the incompletely closed neuroectoderm.13 another theory is that premature detachment of the neural tube allows the neuroectoderm to fold in , allowing mesenchymal cells in the dorsal aspect of the cord .
the ingrowth of mesenchymal tissue is the cause of spinal dysraphism.14 unfortunately , the pathogenesis of nondysraphic spinal cord lipomas is unclear .
treatment options include observation , spinal cord decompression , biopsy , subtotal resection , and gross total resection .
if severe neurologic deficits are observed , decompression with an attempt at resection may be the treatment of choice .
gross total resection may prove to be technically challenging because most of these lipomas are adherent to the spinal cord and do not have a true plane for dissection.5 if the lipoma is found incidentally , observation may be sufficient . due to their benign nature , early literature advocates for conservative treatment measures ;
others argue for a subtotal resection.15
16 functional improvement is anticipated after cord decompression due to their indolent behavior ; however , the adherent nature may cause an intrinsic component of neurologic dysfunction.17
there is literature that discusses this pathology ; however , nondysraphic lipoma was isolated to the cervical cord with an exophytic component . | introduction spinal intradural lipoma is a rare condition , accounting for < 1% of all spinal cord tumors .
spinal cord lipomas are frequently associated with dysraphism and occur in the thoracic spine .
another common finding is that spinal cord lipomas tend to present in the pediatric population
. isolated nondysraphic cervical lipomas are a rare entity .
we discuss a case of nondysraphic cervical lipoma with an exophytic component .
case a 31 year - old woman presented with bilateral numbness in her hands and a burning and aching sensation in her arms for 6 months .
the patient did not have any weakness or myelopathic signs .
magnetic resonance imaging t1 showed a t1 hyperintense , t2 hypointense , non contrast - enhancing mass on the dorsal aspect of the spinal cord with significant compression .
the patient underwent a dorsal cervical laminectomy with subtotal resection of an isolated cervical lipoma with an exophytic component .
the pathology confirmed the diagnosis of a lipoma .
conclusions surgical management of this rare pathology has a wide variety of options . depending on the neurologic deficits ,
observation to gross total resection may be reasonable options . in our case ,
a subtotal resection was achieved with no further worsening of neurologic symptoms . |
severe or profound sensorineural hearing loss causes several deficiencies that are not only sensorial but also social and emotional . characterized by a lack of perception and speech coding , individuals with this degree of hearing loss might need special resources to minimize the consequences of such a loss . with the advancement of medicine and technology , the cochlear implant ( ci ) represents an alternative that allows access to the sounds of speech in individuals with severe to profound hearing loss.1 such improvements can also be observed in the objective methods used to verify the integrity of the cochlear device and the electrophysiologic response of users . neural response telemetry ( nrt ) , developed from the studies by abbas et
al,2 is a fast and easily applicable technique that assesses the response of the peripheral segment of the auditory nerve to electrical stimulation .
the implant used for nrt elicits stimuli and records the electrically evoked compound action potential ( ecap ) .
given that this is a fast and easily applicable technique , nrt is also used in the intraoperative setting . the major contribution of nrt is the confirmation of the physiologic integrity of the auditory nerve.3 it is also useful when establishing the electrodes that might be included in a given map , the best stimulation speeds , the speech coding strategies , and the estimation of the stimulation of t ( minimum stimulation ) and c ( maximum stimulation ) levels.4
composed of a negative peak ( n1 ) followed by a positive peak ( p2 ) , the ecap is analyzed with regard to the amplitude of the response .
the measurement between n1 and p2 yields the wave amplitude , which varies according to the increased stimulation intensity .
the ecap represents the synchrony of a group of neurons , and the amplitude of response is proportional to the number of neurons activated by a stimulus .
consequently , the presence of the ecap allows one to predict a satisfactory postoperative performance,5 which likely corresponds to better synaptic efficiency and synchronization of the neural response.6
knowledge of the physiology and responses of the auditory nerve to electrical stimuli is important in establishing the current level used for stimulation and other programming adjustments.7 in some cases , the ecap is not present during surgery , which might suggest a dysfunction of the cochlear nerve and existing neural structures . in some of these cases , continual stimulation predicts the appearance of neural response , which might be related to the synchronization of nerve fibers.8
cafarelli dees et al9 and van dijk et al10 suggested the rates of postoperative presence of ecap to be 96 and 90% , respectively , whereas guedes et al reported a rate of 80%.3 guedes et al found that the absence of responses was correlated with limited prognosis.11 the continued use of cis has tended to result in the emergence of the action potential after some months of stimulation . even longitudinal studies on neural responses , however , have failed to mention the possibility of the appearance of a response or the average time required for the onset of such a response .
this study aimed at establishing whether the ecap of the auditory nerve , when absent intraoperatively , can appear after continual electrical stimulation .
when nrt responses were absent during surgery , we analyzed the evolution of these responses after at least 3 months of stimulation .
this study was approved by the ethics committee of research projects ( cappesq ) of the clinical board of the clinical hospital and the medicine faculty of the universidade de so paulo , protocol no . 010/11 .
this was a retrospective study conducted through the survey and analysis of the database of the cochlear implant group of the clinical hospital of the medicine faculty of the universidade de so paulo ( hc - fmusp ) .
we selected the clinical records of children who underwent multichannel ci ( nucleus freedom , cochlear ltd . ,
australia , model ci24re ) surgery between january 2009 and january 2010 at the cochlear implant group of the hc - fmusp .
the criteria for sample selection included systematic use of the multichannel ci ( more than 8 h / d ) , the use of the ci24re internal and external units , and nrt records during the intra- and postoperative periods for 6 months after surgery for electrodes 1 , 6 , 11 , 16 , and 22 .
of the 63 children , 51 met the inclusion criteria , with ages at implantation varying between 11 and 187 months . to record the nrt ,
the cochlear implant group of the hc - fmusp used a speech processor , an external magnet antenna , a link wire between the speech processor and the external antenna , a programming interface ( pod ) , and a computer to send and receive the neural data .
all data were analyzed using the nrt software custom sound ep 2.0 ( cochlear ltd . ,
the nrt results were analyzed according to the protocol described by abbas et al2 and van dijk et al.10 ecap comprises a negative peak ( n1 ) with an approximate latency between 0.2 and 0.4 milliseconds , followed by a positive peak ( p2 ) with an approximate latency up to 1 millisecond .
the amplitude of the response ( measurement between n1 and p2 ) is proportional to the increase in the intensity of stimulation , which is measured in current units ( cu ) .
the presence of a visible n1 peak accompanied by the reproducibility of tracing , lack of artifact , or saturation of the amplifier ( figs . 1 and 2 ) was considered a valid neural response.12 the values of positive responses represented the nrt thresholds expressed as cu , which is the smallest amount of current needed to generate an ecap with an amplitude measurable by the software according to the autotelemetry protocol of van dijk et al.10
present response .
( a ) stimulation and recording parameters employed for the measurement of the ecap on electrode 6 and the stimulation level equal to 209 cu .
abbreviations : cmv , cytomegalovirus ; cu , current units ; ecap , evoked compound action potential ; nrt , neural response telemetry .
stimulation and recording parameters employed for the measurement of the evoked compound action potential ( ecap ) on electrode 1 and stimulation level equal to 204 current units ( cu ) .
data regarding the etiology , model of electrodes , chronological age , and length of auditory deprivation were collected from the clinical records .
it should be emphasized that the length of auditory deprivation is equal to the length of time between the onset of hearing loss and implant surgery . according to the intraoperative response ,
one group had positive neural responses in all electrodes , and the other group exhibited an absence of response in at least one electrode .
the nrt records were collected during surgery and at least 3 months after the activation of the ci .
the average length of stimulation was estimated from the record of a postoperative time per individual .
patients returned for follow - up at 3 and 6 months after activation , and the nrt response at the second or third times was used depending on the availability of records in the database .
the values obtained for the threshold of neural responses per electrode ( 1 , 6 , 11 , 16 , and 22 ) were initially analyzed in a descriptive manner . the mann - whitney test was used to establish the relationships between the absence of response and age and the absence of response and length of deprivation .
the wilcoxon matched pairs test was used to analyze the appearance of postoperative nrt responses per electrode .
we selected the clinical records of children who underwent multichannel ci ( nucleus freedom , cochlear ltd . ,
australia , model ci24re ) surgery between january 2009 and january 2010 at the cochlear implant group of the hc - fmusp .
the criteria for sample selection included systematic use of the multichannel ci ( more than 8 h / d ) , the use of the ci24re internal and external units , and nrt records during the intra- and postoperative periods for 6 months after surgery for electrodes 1 , 6 , 11 , 16 , and 22 .
of the 63 children , 51 met the inclusion criteria , with ages at implantation varying between 11 and 187 months .
to record the nrt , the cochlear implant group of the hc - fmusp used a speech processor , an external magnet antenna , a link wire between the speech processor and the external antenna , a programming interface ( pod ) , and a computer to send and receive the neural data .
all data were analyzed using the nrt software custom sound ep 2.0 ( cochlear ltd . ,
the nrt results were analyzed according to the protocol described by abbas et al2 and van dijk et al.10 ecap comprises a negative peak ( n1 ) with an approximate latency between 0.2 and 0.4 milliseconds , followed by a positive peak ( p2 ) with an approximate latency up to 1 millisecond .
the amplitude of the response ( measurement between n1 and p2 ) is proportional to the increase in the intensity of stimulation , which is measured in current units ( cu ) .
the presence of a visible n1 peak accompanied by the reproducibility of tracing , lack of artifact , or saturation of the amplifier ( figs . 1 and 2 ) was considered a valid neural response.12 the values of positive responses represented the nrt thresholds expressed as cu , which is the smallest amount of current needed to generate an ecap with an amplitude measurable by the software according to the autotelemetry protocol of van dijk et al.10
present response .
( a ) stimulation and recording parameters employed for the measurement of the ecap on electrode 6 and the stimulation level equal to 209 cu .
abbreviations : cmv , cytomegalovirus ; cu , current units ; ecap , evoked compound action potential ; nrt , neural response telemetry .
stimulation and recording parameters employed for the measurement of the evoked compound action potential ( ecap ) on electrode 1 and stimulation level equal to 204 current units ( cu ) .
data regarding the etiology , model of electrodes , chronological age , and length of auditory deprivation were collected from the clinical records .
it should be emphasized that the length of auditory deprivation is equal to the length of time between the onset of hearing loss and implant surgery . according to the intraoperative response ,
one group had positive neural responses in all electrodes , and the other group exhibited an absence of response in at least one electrode .
the nrt records were collected during surgery and at least 3 months after the activation of the ci .
the average length of stimulation was estimated from the record of a postoperative time per individual .
patients returned for follow - up at 3 and 6 months after activation , and the nrt response at the second or third times was used depending on the availability of records in the database .
the values obtained for the threshold of neural responses per electrode ( 1 , 6 , 11 , 16 , and 22 ) were initially analyzed in a descriptive manner . the mann - whitney test was used to establish the relationships between the absence of response and age and the absence of response and length of deprivation .
the wilcoxon matched pairs test was used to analyze the appearance of postoperative nrt responses per electrode .
of the 51 children , 40 exhibited positive responses during intraoperative nrt in all of the tested electrodes , whereas 11 children exhibited an absence of response in at least one electrode ( table 1 ) .
regarding the implant model ci24re , all patients received the perimodiolar electrode ( contour advance ) , except for one child who received the ci24re implant with a straight electrode beam . regarding the causes of hearing loss found in the total sample ( fig .
3 ) , both groups exhibited a heterogeneous distribution in which an unknown etiology predominated .
distribution of etiology in the total sample between the group with positive intraoperative neural response telemetry ( nrt ) response in all tested electrodes and the group with absent intraoperative nrt response in at least one tested electrode .
abbreviations : ci , cochlear implantation ; nrt , neural response telemetry ; sd , standard deviation .
although 21% of the children showed an absence of response in at least one electrode , a positive intraoperative neural response was observed in all cases .
the absence of an intraoperative nrt was observed in 16% ( 16 ) of 255 electrodes tested ( table 2 ) .
the absence was most frequent in the basal electrodes , primarily in electrode 1 ( 18% ) compared with electrodes 6 , 16 , and 22 ( 4% ) and electrode 11 ( 2% ) .
after an average of 4.9 months of continual stimulation , the number of individuals in the total sample exhibiting an absence of response decreased from 21% ( 11 ) to 11% ( 6 ) , thus reducing the relative absence / individual ratio by 18% . within the group exhibiting an intraoperative absence of response
, the presence of nrt responses appeared in 45% ( 5 ) of the individuals and 14% ( 8) of the tested electrodes . among the positive responses
, three appeared in patients with hearing loss due to unknown etiology , in one patient with hearing loss due to meningitis , and in one patient with hearing loss due to genetic causes .
the results of the mann - whitney test indicated that age at implantation ( u = 184.500 , z 814 , p < 0.05 and length of deprivation ( u = 177.000 , z 986 , p < 0.05 were not statistically significant between the groups .
table 3 shows the comparison between the thresholds of the intra- and postoperative neural response in all of the tested electrodes in the group exhibiting an intraoperative absence of response and the time when the postoperative response was recorded .
electrode 1 , which exhibited the highest rate of absence ( 82% ) , was excluded from the analysis due to the low number of present responses .
a wilcoxon matched pairs test showed that the postoperative appearance of nrt responses was not statistically significant in any of the tested electrodes .
there was , however , a tendency toward significance in electrode 22 , which might be confirmed by studying a larger sample .
abbreviations : intraop , intraoperatively ; postop , postoperatively ; nrt , neural response telemetry ; sd , standard deviation . note : all responses are given in current units ; indicates the absence of a response . analyzing whether the presence ( or appearance ) of a response is significant in any electrode
periodic nrt testing can be a helpful tool in clinical practice for determining the readaptation and recuperation of the cochlear nerve fibers after continued stimulation . in our study
after an average of 4.9 months of continual stimulation , nrt responses appeared in 45% of individuals who had an absence of response during surgery .
this finding suggests a tendency for the response to appear after continual use of the ci , which might be related to the hypothesis that electrical stimulation causes alterations in the synaptic activity and supplies the auditory nerves with reinforced neurotrophic support.13
14 as a result of such modifications , it is believed that the synchronization of neural fibers occurs more efficiently due to the manner in which stimulation activates the primary neurons as well as causing a possible reduction in the periods of neuronal firing.15
age at implantation and length of hearing loss , which are variables that often interfere with the determination of resources and the programming of the ci in clinical practice , did not exhibit any significant influence on the absence of nrts or in the later appearance of response . in our study , these results can be explained by the fact that the sample was comprised exclusively of children between the ages of 11 and 187 months .
similar studies should be performed with adults to analyze the possible relationship between age at implantation , length of hearing loss , and absence of intra- and postoperative ecap responses . in our case series , hearing loss due to
unknown causes was the prevailing etiology . due to the small number of individuals lacking an nrt response , the results reported in prior studies,3
10 which indicate a prevalence in the absence of response in all electrodes in progressive and ossifying conditions ( meningitis and otosclerosis ) , could not be confirmed .
this is likely because in the present study , only children were analyzed , and otosclerosis is known to be predominantly present in the adult population .
electrodes 1 , 6 , 11 , 16 , and 22 were tested using the default settings in the custom sound ( cochlear ) software , which is programmed to record the nrt responses from the bundle of electrodes allocated in the cochlea . the recorded responses therefore must have originated from every cochlear area to detect possible tonotopic interferences in the ecap responses . only individuals with an impedance telemetry within the normal range ( above 0.7 k and below 20 k ) were analyzed , thus avoiding the integrity of the chain of electrodes from becoming a defining variable in the recording of the nrt responses .
we observed a tendency for the response to be absent at higher current levels in the basal electrodes , which is consistent with previous studies.3
16
17
18 this absence is possibly associated with the position of the electrodes on the basal turn , which becomes more distant from the modiolus ( due to the position of cochleostomy ) and leads to the need for higher levels of energy in this area.19 in this case , the position of cochleostomy would distance the array of electrodes from the modiolus .
it would , however , be interesting to study the influence of electrode position on the threshold of neural responses in implanted patients using the technique of insertion through a cochleostomy and a round window with straight and perimodiolar electrodes . in our case series ,
only one child had a straight electrode implant , and this child exhibited responses in all of the tested electrodes .
other studies have suggested a relationship between residual hearing and the need of lower levels of current required to elicit a response in these areas , possibly explaining the need for higher levels of energy in the basal electrodes ( an area with less auditory residue).2
20
table 3 shows that in individual 22 , the postoperative record analysis showed the appearance of nrt responses in electrodes that were absent from the intraoperative setting ( 1 and 22 ) .
it was , however , possible to observe that two of the electrodes ( 11 and 16 ) began to exhibit absent responses .
the results of our study highlight the importance of conducting intraoperative nrt examinations , which is a fast and simple procedure that can ensure the physiological integrity of the cochlear nerve and allow for the monitoring of the response over time .
in addition , this method can assist in the programming of the speech processor of the ci .
periodic retesting of ecap responses allows for a deeper understanding of the neural physiology of patients presenting with an absence of response and may assist in the development of better coding strategies for the device and more efficient rehabilitation for each individual .
ecaps of the auditory nerve appeared in 45% of individuals with intraoperative absence of response after an average of 4.9 months of continual stimulation . | introduction currently the cochlear implant allows access to sounds in individuals with profound hearing loss .
the objective methods used to verify the integrity of the cochlear device and the electrophysiologic response of users have noted these improvements .
objective to establish whether the evoked compound action potential of the auditory nerve can appear after electrical stimulation when it is absent intraoperatively .
methods the clinical records of children implanted with the nucleus freedom ( cochlear ltd . ,
australia ) ( ci24re ) cochlear implant between january 2009 and january 2010 with at least 6 months of use were evaluated .
the neural response telemetry ( nrt ) thresholds of electrodes 1 , 6 , 11 , 16 , and 22 during surgery and after at least 3 months of implant use were analyzed and correlated with etiology , length of auditory deprivation , and chronological age .
these data were compared between a group of children exhibiting responses in all of the tested electrodes and a group of children who had at least one absent response .
results the sample was composed of clinical records of 51 children . from these , 21% ( 11 ) showed no nrt in at least one of the tested electrodes .
after an average of 4.9 months of stimulation , the number of individuals exhibiting absent responses decreased from 21 to 11% ( n = 6 ) .
conclusion it is feasible that absent responses present after a period of electrical stimulation . in our sample , 45% ( n = 5 ) of the patients with intraoperative absence exhibited a positive response after an average of 4.9 months of continued electrical stimulation . |
periodontium is a group of specialized tissues that surround and support the teeth and maintain them in the maxillary and mandibular bones , and it consists of four principal components : gingiva , periodontal ligament , cementum , and alveolar bone .
the inflammatory state of periodontium is called periodontitis , which is one of the most common oral diseases and is characterized by the loss of connective tissue attachment and bone around the teeth in conjunction with the formation of periodontal pockets due to the apical migration of the junctional epithelium ( gingival inflammation and alveolar bone resorption ) .
periodontitis is generally described as a multifactorial irreversible and cumulative condition , initiated and propagated by bacteria and host factors .
it is known that the oral mucosal surface is the home of numerous normal flora microorganisms ( more than 500 different bacterial species are able to colonize the oral biofilm ) , being the portal of entry for the majority of pathogens .
it is also known that salivary proteins participate in the protection of oral tissues , as well as upper digestive and respiratory tracts , against a number of microbial pathogens .
therefore , potential periodontal and salivary changes may influence the general health [ 27 ] .
almost 4% of global deaths , 4.6% of diseases and injuries , and 30% of health care costs are attributed to alcohol abuse .
alcohol drinking is the world 's third largest risk factor for disease and disability , whereas in middle - income countries it is the greatest risk .
about 2% of the worldwide population is dependent on alcohol . up to 90% of alcohol - dependent patients
the association between alcohol addiction and poor dental state , periodontal state , and oral hygiene is widely known .
it was also found that persons addicted to alcohol with poor dental status have double concentration of acetaldehyde in the saliva , than those addicted to alcohol and taking care of oral hygiene . in alcohol abusers , reduced salivary flow may lead to inflammatory states of the oral cavity , periodontal diseases , or infections .
the association between cigarette smoking and poor oral hygiene is also widely known [ 12 , 15 ] .
cigarette smoking was observed as the strongest of the modifiable risk factors for periodontal disease , second only to bacterial plaque .
analysis of saliva and gingival crevicular fluid ( gcf ) may be especially beneficial in the determination of current periodontal status .
there is a need to develop the noninvasive , cost - effective , and easy - to - use diagnostic tests and to analyze periodontal diagnosis with identification of these markers for the risk of developing periodontitis , active periodontal disease , to manage patient and response to treatment , to reflect the extent of periodontal destruction , and to predict the future disease progression .
so far , some salivary and gcf - derived enzyme markers ( e.g. , elastase , -glucuronidase , -hexosaminidase , and lysozyme ) and nonenzymatic salivary protein markers ( e.g. , platelet - activating factor , growth factors , fibronectin , lactoferrin , and immunoglobulins ) have been identified . however , measuring probing depth and clinical attachment level by calibrated periodontal probe and assessing gingival inflammation by gingival index ( gi ) and papilla bleeding index ( pbi ) are still the most reliable and specific clinical periodontal parameters for periodontal diagnosis .
since -hex b% highly improved the sensitivity of serum -hex , for example , in the diagnosis of alcohol abuse , we investigated the salivary index of -hex a% or -hex b% to be a useful potential method to increase the salivary -hex sensitivity and specificity , as markers of periodontitis .
65 individuals , participated in the study comprised of 33 alcohol - dependent smoking individuals ( 9 females and 24 males ) ( mean age sd ; 44 0.3 ) , recruited from the alcohol detoxification unit of the psychiatric hospital in choroszcz , poland , and the control group consisted of 32 healthy social drinkers ( 10 females and 22 males ) ( mean age 41 7.0 ) with no history of alcohol abuse or smoking .
the chronic alcohol intoxication ranged from 3 to 90 days ( mean 23 ) ; they drank an average of 232 ( 100700 ) g of alcohol / day .
alcohol - dependent individuals met the criteria for the alcohol and nicotine dependence according to icd-10 and dsm - iv criteria .
the average time of dependence was 13 years for the alcohol and 21 years for the smoking .
the study was approved by the bioethical committee of the medical university of bialystok , poland .
informed written consent was obtained from all the subjects after explanation of the nature , purpose , and potential risks of the study .
material from persons admitted to the detoxification unit was collected at the second day of the abstinence period .
the subjects were instructed to refrain from smoking , food , and beverages , except water , for 2 hours before saliva collecting . all salivary samples ( 3 ml of resting whole saliva )
were collected in plastic tubes placed on ice by spitting method , under standardized conditions [ 18 , 19 ] , between 8:00 am and 9:00 am , to minimize the influence of circadian rhythms .
the samples were centrifuged at 3,000 g for 20 minutes at 4c , to remove cells and debris .
the resulting supernatants were divided into 200 l portions , frozen , and kept at 80c , until analyzed .
salivary flow ( sf ) was calculated by dividing the volume of saliva by the time of its collection . in all samples ,
the activity of total -hex , -hex a and -hex b , was assayed in duplicate , and the means of the duplicate results were used as final values .
gingival status was assessed using gingival index ( gi ) and papilla bleeding index ( pbi ) .
gi scores the marginal and interproximal tissues separately on the basis of 0 to 3 .
the criteria are 0normal gingiva ; 1mild inflammation , slight change in color and slight edema but no bleeding on probing ; 2moderate inflammation , redness , edema , and glazing and bleeding on probing ; 3severe inflammation , marked redness and edema and ulceration with a tendency to spontaneous bleeding .
the intensity of any bleeding in pbi is recorded as 0no bleeding ; 1a single discreet bleeding point ; 2several isolated bleeding points , or a single line of blood appears ; 3the interdental triangle is filled with blood shortly after probing ; 4profuse bleeding occurs after probing ; blood flows immediately into the marginal sulcus .
good periodontal health and dental health were interpreted as follows : < 1 for pbi and gi and < 20 for dmft ( decayed , missing , or filled teeth ) .
activities of total -hex and its isoenzymes in supernatants of saliva were determined by the colorimetric method .
the indexes of -hex a% and -hex b% were calculated by dividing , respectively , the activity of -hex a and -hex b by the total activity of -hex .
statistical analysis was performed with statistica version 10.0 ( statsoft , cracow , poland ) .
the comparisons between groups were made using mann - whitney u test with appropriate adjustment to the critical value of p. descriptive characteristics are presented as means 1 sd , whereas the results of the nonparametric analysis are presented as least square means 1 sem ( standard error of the mean ) .
spearman 's rank correlation coefficient was used to measure the statistical dependence between two variables in alcohol - dependent persons .
a receiver operating characteristic ( roc ) analysis , calculations of specificity , sensitivity and area under the roc curve ( auc ) , and auc comparisons ( for paired samples ) were performed using statistica rapid deployment of predictive models .
the mean -hex a% values in c and as groups were , respectively , 11.5 19.6 and 45.5 16.8 . in as group , -hex a% was significantly higher than in c group ( p < 0.001 ) .
the mean -hex b% values in c and as groups were , respectively , 87.9 19.5 and 54.8 17.3 . in as group , -hex b% was significantly lower than that in c group ( p < 0.001 ) ( figure 1 ) .
we did not find any significant correlations between amount / duration - time of alcohol drinking / smoking ( or time of alcohol / smoking dependence ) and -hex a% or -hex b% .
there were significant correlations between -hex a% and gi ( r = 0.479 , p = 0.001 ) and inverse correlations between -hex b% and dmft index ( r = 0.309 , p = 0.038 ) as well as between -hex b% and gi ( r = 0.433 , p = 0.005 ) .
we found also a significant inverse correlation between -hex a% and sf ( r = 0.501 , p < 0.001 ) and a positive correlation between -hex b% and sf ( r = 0.526 , p < 0.001 ) .
an inverse correlation was also found between -hex a% and -hex b% ( r = 0.987 , p < 0.001 ) .
we found significant correlations between gi and pbi ( r = 0.840 , p = 0.0000 ) , as well as between dmft index and age ( r = 0.448 , p = 0.021 ) .
there were significant correlations between -hex b% and pbi ( r = 0.409 , p = 0.037 ) and gi ( r = 0.421 , p = 0.031 ) .
we found significant correlations between dmft index and pbi ( r = 0.522 , p = 0.021 ) , the time period of denture wearing ( r = 0.463 , p = 0.022 ) , and age ( r = 0.448 , p = 0.021 ) .
we found also significant correlations between gi and the time period of denture wearing ( r = 0.743 , p = 0.0004 ) , dmft index ( r = 0.652 , p = 0.002 ) , pbi ( r = 0.721 , p = 0.0007 ) , and the amount of smoked cigarettes per day ( r = 0.627 , p = 0.009 ) .
there was also a significant correlation between pbi and the time period of denture wearing ( r = 0.530 , p = 0.023 ) . obtained by roc analysis , -hex a% cut - off values showed that the measurement of -hex a% , in the saliva of smoking alcoholics , at 0.23 has excellent sensitivity ( 96% ) and specificity ( 91% ) .
the auc for -hex a% was of high diagnostic value ( 0.937 0.029 ) ( figure 2 ) .
the salivary flow ( sf ) was significantly lower in as group than in the controls ( table 1 ) .
the mean decayed , missing , and filled teeth index ( dmft ) had a tendency to be higher , and gingival index ( gi ) and papilla bleeding index ( pbi ) were significantly higher in smoking alcoholics ( as ) than in controls ( c ) .
during periodontitis state , white blood cells , of which the most troublesome and abundant are polymorphonuclear leukocytes ( pmns ) , signal for more and more white cells to be recruited to the bacteria site .
the congregation of too many pmns leads to overproduction of noxious agents which are intended to fight the bacteria but overinduce inflammation of healthy tissue and encourage the progression of bone loss and periodontal disease .
neutrophils play a destructive role in the periodontal tissue breakdown process due to high levels of lysosomal enzymes , generation of superoxides , and reactive oxygen species ( ros ) . in periodontitis ,
bacterial virulence factors result directly in degradation of host tissues or cause the release of biologic mediators from host tissue cells ( proteinases , cytokines , and prostaglandins ) that lead to host tissue destruction .
ethanol , which is directly toxic to the mucosa of the oral cavity ( including periodontium ) , mouth , throat , esophagus , and the stomach , diffuses rapidly into saliva and oral tissues and immediately after drinking its salivary concentration is temporarily much higher than that in plasma [ 21 , 22 ] . after alcohol intake
, acetaldehyde is locally formed in the oral cavity oxidation system through oral mucosal alcohol dehydrogenase and oral microflora . as further metabolism of acetaldehyde to acetate via oral bacteria
is limited , the level of salivary acetaldehyde is 10100 times higher than that in the blood .
acetaldehyde in smoking alcohol - dependent persons comes not only from the metabolism of ethanol but also from tobacco smoke . besides acetaldehyde ,
tobacco smoke is a source of oxidative stress and contains up to 3000 different toxic substances such as nicotine , nitrosamines , carbon monoxide , and other aldehydes that may potentially damage the oral tissues [ 22 , 23 ] . in addition , ros generated during drinking and smoking , as well as nonoxidative metabolites of ethanol ( e.g. , fatty acid ethyl esters , faees ) and the ethanol - water competition mechanism , might potentially be involved in the oral ( periodontal ) tissue damage [ 8 , 12 , 2226 ] .
it is generally known that chronic ethanol consumption leads to fat accumulation in salivary glands , acinar cell swelling , and atrophy and to reduction in weight and protein content of the parotid gland .
chronic drinking also reduces salivary flow rate , some of salivary proteins and glycoproteins levels ( e.g. , amylase ) , and sodium , bicarbonate , and chlorine concentrations [ 12 , 2730 ] . a diffuse infiltration of salivary gland stroma by mononuclear inflammatory cells and their concentration around the salivary ducts were also reported .
epithelial atrophy was also observed with a decrease in basal cellular size following chronic ethanol consumption . as compared to transient oral mucosa damage depending on the concentration of alcohol in acute alcohol ingestion ,
chronically intoxicated mucosa by the alcohol had dysplastic changes with the keratosis ( atrophy with associated hyperregeneration ) [ 21 , 29 ] . in chronic cigarette smokers , swollen secretory cells of the salivary glands and
decreased secretion of total salivary proteins were described [ 26 , 32 , 33 ] .
acetaldehyde from cigarette smoke has been shown to inactivate the most important antioxidant enzyme in the saliva - peroxidase . even in people chewing and snuffing tobacco , decreased levels of lysozyme and lactoferrin and increased secretion of immunoglobulin a in the saliva were found .
although decreased secretion of total salivary proteins was described in smokers , significantly higher stimulated secretion of the saliva in smokers is due to the irritating effect of tobacco smoke on the oral mucosa . in alcoholics who were active smokers , there were described decreased salivary flow and output of salivary acquired and innate immune proteins [ 8 , 26 , 36 , 37 ] .
salivary enzymes , including exoglycosidases , come from gland cells , oral microorganisms , pmns , and macrophages entering the gingival sulcus , as well as from epithelial cells , plasma , and dietary constituents .
in particular , gcf outflow to the oral cavity is an important contributor to the enzyme pool in the whole saliva [ 7 , 38 ] .
-hex is the most active of exoglycosidases that degrades tissue glycoconjugates including gingival tissue glycoconjugates .
it was observed that the activity of -hex , which hydrolyses glycosidic linkages , decreased as a result of periodontitis treatment [ 4 , 6 ] .
another exoglycosidase , -glucuronidase , that is a widely accepted periodontitis marker has a total predictive value that ranged from 83% to 88% , with sensitivity ranged from 65% to 77% and specificity ranged from 85% to 89% .
our study showed the significant increase in the salivary index of -hex a% and decrease in -hex b% in smoking alcoholics , as compared to the control group .
as the activity of -hex a in body fluids is an inflammatory mediator , secreted into extracellular space by the inflammatory cells such as mast cells , basophils , macrophages , eosinophils , and neutrophils [ 29 , 39 , 40 ] , increased -hex a% ( -hex a / total -hex ratio ) in our study seems to be due to the gingivitis .
it is consistent with other studies in which the salivary -hex activities correlated with the degree of periodontal inflammation and decreased along with periodontal healing after treatment [ 46 ] . increased -hex a% ratio may also be due to the inflammatory state of the whole mucosa of the oral cavity and/or salivary glands and their subsequent hyperregeneration .
a diffuse infiltration of the salivary gland stroma by mononuclear inflammatory cells and their concentration around the salivary ducts were earlier reported .
the periodontal inflammatory etiology of the high -hex a% levels seems also to be confirmed by the significantly worse periodontal state in smoking alcoholics , reflected not only by higher periodontal inflammatory index , gi but also by higher papilla bleeding index , pbi , than in controls ( table 1 ) .
the increased ratio of -hex a% may also be due to the decrease in the salivary flow ( sf ) in alcoholics , as we found significant inverse correlations between sf and -hex a% .
salivary flow ( sf ) was significantly lower than that in the control group , which confirms the literature data of chronic alcohol studies [ 27 , 41 ] .
mostly , morphological changes of the salivary glands are considered to be involved in the sf reduction in people addicted to alcohol ; other mechanisms involve risen liquidity of cell membranes , decreased secretory cell function , severe stress of the abstinence syndrome with accompanying sympathetic system arousal , hypercortisolemia , fluid diet and reduced chewing stimulation , decreased amount of muscarinic receptors , or deregulated secretion in the paracrine transmitters and gastrointestinal hormones , for example , cholecystokinin , somatostatin , and pp peptide [ 12 , 26 ] .
a higher secretion of stimulated saliva in smokers is known [ 12 , 35 ] ; hence , the sf reduction in alcohol - dependent smokers in our study seems to be rather the result of alcohol consumption than smoking .
increased ratio of -hex a / total -hex ( -hex a% ) indicates the increase in the -hex a isoenzyme activity , as shown in earlier study .
the increased -hex activity in the body fluids including saliva may occur in smoking alcoholics via many mechanisms , for example , increased lysosomal membrane permeability , delayed removal of the enzymes from the fluid , impaired trafficking of lysosomal hydrolases to organelles , enhanced synthesis of the enzyme by activated leucocytes , or leakage from damaged cells [ 12 , 24 , 25 , 39 ] .
therefore , during periodontitis , the lysosomal and cellular membranes may be damaged , and proteases , together with glycosidases ( including -hex ) , may be translocated into the cytosol fraction , the intercellular space , and to the body fluids ( e.g. , to the saliva ) [ 6 , 42 , 43 ] .
very high salivary levels of acetaldehyde derived from ethanol ( 10100 times higher than in the blood ) and cigarettes may be the reason of the lack of correlation of drinking or smoking with -hex b% or -hex a% . besides acetaldehyde
, many other toxic substances such as nicotine , nitrosamines , carbon monoxide , and other aldehydes , [ 12 , 22 , 23 ] in saliva of smoking alcohol - dependent persons may potentially be the reason of decreased ratio of -hex b% due to -hex b inactivation .
although -hex a is a generally more labile isoenzyme than -hex b for high temperature , -hex b may be potentially more sensitive to highly concentrated acetaldehyde or other toxic compounds of the cigarette smoke .
however , high correlation between gi and amount of smoked cigarettes per day may point to smoking as the main inductor of the bad gingival ( periodontal ) state .
the high value of the area under the curve ( auc ) for -hex a% index ( 0.937 ) in as group , excellent sensitivity ( 96% ) and specificity ( 91% ) of -hex a% , and its correlation with gi point to the high value of -hex a% as an excellent periodontitis marker . since
a spectrophotometric method gives the same results as an immunoassay method to distinguish the activities of the -hex isoenzymes , the salivary -hex a% determination may be a cheap and useful method for the detection of periodontitis in smoking alcoholics .
beta - hex a% index may be even a possible marker of periodontitis in a vast majority of the population , as we found correlations between -hex a% and gi in smoking alcoholics together with controls .
although there are some limitations for our study , such as a lack of equal female - male representation in c and as groups and no group of periodontitis patients without history of alcohol and nicotine dependence , our study clearly shows that the salivary -hex a% that correlates with gingival index has an excellent sensitivity and specificity .
however , further studies need to check if there are similar high sensitivity and specificity of -hex a% as a periodontitis marker in patients suffering from clear periodontitis ( without substance dependence syndrome ) and other pathological conditions . as we noticed a tendency to the worse dental state ( reflected by lower dmft index ) and significantly worse periodontal state ( reflected by high gi and pbi indexes ) ( table 1 ) in smoking alcoholics than in the controls , according to earlier studies , we confirmed the association between alcohol and nicotine addiction and poor dental state , periodontal state , and oral hygiene [ 1215 ] .
we also confirmed an earlier study stating that cigarette smoking is a strong modifiable risk factor for periodontal disease . as we found a correlation of dmft index and age , we confirmed data from the literature that the tooth loss is associated with increasing age .
it is also known that bad periodontal state worsens dental state , which we confirmed by a correlation of pbi and gi with dmft index .
therefore , denture wearing seems to be a natural consequence of bad periodontal and subsequently bad dental states .
we found high significant correlations between the time period of denture wearing and gi , pbi , and dmft indexes .
however , wearing of dentures is also a known major risk factor of periodontitis and caries , due to the poor oral hygiene , increased plaque and calculus accumulation , and transmission of excessive forces to the periodontal structures from occlusal surfaces of the framework of dentures [ 45 , 46 ] . in conclusion
, the increased salivary -hex a% index may be a possible excellent marker of the periodontitis .
smoking alcohol - dependent persons have worse periodontal state than controls , mostly due to the smoking - induced inflammatory state of the periodontium , as reflected by the high salivary -hex a% index . | background . severe periodontitis leading to tooth loss is found in 515% of most populations worldwide .
aim .
the applicability of salivary -hexosaminidase ( -hex a% , percentage of -hex a isoenzyme to total -hex ) and -hex b% ( -hex b/-hex ) indexes was investigated as a possible marker of periodontitis .
methods . thirty three alcohol - dependent smokers ( as ) and 32 healthy controls ( c ) were enrolled in the study .
the activity of -hex was measured spectrophotometrically .
results .
-hex
a% was significantly higher and -hex b% was lower in as than in c group .
we found a significant correlation between -hex a% and gingival index ( gi ) and an inverse correlation between -hex a% and salivary flow ( sf ) , in all groups .
salivary -hex a% index in smoking alcoholics at 0.23 had excellent sensitivity ( 96% ) and specificity ( 91% ) ; the auc for -hex a% was high ( 0.937 ) .
there were no correlations between amount / duration - time of alcohol drinking / smoking and -hex a% or -hex b% .
we found significant correlations between the time period of denture wearing and gi , papilla bleeding index ( pbi ) , and decayed missing filled teeth index ( dmft ) and between gi and the amount of smoked cigarettes per day .
conclusion .
bad periodontal state was most likely due to the nicotine dependence .
salivary -hex a% is a promising excellent marker for the diagnosis of periodontitis . |
osteoimmunology . an overactive immune response may initiate the onset of inflammatory bone disorders , such as rheumatoid arthritis ( ra ) and periodontitis , which can be significant sources of covert systemic inflammation . because the prevalence of ra and periodontitis increases with age
, it is important to recognize the contribution of these inflammatory bone disorders in the increasing elderly population worldwide . on the other hand ,
alzheimer 's disease ( ad ) , the most common form of dementia , is known to be the most common cause of disability in elderly subjects .
although the molecular mechanisms involved in the etiology and pathogenesis of ad have not been completely elucidated , the accumulation of -amyloid ( a ) and hyperphosphorylation of tau in the brain is the hallmarks of ad , and microglia - mediated neuroinflammation is well known to be related to the onset and progression of ad due to the release of proinflammatory mediators [ 2 , 3 ] .
interleukin-1 ( il-1 ) is the key molecule involved in the neuroinflammation observed in cases of ad , as il-1 drives the release of multiple inflammatory mediators by activated microglia , leading to a self - propagating cycle of neuroinflammation , which results in direct neurotoxicity and contributes to promoting the formation of dystrophic neurites .
it is well known that chronic systemic inflammation can alter the degree of neuroinflammation in the brain [ 5 , 6 ] .
ra and periodontitis , both chronic systemic inflammatory diseases , not only are associated with other systemic inflammatory diseases , such as atherosclerosis and diabetes , but also directly initiate or hasten the rate of progression of ad .
an increasing number of clinical studies have demonstrated the impact of ra and periodontitis on ad , and recent experimental studies have clarified the route of transduction of inflammatory signals from ra and periodontitis to the brain .
we herein review the current understanding of the link between inflammatory bone disorders and ad .
a reduced prevalence of ad was described in ra patients who were long - term users of nonsteroidal anti - inflammatory agents ( nsaids ) analyzed in a postmortem survey , and a meta - analysis including 17 epidemiological studies demonstrated that nsaid use is a protective factor for ad onset .
furthermore , a prospective study of 7,000 healthy subjects using nsaids for joint symptoms , including ra , showed that the long - term use of nsaids protects against ad .
another systematic review of multiple prospective and nonprospective studies further showed that nsaid exposure is associated with a decreased risk of ad .
more recently , an increased risk of cognitive impairment in patients with midlife ra was confirmed based on a 21-year follow - up of the association between ra or arthritis and dementia / ad in several case - control and hospital- and register - based studies , which indicates that the presence of joint disorders , especially ra , in midlife appears to be associated with a worse cognitive status later in life .
the first hypothesis of a positive link between periodontitis and ad was raised in 2008 .
proposed that periodontitis induces systemic inflammation , which stimulates the production of a and tau protein in the brain , leading to alzheimer 's neuropathology .
in addition to the effects of low - grade chronic inflammation itself , periodontitis causes or promotes other chronic systemic inflammatory diseases , including atherosclerosis , cardiovascular disease , and diabetes , indicating that periodontitis is a significant source of systemic inflammatory molecules . based on the contribution of periodontitis to systemic inflammation , and the potential role of systemic inflammation in the onset of neuroinflammation
, it is reasonable to consider that chronic periodontitis is a risk factor for the incidence and progression of ad .
there is growing clinical evidence that chronic periodontitis is closely linked to the initiation and progression of ad .
noble et al . identified a cross - sectional association between a serologic marker of a common periodontitis pathogen , porphyromonas gingivalis ( p. gingivalis ) , and poor cognitive test performance among patients older than 60 years in the third national health and nutrition examination survey ( nhanes - iii ) .
this preliminary study suggested that periodontitis is associated with cognitive impairment , especially in the elderly .
furthermore , stein et al . examined the serum levels of antibodies against periodontal bacteria in participants who were eventually diagnosed with ad in comparison with that observed in non - ad controls and found elevated levels of antibodies to periodontal bacteria years before the onset of cognitive impairment , suggesting that periodontitis potentially carries a risk of ad development and/or progression .
more recently , poole et al . assessed the presence of the major three periodontal bacteria , the so - called red complex including treponema denticola , tannerella forsythia , and p. gingivalis and/or bacterial components in the brain tissue of individuals with and without dementia .
the authors obtained statistically significant evidence of the presence of lipopolysaccharide ( lps ) from p. gingivalis in the ad cases , thus confirming that lps from periodontal bacteria can access the ad brain during life .
moreover , riviere et al . detected oral treponema in the trigeminal ganglia , brain stem , and cortex and hippocampus of ad patients using molecular and immunological techniques .
taken together , clinical evidence suggests that the chronic inflammation associated with inflammatory bone disorders may have an important role in increasing the risk of cognitive impairment in cases of ad ( figure 1 ) .
however , the exact route by which these disorders transduce systemic inflammatory signals into the brain remains unclear .
receptor activator of nuclear factor-b ligand ( rankl ) is a critical factor for the pathogenesis of inflammatory bone disorders due to its requirement for both the formation and activation of osteoclasts [ 20 , 21 ] .
for this reason , inhibition of the rankl expression represents an innovative therapeutic target for controlling osteoclast formation in inflammatory bone disorders .
the nuclear factor-b ( nf-b ) pathway is central for regulating the rankl - dependent osteoclast formation , function , and survival [ 2224 ] , which explains the inhibitory effects on osteoclast formation induced by the prevention of nf-b activation [ 22 , 25 ] .
furthermore , reactive oxygen species ( ros ) act as intracellular signaling molecules involved not only in the regulation of rankl - dependent osteoclast formation but also in the peroxidation of lipids and oxidative damage to proteins and dna [ 26 , 27 ] as well as rankl in turn to increase the ros levels .
it has been identified that a decrease in the ros levels results in a reduction in osteoclast formation and attenuation of bone destruction , as found in both in vitro and in vivo models .
this raises the possibility that antioxidants may be therapeutic targets for the treatment of inflammatory bone disorders .
oxidative stress - induced cell damage is the major component of harmful cascades activated in the development of aging - related neurodegenerative disorders , including ad [ 2830 ] .
numerous reports have provided the direct morphological and biochemical evidence indicating a connection between oxidative stress and cell death in the brain of patients with ad [ 3133 ] .
furthermore , ros accumulate unfolded or misfolded proteins in the ad brain , and increasing evidence supports the role of ros in the pathogenesis of ad , as a oligomers directly generate hydrogen peroxide . moreover , the overproduction of ros under condition of oxidative stress acts as a second messenger in signal transduction cascades leading to the activation of mitogen - activated protein kinases ( mapk ) , as the intracellular redox state of cells regulates cellular signaling pathways [ 36 , 37 ] .
the major mapk subfamilies , c - jun n - terminal kinase , p38 , and extracellular signal - regulated kinase 1/2 are known to be cell death factors produced in response to oxidative stress [ 38 , 39 ] .
indeed , the levels of phosphorylated mapks are increased in the postmortem brains of ad patients [ 4042 ] .
therefore , antioxidant therapy is considered to be a promising approach for the prevention and clinical management of ad [ 43 , 44 ] .
the modulation of endogenous cellular defense mechanisms represents an innovative approach to providing therapeutic interventions for diseases causing chronic tissue damage , including inflammatory bone disorders as well as ad .
cellular stress response consists of prosurvival pathways controlled by cytoprotective genes called vitagenes that stimulate the production of molecules endowed with antioxidant and antiapoptotic potential .
vitagenes include members of the heat shock protein ( hsp ) family , such as heme oxygenase-1 and hsp72 , sirtuins , and the thioredoxin / thioredoxin reductase system [ 47 , 48 ] .
increasing evidence suggests that the hsps promotes cytoprotective conditions in the human disease states , such as chronic inflammation , bone diseases , and ad , as thoroughly reviewed by calabrese et al .
furthermore , previous studies have indicated that sirtuin 1 is a potent inhibitor of nf-b transcription .
bone is sensitive to an overactive immune responses , which are mediated by macrophages , dendritic cells , and t cells , for diverting proinflammatory mediators in the bone microenvironment . in particular ,
the dramatic impact of proinflammatory mediators on bone cells , including osteoclasts and osteoblasts , is a key component of the pathogenesis of inflammatory bone disorders .
il-1 is considered to be a critical inducer of the pathogenesis and tissue damage observed in cases of inflammatory bone disorders , including ra and periodontitis , as il-1 impairs the migration of osteoblasts and upregulates the rankl expression induced by osteocytes .
furthermore , il-1 is used as a biomarker to assess the therapeutic outcomes of patients with chronic periodontitis .
therefore , il-1 has become a focus of research due its potential as an attractive therapeutic target for inflammatory bone disorders .
the processing and secretion of il-1 are tightly regulated by a two - step mechanism .
the first step consists of the transcription and production of pro - il-1 which depends on the activation of nf-b by toll - like receptors ( tlrs ) , and the second step consists of the activation of inflammasomes , which in turn activate procaspase-1 .
inflammasomes are multiprotein oligomers consisting of the nod - like receptor ( nlr ) family , including the pyrin domain - containing 3 ( nlrp3 ) , and the non - nlr family proteins , melanoma 2 receptor , and procaspase-1 . upon activation ,
the nlrp3 inflammasome binds to an adaptor protein , asc , which in turn recruits procaspase-1 for autoactivation .
finally , caspase-1 cleaves pro - il-1 , which is then secreted in a mature form .
recent studies have provided evidence that the nlrp3 inflammasome is involved in the pathogenesis of both ra and chronic periodontitis .
showed that the negative regulation of nlrp3 inflammasome activation induced by a20 markedly protects against the onset of ra - associated inflammation and cartilage destruction , highlighting the contribution of the nlrp3 inflammasome to the pathology of ra .
found significantly higher levels of the nlrp3 inflammasome and caspase-1 in the gingival tissues of patients with chronic periodontitis compared to that observed in healthy controls .
furthermore , the activation of both the nlrp3 and aim2 inflammasomes is necessary for il-1 secretion after stimulation with p. gingivalis , the main bacteria that induces periodontitis .
peripheral nervous fiber sprouting is observed in inflammatory bone disorders and considered to be associated with pain , the major symptom of inflammatory bone disorders .
however , our previous studies using an animal model of ra , adjuvant arthritic ( aa ) rats , showed that sprouting of peripheral sensory fibers is closely linked with bone remodeling and local inflammatory processes .
infiltrated macrophages and cd4 t cells produce nerve growth factor ( ngf ) and express its high - affinity receptor , trka , in the inflamed synovial tissues .
however , a dense network of its low - affinity receptor , p75-positive nerve fibers , with numerous terminal varicosity , is also observed .
these findings suggest that infiltrating mononuclear cells secrete ngf in an autocrine or paracrine manner in the inflamed synovium to promote sensory nerve fiber sprouting , as most of new sprouting gap-43-positive fibers are calcitonin gene related peptide ( cgrp)-positive sensory fibers located around osteophytes
. however , the surgical reduction of cgrp - positive sensory fibers via resection of the sciatic nerves prior to adjuvant injection suppresses the size of osteophytes and delays the recovery of inflammation due to the effects of prolonged infiltration of th1 cells in the synovial tissues in aa rats .
these observations suggest the involvement of sporting sensory fibers in the bone remodeling and local inflammatory processes observed in inflammatory bone disorders .
more recently , the function of sprouting sympathetic fibers in inflammatory bone disorders has also been noted .
longo et al . showed that the sprouting of sympathetic fibers into the upper dermis of the skin , which results from increases in the mature ngf levels in the skin following joint and bone damage , is present in aa rats .
the pharmacological suppression of the sympathetic fiber function with systemic guanethidine significantly decreases the pain - related behavior associated with arthritis in these animals , thus suggesting that sprouting sympathetic fibers contribute to the pain - related behavior associated with inflammatory bone disorders .
furthermore , the dense sympathetic innervation of joints suggests the importance of this phenomenon in the onset of inflammatory bone disorders , as the sympathetic system controls the blood flow in the joint as well as the degree of vascular permeability and thus influences the extent of inflammation , as immune cells exhibit adrenergic signaling pathways .
therefore , the sprouting of sympathetic fibers may have local effects on osseous tissues in addition to effects on the local and systemic immune functions in cases of inflammatory bone disorders .
taken together , these findings indicate that inflammatory bone disorders are a source of chronic systemic inflammation .
it is well known that chronic systemic inflammation has causal links to neuroinflammation via the actons of systemically released proinflammatory mediators including il-1 and that microglia are the primary brain cells responding to systemic inflammation [ 5 , 6 ] .
repeated lps - induced chronic systemic inflammation in mice results in prolonged il-1 production as well as microglial activation in the brain .
furthermore , mice subjected to systemic inflammatory challenges in late gestation are predisposed to develop ad - like neuropathology during the course of aging .
these mice also display chronic elevation of il-1 , an increased expression of hippocampal amyloid precursor protein and its proteolytic fragments and enhanced tau phosphorylation , thus resulting in a significant impairment of working memory in old age .
more importantly , this phenotype is strongly exacerbated when the prenatal infection is followed by a second systemic inflammatory challenge in adulthood , further suggesting that systemic inflammation represents a major risk factor for the development of ad .
several routes by which systemic immune signals may be transmitted to the brain have been studied intensively [ 5 , 6 ] .
first , the direct pathway involves the circumventricular organs , specialized regions lacking a contiguous blood - brain barrier . in circumventricular organs , pathogen - associated molecular patterns
induce the production and release of proinflammatory mediators stimulated by macrophage - like cells expressing tlrs .
a second route involves the il-1 receptors located on the perivascular macrophages and endothelial cells of brain capillaries .
the activation of il-1 receptors by circulating mediators initiates the release of cytokines into the brain , without the physical entry of constituents across the blood - brain barrier .
a third route comprises the overflow of cytokine transporters into the systemic circulation , which then allows cytokines to gain access to the brain through these transport systems .
a fourth route involves the transmission of systemic immune signals via the autonomic nervous system .
systemic cytokines directly stimulate primary afferent nerves , such as the vagus nerve , which in turn activate central pathways involved in sickness behavior .
in addition to these four classical routes , we further found that a leptomeningeal pathway may be involved .
the leptomeninges covering the surface of the brain parenchyma provide a physical boundary at the cerebrospinal fluid - blood barrier .
the activation of leptomeningeal cells by circulating cytokines induces the production and release of proinflammatory cytokines into the brain [ 64 , 65 ]
. therefore , leptomeningeal cells are able to transmit signals from systemic immune cells into the brain - resident microglia ( figure 1 ) .
it is interesting to note that inflammatory bone disorders induce age - dependent differential responses in microglia .
using a model of ra , rat aa , we found that activated microglia in the proximity of the leptomeninges produce anti - inflammatory cytokines , such as il-10 and transforming growth factor-1 ( tgf-1 ) , in young adult aa rats [ 6 , 66 , 67 ] .
in contrast , activated microglia in close proximity to the leptomeninges in middle - aged aa rats produce il-1 and exhibited an increased expression of prostaglandin e2 ( pge2 ) synthesizing enzymes , such as cyclooxygenase-2 and microsomal prostaglandin synthase-1 . in cultured leptomeningeal cells , il-1 and pge2 , respectively , caused the marked loss of occludin and zo-1 , two major tight junction proteins : pretreatment with il-10 and tgf-1 significantly antagonizes these effects .
therefore , chronic systemic inflammation induces age - dependent phenotypic changes in microglia , yielding an anti - inflammatory cell phenotype in young rats and a proinflammatory cell phenotype in middle - aged rats .
furthermore , age strongly influences the barrier functions of the leptomeninges as a result of the age - dependent differential microglial responses in the setting of inflammatory bone disorders .
these observations prompted further investigation of the functional outcomes of the resultant differential microglial phenotypic changes noted under condition of chronic systemic inflammation .
we therefore examined the effects of chronic systemic inflammation on long - term potentiation ( ltp ) in the hippocampus in young adult and middle - aged rats , as ltp is the cellular substrate for learning and memory .
consequently , the incidence of ltp in the schaffer collateral - ca1 synapses was not affected in the young adult aa rats , whereas the formation of hippocampal ltp was significantly impaired in the middle - aged aa rats .
the systemic administration of minocycline , a known inhibitor of microglial activation , significantly restored the magnitude of ltp in middle - aged aa rats .
these observations suggest that chronic systemic inflammation induces deficits in the hippocampal ltp in middle - aged rats via the effects of neuroinflammation , which is primarily induced by brain - resident microglia .
therefore , it is considered that microglia may be primed during aging , even by middle age .
primed microglia are hyperresponsive to secondary stimuli and can thus produce an exaggerated inflammatory response in the brain .
it is also considered that age - dependent autophagic and lysosomal dysfunction allows for the dominance of ros - hypergenerating older mitochondria in microglia .
the increased levels of intracellular ros , in turn , activate redox - sensitive transcription factors , such as nf-b , to provoke an exaggerated inflammatory response . therefore , increased oxidative stress and the resultant activation of redox - sensitive transcription factors observed during aging may drive the emergence of senescent - type microglia ( microglia aging ) .
this may explain why a , which is not capable of sufficiently activating nf-b , is able to induce the secretion of il-1 by activated microglia isolated from the aged mouse brain but can not induce il-1 secretion from the young adult mouse brain .
it is important to note that the inflammatory bone disorders ra and periodontitis are generally found in the middle - aged and older populations , thus further indicting the risk association between inflammatory bone disorders and ad .
latz 's group has proposed a model for the activation of the nlr family , nlrp3 inflammasomes . according to this model
, the phagocytosis of various molecules , including fibrillar a42 and silica crystals , by lps - primed microglia / macrophages causes phagosomal destabilization and lysosomal rupture .
the subsequent secretion of catb into the cytoplasm triggers the activation of the nlrp3 inflammasome directly or indirectly , thereby leading to the production and secretion of mature il-1 [ 66 , 67 ] .
recently , catb was found to directly interact with the leucine - rich - repeat domain of nlrp3 .
after activation , the nlrp3 inflammasome mediates procaspase-1 activation to promote the processing and secretion of il-1. therefore , caspase-1 is an essential enzyme for il-1 production , as it is required for the processing of inactive precursors into mature , active forms that can then be secreted from cells ( figure 1 ) .
latz 's group also recently reported that nlrp3-deficient mice carrying mutations associated with familial ad show improvements in spatial memory deficits , reductions in the expression of caspase-1 and il-1 in the brain , and enhanced a clearance .
these observations suggest that a activates microglia surrounding plaques to induce il-1 production via the nlrp3-catb pathway .
in addition to a , cga , a neurosecretory acidic glycoprotein , is found in 3040% of ad neuritic plaques , and cga - positive plaques are surrounded more frequently by hyperactivated microglia in comparison to that observed in the case of a-positive neuritic plaques .
more importantly , cga alone is capable of inducing il-1 production by microglia , whereas a induces the il-1 production only by lps - primed or senescent - type microglia .
there is evidence suggesting that catb is associated with the maturation of pro - il-1 in the endosomal / lysosomal system , as catb can effectively cleave procaspase-1 in a cell - free system only at an acidic ph [ 75 , 76 ] .
we recently demonstrated that catb and caspase-1 are colocalized and that ca-074me markedly inhibits the caspase-1 expression in the cga - induced proteolytic processing of procaspase-1 to its mature form in the lysosome - related vesicles of microglia , which contain inactive forms of il-1 [ 71 , 77 ] .
furthermore , there are no signs of any leakage of catb in microglia following treatment with cga .
the typical size of primary lysosomes is less than 1 m in diameter , whereas the mean diameter of catb - containing enlarged lysosomes in cga - stimulated microglia is 4.2 m . these findings are consistent with previous observations showing that il-1 and catb are colocalized in phagolysosomes and that the secretion of il-1 involves the exocytosis of phagolysosomes in lps - activated human monocytes [ 77 , 78 ] ( figure 1 ) .
however , the possibility that catb is indirectly involved in the activation of caspase-1 via the proteolytic maturation of caspase-11 can not be totally ruled out , as caspase-11 can activate procaspase-1 .
furthermore , cga leaked from damaged neurons also activates microglia surrounding plaque to induce il-1 production at an earlier age than fibrillar a via the phagolysosome - catb pathway .
our preliminary experiments showed that aa and chronic systemic treatment with p. gingivalis lps induces the intracellular accumulation of a and cga in hippocampal pyramidal neurons , leading to memory impairment in middle - aged animals ( wu et al .
, unpublished observations , figure 1 ) . however , further studies are needed to clarify the mechanism underlying the accumulation of a and cga in presence of inflammatory bone disorders .
it is noteworthy that the il-1 secreted from activated microglia further accelerates tangle formation in cortical neurons via tau hyperphosphorylation , thus indicating that activated microglia may also play important roles in tau pathology in ad .
recent evidence indicates that inflammatory bone disorders are potential risk factors for ad . in individuals with chronic inflammatory bone disorders , proinflammatory blood cells and bacterial components , including lps ,
activate the receptors localized on the surface of leptomeningeal cells , which in turn activate brain - resident microglia to evoke neuroinflammation ( figure 1 ) .
the intense neuroinflammation evoked by senescent - type microglia may contribute to the initiation and progression of ad , resulting in cognitive impairment .
therefore , providing early treatment of inflammatory bone disorders may delay the onset and limit the severity and/or progression of ad .
more importantly , microglia undergo several morphological and functional changes involving the induction of exaggerated neuroinflammation in response to systemic inflammation during normal aging . therefore
, pharmacological approaches aimed at rejuvenating senescent - type microglia may also constitute a promising avenue for future research to reduce the risk of ad . | bone is sensitive to overactive immune responses , which initiate the onset of inflammatory bone disorders , such as rheumatoid arthritis and periodontitis , resulting in a significant systemic inflammatory response . on the other hand ,
neuroinflammation is strongly implicated in alzheimer 's disease ( ad ) , which can be enhanced by systemic inflammation , such as that due to cardiovascular disease and diabetes .
there is growing clinical evidence supporting the concept that rheumatoid arthritis and periodontitis are positively linked to ad , suggesting that inflammatory bone disorders are risk factors for this condition .
recent studies have suggested that leptomeningeal cells play an important role in transducing systemic inflammatory signals to brain - resident microglia .
more importantly , senescent - type , but not juvenile - type , microglia provoke neuroinflammation in response to systemic inflammation .
because the prevalence of rheumatoid arthritis and periodontitis increases with age , inflammatory bone disorders may be significant sources of covert systemic inflammation among elderly people .
the present review article highlights our current understanding of the link between inflammatory bone disorders and ad with a special focus on microglia aging . |
treatment of adhesions can be expensive , and in some cases , life - threatening . in spite of the large number of surgical operations performed daily , the mechanism of peritoneal adhesion is not well understood .
previous reports have shown that peritoneal injury is triggered by leakage of plasma proteins , followed by formation of fibrinous deposits and proliferation of fibroblasts .
rapid and transient influx of neutrophils into the peritoneal cavity also occurs , followed by the accumulation of mononuclear cells , usually macrophages . cd4
t cells and the t cell - derived proinflammatory cytokine interleukin ( il)-17 also play significant roles in peritoneal adhesion .
we previously found that peritoneal macrophages ( pmf ) form a large aggregated mass that packs the serosal side of the perforating ulcer with adhesion of visceral tissues in a colitis model in mice .
transcripts encoding chemokine c - c motif receptor 8 ( ccr8 ) were upregulated in the aggregating cells , while other chemokine receptors were downregulated compared with nave pmf . of note ,
chemokine c - c motif ligand 1 ( ccl1 ) , the ligand for ccr8 , was also produced by macrophages and mesothelial cells in response to inflammatory stimuli .
the presence of ccl1-induced formation of cell aggregates by pmf and mesothelial cells in vitro , and neutralization of ccl1 prevented formation of peritoneal membranous adhesion in vivo .
thus , autoactivation of peritoneal macrophages via chemokines was the initial trigger for peritoneal adhesion .
these results from the mouse system prompted us to investigate similar chemokine-/cytokine - driven mechanisms of peritoneal adhesion in humans . in this study , we collected peritoneal lavage fluid at the beginning and the end of laparotomy , and quantified the presence of 27 cytokines and chemokines in an effort to identify potential molecules that could be targeted to prevent adhesion formation in humans .
this study was performed with approval from the ethics committees of the national center for global health and medicine and jichi medical university .
seven patients ( table 1 ) who had laparotomy for colorectal resection of colorectal cancer were recruited in this study with informed consent .
these patients had not had a previous major laparotomy , and none of them suffered from inflammatory bowel disease .
immediately after incision at the beginning of the operation ( preoperative ) and before closure at the end ( postoperative ) , 1 l of saline was poured into the peritoneal cavity , and as much fluid as possible was aspirated back into a bottle .
the recovered fluid volume was approximately 777934 ml [ 857 76 ml , mean standard deviation ( sd ) ] .
after removal of visible fat debris , exudate cells were separated by centrifugation at 600 g. supernatant aliquots were maintained at 80c until use , and were subjected to cytokine assays using the bio - plex suspension array system ( bio - rad japan , tokyo , japan ) .
the assay tested for the presence of il- , il-1 receptor antagonist ( il-1ra ) , il-2 , il-4 , il-5 , il-6 , il-7 , il-9 , il-10 , il-12 ( p70 ) , il-13 , il-15 , il-17 , interferon ( ifn)- , tumor necrosis factor ( tnf)- , basic fibroblast growth factor ( fgf basic ) , granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage colony - stimulating factor ( gm - csf ) , platelet - derived growth factor ( pdgf)-bb , vascular endothelial growth factor ( vegf ) , eotaxin ( ccl11 ) , ip-10 [ chemokine c - x - c motif ligand 10 ( cxcl10 ) ] , mcp-1 ( ccl2 ) , mip1 ( ccl3 ) , mip1 ( ccl4 ) , rantes ( ccl5 ) , and il-8 ( cxcl8 ) .
preoperative peritoneal exudate cells from five patients ( cases 13 , 6 , and 7 ) were cultured in a 24-well plate at 5.8 10 cells / ml per well , with or without 100 ng / ml lipopolysaccharide ( lps ; from salmonella minnesota , sigma - aldrich , st .
louis , mo , usa ) for 20 h. supernatant of duplicated cultures was harvested and subjected to the assay described above . peripheral blood mononuclear cells ( pbmcs ) obtained from a healthy volunteer using ficoll - paque plus ( ge healthcare japan , tokyo , japan )
were prestained for 30 min at 37c with 3 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetomethyl ester ( bcecf - am , molecular probes , eugene , or , usa ) and then suspended at 2 10 cells / ml in hank 's balanced saline solution containing 0.5% bovine serum albumin and 20 mm hepes .
chemotaxis assay was performed using a chemo tx-96 chemotaxis plate ( neuroprobe , inc . ,
after washing , 65 l of cell suspension was loaded onto the membrane plate and placed onto a flat - bottomed microtiter plate with 96 wells containing 30 l of recombinant ccl5 ( r & d systems , minneapolis , mn , usa ) solution in each well or peritoneal lavage fluid diluted with phosphate buffered saline ( 1:1 ) .
the plate was then incubated at 37c for 120 min , and cells that had undergone migration were collected .
these collected cells were lysed with 0.1% triton x-100 and counted using a fluorescence microplate reader ( flexstation , molecular device japan , tokyo , japan ) .
anti - human ccl5 and anti - human ccl2 antibodies were purchased from r & d systems .
the results were statistically analyzed by the two - tailed paired t - test using prism 4 software ( graphpad software , inc .
, lajolla , ca , usa ) . when pvalues were less than 0.05 , the results were considered as a significant difference .
this study was performed with approval from the ethics committees of the national center for global health and medicine and jichi medical university .
seven patients ( table 1 ) who had laparotomy for colorectal resection of colorectal cancer were recruited in this study with informed consent .
these patients had not had a previous major laparotomy , and none of them suffered from inflammatory bowel disease .
immediately after incision at the beginning of the operation ( preoperative ) and before closure at the end ( postoperative ) , 1 l of saline was poured into the peritoneal cavity , and as much fluid as possible was aspirated back into a bottle .
the recovered fluid volume was approximately 777934 ml [ 857 76 ml , mean standard deviation ( sd ) ] .
after removal of visible fat debris , exudate cells were separated by centrifugation at 600 g. supernatant aliquots were maintained at 80c until use , and were subjected to cytokine assays using the bio - plex suspension array system ( bio - rad japan , tokyo , japan ) .
the assay tested for the presence of il- , il-1 receptor antagonist ( il-1ra ) , il-2 , il-4 , il-5 , il-6 , il-7 , il-9 , il-10 , il-12 ( p70 ) , il-13 , il-15 , il-17 , interferon ( ifn)- , tumor necrosis factor ( tnf)- , basic fibroblast growth factor ( fgf basic ) , granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage colony - stimulating factor ( gm - csf ) , platelet - derived growth factor ( pdgf)-bb , vascular endothelial growth factor ( vegf ) , eotaxin ( ccl11 ) , ip-10 [ chemokine c - x - c motif ligand 10 ( cxcl10 ) ] , mcp-1 ( ccl2 ) , mip1 ( ccl3 ) , mip1 ( ccl4 ) , rantes ( ccl5 ) , and il-8 ( cxcl8 ) .
preoperative peritoneal exudate cells from five patients ( cases 13 , 6 , and 7 ) were cultured in a 24-well plate at 5.8 10 cells / ml per well , with or without 100 ng / ml lipopolysaccharide ( lps ; from salmonella minnesota , sigma - aldrich , st .
louis , mo , usa ) for 20 h. supernatant of duplicated cultures was harvested and subjected to the assay described above .
peripheral blood mononuclear cells ( pbmcs ) obtained from a healthy volunteer using ficoll - paque plus ( ge healthcare japan , tokyo , japan ) were prestained for 30 min at 37c with 3 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetomethyl ester ( bcecf - am , molecular probes , eugene , or , usa ) and then suspended at 2 10 cells / ml in hank 's balanced saline solution containing 0.5% bovine serum albumin and 20 mm hepes .
chemotaxis assay was performed using a chemo tx-96 chemotaxis plate ( neuroprobe , inc . ,
after washing , 65 l of cell suspension was loaded onto the membrane plate and placed onto a flat - bottomed microtiter plate with 96 wells containing 30 l of recombinant ccl5 ( r & d systems , minneapolis , mn , usa ) solution in each well or peritoneal lavage fluid diluted with phosphate buffered saline ( 1:1 ) .
the plate was then incubated at 37c for 120 min , and cells that had undergone migration were collected .
these collected cells were lysed with 0.1% triton x-100 and counted using a fluorescence microplate reader ( flexstation , molecular device japan , tokyo , japan ) .
anti - human ccl5 and anti - human ccl2 antibodies were purchased from r & d systems .
the results were statistically analyzed by the two - tailed paired t - test using prism 4 software ( graphpad software , inc . ,
when pvalues were less than 0.05 , the results were considered as a significant difference .
we measured the amounts of various cytokines and chemokines in the peritoneal fluid from patients prior to and following a colorectal operation in an effort to identify molecules that may be involved in the formation of adhesions in the gut .
many molecules were drastically increased in the peritoneal lavage fluid after the laparotomy procedure ( table 2 ) .
the concentration of il-6 was increased 11-fold , while that of il-1 , pdgf , and il-1ra showed more than a fivefold increase .
furthermore , ifn- , il-13 , il-4 , il-10 , and vegf were increased more than twofold in postoperative samples .
when the percent composition of each cytokine in the total sample was evaluated , we observed that fgf basic , gm - csf , and il-6 were dominant in the preoperative samples . however , only il-6 increased to account for more than 50% of the measured cytokines in the postoperative samples ( figure 1a ) . for the chemokine response , we observed that ccl5 , ccl3 , ccl2 , cxcl8 , and ccl4 were increased after the operation .
however , in terms of absolute amount of protein , ccl5 , ccl2 , cxcl8 , and ccl4 were the major secreted chemokines .
as for percent composition , increases in ccl5 were the most obvious , while the relative amount of other chemokines did not significantly change , except for cxcl10 ( figure 1b ) .
one patient , case 5 in table 1 , who underwent lobectomy of the liver with relatively long operation time was included in our study .
since this case might have different conditions of the peritoneal cavity from other cases , we reviewed individual data .
however , this patient did not show a particular secretion pattern of ccl5 , il-6 , or other cytokines and chemokines when compared with other cases ( figure 1c ) .
since we had found that the ccl1 / ccr8 system is critical in the formation of peritoneal adhesions in mice , we attempted to measure the amount of ccl1 present in the samples , but the concentration in the original peritoneal lavage fluid was less than the sensitivity of the assay .
we detected 10 pg / ml ccl1 after removal of albumin , with a 10-fold greater concentration of the fluid in the preoperative sample from one individual , but this patient had severe peritoneal adhesion and was therefore excluded from this study .
concentration of cytokines and chemokines ( pg / ml ) in peritoneal lavage fluid data are shown as an average of seven cases with a standard deviation . statistically significant difference ( p < 0.05 ) between pre- and postoperative samples .
the majority of cells harvested from the peritoneal lavage fluid prior to the operation were cd33cd14 macrophage - type cells ( data not shown ) and lymphocytes as reported previously . in order to know how these cells respond to inflammatory stress that possibly occurs during colorectal operation , we tested if the treatment of these cells with lps , a potent activator of macrophage type cells , would enhance the chemokine / cytokine response .
as expected , secretion of inflammatory cytokines , which were upregulated during operation including tnf- and il-6 , were significantly augmented in the presence of lps ( figure 2 ) . in spite of the striking increase in ccl5 in the postoperative lavage samples , the amount secreted by cultured exudate cells was low and
further , to support the inflammatory process that may be mediated by ccl5 in the peritoneal cavity , we tested pbmcs if they have chemotaxis potential in response to ccl5 .
as shown in figure 3 , recombinant ccl5 as well as peritoneal fluid was able to induce chemotaxis of pbmcs .
anti - ccl5 antibody but not anti - ccl2 antibody partially inhibited the chemotaxis induced by peritoneal lavage fluid .
relative amount of measured cytokines ( a ) and chemokines ( b ) listed in table 1 .
percentage was calculated by dividing the concentration of cytokines or chemokines ( pg / ml ) with sum of the concentrations ( total concentration ) of measured cytokines or chemokines ( pg / ml ) .
the case number indicates the patient i d in table 1 ( color figure available online ) .
peritoneal exudate cells harvested from preoperative lavage fluid were cultured with ( lps ) or without lps ( none ) .
culture supernatant was subjected to the assay for tnf- ( a ) , il-6 ( b ) , and ccl5 ( c ) .
this study reports the comprehensive analysis of the secretion of cytokines and chemokines in the peritoneal cavity during laparotomy .
prior studies have investigated individual cytokines in peritoneal fluid after operation , but the total composition of cytokines present in the peritoneal cavity before and after operation has not been addressed until now .
importantly , assaying both the pre- and postoperative lavage fluid from the same individuals enabled us to quantify the cytokine levels under conditions close to the nave status of each patient , as well as quantify the net secretion during the operation .
although the stage of the disease in the tested cases varied , the result was quite constant .
for example , il-1 , il-6 , il-10 , il-12 , il-13 , il-15 , and il-17 were detected in the preoperative fluid . in combination with the high amount of gm - csf , this array of cytokines may work to maintain the steady - state nature of the peritoneal cavity and its resident cells .
after operation , increases in il-6 were the most striking , though those of il-13 and il-15 were also statistically significant .
a previous study reported that adhesion formation 48 h after laparoscopy was associated with a high concentration of il-6 .
though most studies have not addressed il-13 and il-15 under these conditions , they are most likely involved in the response to surgical stress in the peritoneal cavity .
since il-15 is known to activate t cells , which are involved in adhesion formation , it is possible that it could trigger the reaction of these cells to induce further immune responses .
il-13 may induce alternative ( m2 type ) activation of macrophages , and m2 type macrophages have reduced abilities to stimulate t cell proliferation and th1 differentiation , with the production of large amounts of il-10 . therefore , the balance of il-15 and il-13 may be critical for avoiding excess immune reactions . in the mouse ,
il-17 is associated with adhesion formation ; however , we found that in humans , the amount of il-17 either decreased or remained unchanged after the operation .
it is possible that il-17 may not play a role in this process until a later time .
the amount of ifn- was not very high in the peritoneal fluid prior to the operation , but the fact that it increased by 3.2-fold following laparotomy may support the report that recommends ifn- as a target for prevention of adhesion in a mouse model . although differences were not statistically significant , elevation of il-1ra and il-10 may be important , as both have antagonizing effects on the inflammatory response .
( a ) chemotaxis of pbmcs induced by recombinant ccl5 was measured in the presence of indicated concentrations of anti - ccl5 antibody ( mg / ml ) .
( b ) pre- ( blank ) and postoperative ( solid ) peritoneal fluid from case 1 was tested for the chemotactic potential of human pbmcs in the presence of 0.1mg / ml of anti - ccl5 antibody or anti - ccl2 antibody .
ccl5 is a chemoattractant for t cells , monocytes / macrophages , eosinophils , basophils , natural killer cells , granulocytes , and dendritic cells , and therefore induce cascade reactions following immune responses in the peritoneal cavity together with ccl3 , ccl4 , and cxcl8 , eventually leading to peritoneal adhesion .
the exudate cells did not produce a significant amount of ccl5 during the overnight culture in the presence of lps .
this result suggests that peritoneal exudate cells may be not the source of ccl5 during operation .
indeed , human peritoneal mesothelial cells are reported to produce ccl5 in response to il-1 or tnf- , which results in significant up - regulation of ccl2 and ccl5 protein secretion .
these results indicate it is possible that ccl5 levels present in a sample may be indicative of mesothelial cell injury .
in our previous study using a mouse model , inhibition of macrophage migration and aggregation was an effective way of preventing peritoneal adhesion . in mice
, we found approximately 0.51.5ng / ml ccl1 in 2ml peritoneal lavage fluid in mice with peritoneal adhesion , which was undetectable in our elisa system in the peritoneal cavities of nave mice .
this indicated that around 0.09 ng / g body weight ( mouse body weight was estimated as 22 g ) of ccl1 was secreted in mice when adhesion was induced .
in contrast , in humans , only 0.00017 ng / g ( body weight was estimated as 60 kg ) of ccl1 was detected as mentioned above .
although we failed to detect a significant amount of ccl1 during the operation in humans , which needs further study , we did observe dramatic changes in ccl5 instead .
ccl5 was produced at the level of 0.04 ng / g body weight in humans after laparotomy , which was comparable to the level of ccl1 in mice .
thus , we postulate that ccl5 may be a component of a chemokine system unique to the human peritoneal cavity .
importantly , both ccl1 in mice and ccl5 in humans are primarily produced by mesothelial cells .
ccl5 induces recruitment of a wide range of cells including monocytes , t cells , eosinophils , mast cells , and basophils to sites of inflammation .
indeed , we confirmed the chemoattractant activity of peritoneal lavage fluid , which was partially mediated by ccl5 .
in addition to its chemotactic activity , ccl5 promotes angiogenesis and cancer metastasis , which may be related to the formation of postoperative adhesion .
in addition , increase of ccl5 may promote postoperative cancer cell dissemination in the peritoneal cavity .
since major cytokines in the peritoneal cavity are produced by exudate cells , blocking their migration to mesothelial cells may be an effective way to prevent peritoneal adhesion .
however , it is necessary to only block molecules that function specifically in peritoneal exudate and mesothelial cells , without affecting systemic immunity against infection , tissue repair , or coagulation and fibrinolytic systems . as such , inhibition of chemokines that specifically recruit immune cells
although further investigation is required , we propose that targeting ccl5 may prevent postoperative peritoneal adhesion as well as cancer metastasis .
during laparotomy , acute secretion of chemokines and cytokines , which are involved in the recruitment and activation of macrophages , was induced .
increase of ccl5 is the major primary response to surgical stress in the peritoneal cavity and is possibly involved in the mechanism of postoperative adhesion in humans . | we recently found that chemokine - driven peritoneal cell aggregation is the primary mechanism of postoperative adhesion in a mouse model . to investigate this in humans , paired
samples of peritoneal lavage fluid were obtained from seven patients immediately after incision ( preoperative ) and before closure ( postoperative ) , and were assayed for the presence of 27 cytokines and chemokines using multiplex beads assay . as a result , il-6 and
ccl5 showed the most striking increase during operation .
recombinant ccl5 or lavage fluid induced chemotaxis of human peripheral blood mononuclear cells . we propose that ccl5 is possibly involved in the mechanism of postoperative adhesion in humans . |
the cell cycle is a fundamental cellular process that governs the ability of cells to divide .
control of the cell cycle is crucial for the generation of tissues from dividing stem cells , such as the development of the nervous system .
exit from the cell cycle is associated with the control of differentiation because differentiated cells tend to be postmitotic .
re - entry of differentiated neurons into the cell cycle leads to apoptosis ( folch et al . ,
2012 ) , although there are some examples where the cells have been shown to differentiate even if cell - cycle exit is prevented ( lobjois et al . , 2008 ) .
recently , not only cell - cycle exit , but also the length of the cell cycle , in particular the g1 phase length , has been associated with the decision of cells to differentiate . during mouse
cortical development , elongation of the g1 phase by inhibiting the g1 cyclin - dependent kinases ( cdk4/6 ) promotes neurogenesis , whereas shortening of g1 by overexpressing g1 kinase cdk4/cyd1 promotes proliferative divisions ( lange and calegari , 2010 ; lange et al . , 2009 ) .
a correlation between cell cycle / g1 length and the propensity for differentiation has also been documented in embryonic stem cells ( roccio et al . , 2013 ; coronado et al . , 2013 ) and neural progenitors in the chicken spinal cord ( wilcock et al . , 2007 ) .
apart from g1 , the other two phases of interphase , g2 and s , have also been linked to neuronal differentiation .
expanding progenitors in the mouse cortex have been shown to have a longer s phase ( arai et al . , 2011 ) and
elongation of the g2 phase has also been shown to promote progenitor proliferation ( peco et al . , 2012 ) , although in the latter case , the effect has been linked back to the shortening of the g1 phase .
this is not surprising because g1 is the key cell - cycle phase where both intrinsic and extrinsic signaling pathways impinge to instruct the cell whether to go for another round of division or differentiate ( hindley and philpott , 2012 ) . why do some progenitors have a longer cell - cycle / g1 phase ?
an intriguing observation is that in the cortex , progenitors with a longer g1 phase are nonpolar ( basal progenitors ) whereas progenitors with shorter g1 are apicobasally polarized ( apical progenitors ; arai et al . , 2011 ) .
it is also known that in many systems such as drosophila neuroblasts , and mouse , xenopus , and chicken neuroepithelium , polarized progenitors have a lower propensity to differentiate than their nonpolar daughter cells ( sabherwal and papalopulu , 2012 ) .
these results point to a link between polarity and the cell cycle , however , a direct mechanistic link between these two biological processes is not known .
any such mechanism is likely to involve key regulators of cell - cycle progression , such as the cyclin / cyclin - dependent kinase ( cy / cdk ) complexes and their inhibitors ( cyclin - dependent kinase inhibitors / cdkis ) .
inhibition of cdk activity by stronger association with cdkis has been shown to trigger neuronal differentiation ( kranenburg et al . , 1995 ) .
conversely , loss of cdkis has been shown to inhibit differentiation ( carruthers et al .
, 2003 ; mairet - coello et al . , 2012 ; vernon et al . ,
, we have used the neuroectoderm of the xenopus embryo to investigate how the apicobasal polarization of neural progenitor cells affects cell - cycle kinetics and consequently , neuronal differentiation .
xenopus neuroectoderm exhibits a clean segregation of polarized and nonpolar progenitors into two distinct layers and provides a simple and accessible model for answering such fundamental questions . in xenopus ,
deep ( inner ) nonpolar cells have a high propensity to differentiate , whereas superficial ( outer ) apicobasally polarized neuroepithelial cells are intrinsically resistant to primary neuronal differentiation ( chalmers et al . , 2002 ) .
using this system , we have previously shown that the key regulator of polarity , the atypical ( serine - threonine ) protein kinase c ( apkc ) has an instructive role in promoting proliferation and suppressing differentiation in polarized progenitors ( sabherwal et al . , 2009 ) .
in this paper , we show that , first , the cell - cycle kinetics of polarized and nonpolar neuroectodermal cells differ significantly , with polarized cells having shorter total cell - cycle length and shorter s and g1 phases than nonpolar cells .
overexpression of p27xic1 , a member of the cip / kip cdki family , elongates the g1 phase of the cell cycle and promotes terminal differentiation during xenopus primary neurogenesis , a phenotype opposite to that of overexpression of an activated membrane - targeted form of apkc , apkc - caax .
apkc - caax overexpression rescues the increased neuronal differentiation phenotype of p27xic1 overexpression , as would be expected if apkc counteracted the activity of p27xic1 .
then , we show that apkc directly phosphorylates p27xic1 in the n terminus of the protein .
phosphomimetic p27xic1 shows reduced binding to the g1-s related cyclin - dependent kinase 2 ( cdk2 ) , resulting in reduced inhibition and higher kinase activity , which in turn causes a faster cell cycle .
this study thus identifies a direct mechanistic link between apicobasal cell polarity and the cell cycle .
dual - pulse s phase labeling ( dpsl ) analysis on outer layer apicobasally polarized progenitors and inner layer nonpolar neural progenitors ( both sox3 + ) , showed that polarized progenitors have a significantly shorter cell cycle length and a shorter s phase length than nonpolar progenitors at open neural plate stage , nf13 ( tc , polarized versus nonpolar , mean sem , 282 14 min versus 411 21 min , and ts , polarized versus nonpolar , 40 4 min versus 141 14 min , figure 1a ) .
we also analyzed these progenitors for percentage of labeled mitoses ( plm ) to estimate g2 + 1/2 m phase length and compared them for their mitotic indices ( figure 1b ) .
putting the numbers together from these experiments ( see supplemental experimental procedures available online for details ) showed that polarized progenitors have a shorter g1 phase ( tg1 , 94 min versus 160 min for nonpolar cells ) but a longer g2 phase than the nonpolar progenitors ( tg2 , 132 min versus 89 min ) .
the lengths of mitoses were marginally different between the two layers ( tm , 16 min versus 21 min ) ( figure 1c ) .
thus , establishing the cell - cycle kinetic parameters for polarized and nonpolar progenitors showed that they differ significantly and that polarized progenitors cycle faster .
overexpression of constitutively active , membrane - targeted apkc ( apkc - caax ) suppressed neuronal differentiation , as judged by the reduction in the expression of a terminal differentiation marker n - tubulin .
conversely , nuclear dominant - negative apkc ( nls - apkc - nt ) promoted neuronal differentiation as n - tubulin was enhanced ( figure s1 available online ) . extending these observations
further , we found that on apkc - caax overexpression , elrc , a marker of committed neural progenitors ( carruthers et al . ,
2003 ) , was also suppressed , whereas cells expressing the neural progenitor marker sox3 showed expansion ( sox3 + area was increased on the injected side as shown by in situ hybridization ) ( figure s1 ) .
this suggested that apkc - caax suppressed neuronal differentiation by promoting neural progenitor expansion . in apkc - caax - overexpressing embryos , the number of sox3 + progenitors on the injected side was significantly higher ( shown by immunostaining , figure 2a ) and cells on the injected side had significantly shorter tc and ts , than on the noninjected side , calculated by the dpsl technique .
control embryos overexpressing gfp - caax exhibited no such differences ( figure 2a ) . to further see the effects of apkc on different phases of the cell cycle
, we treated and imaged hela fucci cells ( sakaue - sawano et al . , 2008 ) for 4860 hr in the presence of a myristoylated , cell - permeable inhibitor specific against apkc ( sajan et al . , 1999 ) .
cells inhibited for apkc showed significant lengthening of the total cell - cycle time ( tc , 25.32 0.58
this was mainly attributed to the lengthening of the g1 phase ( tg1 , 17.04 0.47 hr versus 11.79 0.51 hr for controls ) , with small effects observed in the early s phase ( ts , 5.68 0.78 hr than 3.38 0.40 hr for controls ) .
the effect on the length of s , g2 , and m together ( tsg2 m , 14.15 0.67 hr versus 10.92 0.43 hr for controls ) indicated that the g2 phase was also largely unaffected by the inhibitor treatment ( figure 2b and movies s1a and s1b ) .
the pseudosubstrate myristoylated inhibitor used here is highly specific and has no effect on classical and novel pkcs ( standaert et al . , 1999 ) .
nevertheless , these data were further substantiated by the experiment with a chemical inhibitor against apkc , bisindolylmaleimide g6983 .
this inhibitor has been used previously to show that apkc is involved in te formation ( eckert et al . , 2004 ) and can maintain es cells in an undifferentiated state in the absence of lif , through the inhibition of pkc ( dutta et al . , 2011 ) .
g6983 showed similar effects on cell - cycle kinetics as shown by myr inhibitor against apkc ( figure 2b and movies s1a and s1c ) .
the fucci data along with dpsl data suggested that both gain and loss of apkc signaling activity affects cell cycle length via g1 and s phases , with g2 and m phases remaining largely unaffected .
these data suggested that the effects of apkc on progenitors proliferation might be mediated via its effects on cell - cycle kinetics . to test this further , we investigated the interaction of apkc with cell - cycle regulators .
g1 kinases ( cdk4/6 ) and g1/s kinase ( cdk2 ) are positively regulated by cyd and cye / a , respectively , and negatively regulated by a cip / kip family cyclin - dependent kinase inhibitor ( cdki ) p27xic1 ( ohnuma and harris , 2003 ) . because cyclins d , e , a , and p27xic1 are regulated by posttranslational modifications , mainly phosphorylations ( alberts et al . ,
in vitro kinase assays showed that only bacterially expressed p27xic1 is a direct phosphorylation target of gst - apkc ( commercially supplied ) ( figure 3a and data not shown ) .
the specificity of this in vitro phosphorylation was confirmed by performing the kinase assay in the presence of a pseudosubstrate inhibitor specific against apkc ( sajan et al . , 1999 ) .
this reduced the kinase signal in a dose - dependent manner ( figure 3b ) . to see if apkc can phosphorylate p27xic1 in a complex embryonic milieu as well , we performed in vitro kinase assay using embryonic lysate , instead of kinase buffer , as the medium for interaction between exogenous gst - p27xic1 ( bacterially expressed ) and his - apkc ( commercially supplied ) .
the result demonstrated that phosphorylated gst - p27xic1 significantly increases in the presence of apkc , whereas it drops down to basal level in the presence of pseudosubstrate inhibitor against apkc ( figure 3c ) . to further substantiate these observations in vivo
, we performed in vivo kinase assay measuring the incorporation of -p32-labeled orthophosphate by p27xic1 , overexpressed in hela cells along with apkc - caax or in the presence of myristoylated pseudosubstrate apkc inhibitor .
the experiment showed that p27xic1 incorporated more orthophosphate when expressed along with apkc - caax ; the incorporation was reduced when the same cells were cultured in the presence of the inhibitor ( figure 3d ) .
p27xic1 and apkc - caax also showed direct physical interaction in coimmunoprecipitation ( coip ) assays .
first , flag - p27xic1 and ha - apkc - caax constructs were co - overexpressed in hela cells and ha - apkc - caax was detected after ip of flag - p27xic1 using anti - flag beads ( figure 3e ) .
more importantly , the endogenous p27xic1 and apkc from embryonic lysate also showed direct physical interaction in coip experiments where either p27xic1 or apkc is immunoprecipitated with antibodies and apkc or p27xic1 is detected by western blot in the pull - down , respectively ( figures 3f and s2 ) .
the effect of apkc inhibitors on hela fucci cell - cycle length ( figure 2b ) suggested that human p27kip1 might also be negatively regulated by apkc and may be a phosphorylation target of apkc .
however , our in vitro kinase assay using immunoprecipitated p27kip1 from hela cells showed that human p27kip1 is not a direct phosphorylation target of apkc ( data not shown ) .
this suggests that apkc negatively regulates p27kip1 in an indirect manner . to gain some insight into where the interaction of apkc and p27xic1 may be taking place , we performed immunostaining on neurula stage embryos overexpressing flag - p27xic1 and ha - apkc - caax .
these showed that p27xic1 was largely in the nucleus of the cells , and a portion of apkc - caax , which was mostly localized to the cell cortex , was also found in the nucleus ( figure s3 ) .
these findings are consistent with previous reports showing nuclear enrichment of p27xic1 ( chuang and yew , 2001 ; chuang et al . , 2005 ) and some nuclear localization of apkc - caax ( sabherwal et al .
, 2009 ) and lend support to the idea that apkc interacts with p27xic1 in the nucleus . in our previous publication ( sabherwal et al . , 2009 ) , we showed that apkc - caax is more active and nuclear than apkc and hypothesized that after getting activated in the membrane , a small proportion of apkc - caax is translocated to the nucleus .
this suggests that although apkc - caax is predominantly membrane localized and weakly nuclear , it is highly active and sufficient to influence nuclear events like interacting with p27xic1 and modulating its activity . to identify domains of p27xic1 phosphorylated by apkc
as shown in figure 4a , the n - terminal and middle parts of the protein showed positive kinase signal whereas the c - terminal fragment showed no sign of phosphorylation during in vitro kinase assays .
phosphosite identification using liquid chromatography / tandem mass spectrometry ( lc / ms / ms ) analysis on flag - p27xic1 phosphorylated by apkc in vitro ( i.e. , in vitro kinase reaction on immunoprecipitated / iped flag - p27xic1 from hela cells ) identified multiple phosphorylation sites with significant ascore values ( 13 , ascore is a measurement of the confidence of phosphorylation ; beausoleil et al . , 2006 ) within the n - terminal , cdk - binding domain ( figure 4a ) . to see if the sites identified correspond to the phosphosites in vivo , similar lc / ms / ms
analysis was carried out on flag - p27xic1 iped from hela cells co - overexpressing it with ha - apkc - caax .
this analysis identified a phosphosite ( t68 ) with a significantly high ascore ( ascore = 27 ; figure 4a ) , located within the cdk interaction domain of p27xic1 ; another site ( t99 , located immediately after the cdk interaction domain ) was picked with a low ascore value ( ascore = 13 ; figure 4a ) .
none of these sites was identified on flag - p27xic1 iped from hela cells overexpressing it alone .
xenopus p27xic1 is 44% identical to human p27kip1 and 40% identical to human p21cip1 in the conserved n terminus .
it also possesses a pcna - like binding site characteristic of p21cip1 in the c terminus ( su et al . , 1995 ) .
the apkc phosphosite in p27xic1 ( t68a ) is not conserved in mammalian p27kip1 but is conserved in mammalian p21cip1 ( although it has not been identified as an apkc phosphosite by others ) and p27xic1 from zebrafish ( figure 4b ) , suggesting some degree of functional conservation linking apkc activity and cell - cycle regulation ; the other phosphosite identified ( t99 ) shows no conservation with other members of cip / kip cdkis .
identification of apkc phosphorylation sites prompted us to check if these phosphorylation events are functionally important by generating phospho ( s / t to a ) or phosphomimetic ( s / t to e ) mutants .
initial protein abundance experiments showed that the level of flag - p27xic1 protein is decreased in embryos injected with ha - apkc - caax and increased in embryos injected with the dominant - negative ha - apkc - nt ( figure s4a ) .
protein stability experiments using cycloheximide showed that the half - life of overexpressed flag - p27xic1 was reduced when it was co - overexpressed in hela cells along with ha - apkc - caax ( figure s4b ) .
however , nonphosphorylatable mutants ( like t68a ) were also destabilized in comparison to the wild - type protein , whereas phosphomimetic mutants ( like t68e ) of p27xic1 appeared more stable ( see figures s4c and s4d for examples ) , suggesting that the effect of apkc phosphorylation on p27xic1 stability is complex and can not be reproduced by single amino acid changes .
not only the stability of cdkis , but also the inhibition of cdk kinase activity by cdkis has been shown to promote differentiation ( hasan et al . , 2013 ) .
we tested whether the binding of p27xic1 to cdks is affected by apkc phosphorylation , which could explain the shortening of g1 and s phases of cell cycle . p27xic1
interacts with cdk2 and cdk4 and inhibits their kinase activities ( finkielstein et al . , 2001 ) .
coip assays using hela cells co - overexpressing flag - p27xic1 along with either ha - cdk2 or ha - cdk4 showed that p27xic1 binds to cdk2 much stronger than to cdk4 ( data not shown ) .
similar coip assays showed that phosphomimetic mutant t68e showed almost 50% reduction in its binding to cdk2 in comparison to the wild - type p27xic1 ( figures 4c , 4e , and s5 ) , whereas the phosphomutant t68a showed enhanced binding ( increased by almost 140% ) to cdk2 ( figures 4d , 4e , and s5 ) .
other mutants showed binding similar to the wild - type p27xic1 ( figures 4c , 4d , and s5 ) .
these assays suggested that a single phosphorylation event at amino acid t68 of p27xic1 by apkc is enough to affect its interaction with cdk2 . using histone h1 as a substrate for active cdk2/cyclina
, we found that in comparison to the wild - type p27xic1 , phosphomutant t68a abolished the kinase activity of cdk2 almost completely , while the phosphomimetic mutant t68e had a negligible effect on cdk2 activity in the same assay ( figure 4f ) .
taken together , these results mean that phosphorylation of p27xic1 by apkc essentially inhibits the activity of p27xic1 by reducing its binding to cdk2 , which in turn results in failure to negatively regulate the cdk2 kinase activity .
p27xic1 overexpression by injecting mrna has been shown to promote neurogenesis ( vernon et al . ,
2003 ) , while its morpholino - mediated knockdown blocked neurogenesis and promoted progenitor proliferation ( carruthers et al . , 2003 ) . here , we have used dna injections , because in xenopus the g1 phase appears in post - mid - blastula transition cell cycles .
we found that such p27xic1 dna injections promoted neurogenesis ( figure 5a ) in a cell autonomous fashion ( figure s6 ) and suppressed neural progenitor proliferation , consistent with results obtained previously with rna overexpression .
a subset of p27xic1-overexpressing embryos was used for fluorescence - activated cell sorting ( facs ) cell - cycle profiling on isolated nuclei .
experimental embryos showed significantly higher percentage of nuclei in g1 phase than the control embryos ( 35.4% versus 27.4% ) , with a concomitant decrease of nuclei in s phase ( 40.7% versus 49.7% for control ) and negligible effect on the proportion of nuclei in g2 phase ( 23.9% versus 22.9% for control , figure 5b ) , indicating that g1 phase elongates and proliferation decreases upon p27xic1 overexpression .
whereas 89% of p27xic1-injected embryos showed enhanced staining for elrc , the percentage dropped to 33% with a milder phenotype when p27xic1 was coinjected with apkc - caax ( figure 5c ) . thus , p27xic1 s overexpression phenotype of promoting neuronal differentiation was rescued by unilateral injection of apkc - caax .
the corresponding numbers for n - tubulin staining were 88% and 20% after overexpression of p27xic1 alone or with apkc - caax ( data not shown ) .
this supported the idea that apkc promotes progenitor proliferation , at the expense of neuronal differentiation upstream of p27xic1 , by inhibiting its activity .
the idea was further supported by the observation that the phosphomutant of p27xic1 ( t68a ) could promote neuronal differentiation similar to p27xic1 , but apkc - caax could not effectively rescue this effect ( figure 5c ) . to understand if the effects of apkc and p27xic1 activities on neuronal differentiation were mediated via their effects on the cell - cycle length , we manipulated the cell cycle of embryos by nongenetic , chemical means using olomoucine ( olo ) , an inhibitor that lengthens the g1 phase by inhibiting the activity of g1 kinases ( calegari and huttner , 2003 ) .
facs analysis of nuclei from embryos treated with olo showed that experimental embryos had more nuclei in g1 and g2 phases than control embryos , with a corresponding decrease in the proportion of nuclei in s phase , suggesting a decrease in proliferation and an elongation of growth phases ( figure 6a ) .
experimental embryos also showed a significantly higher number of myt1-positive cells ( a marker of differentiated neurons , 12.53 2.44 per section ) than the dmso controls ( 8.75 0.38 ) ( figures 6b and 6c ) , supporting the idea that elongating g1 phase promotes differentiation .
cell polarization and cell division are two fundamental biological processes that have been independently linked to cellular differentiation . in this work
, we showed that apicobasal polarity and cell - cycle control are directly linked in neural progenitor cells in a way that leads to distinct differentiation potential of polarized versus nonpolar progenitors .
our work has uncovered a remarkable similarity in the endogenous cell - cycle kinetics of apicobasally polarized and nonpolar progenitors between xenopus neuroectoderm and the mouse embryonic cortex ( arai et al . , 2011 ) .
in both cases , the total length of the cell cycle and the length of g1 in apicobasally polar progenitors ( apical progenitors in the mouse , superficial progenitors in xenopus ) are shorter than those found in nonpolar ones ( basal progenitors in the mouse , deep progenitors in xenopus ) .
in addition , in both cases , the basal nonpolar progenitors have a higher propensity to differentiate than the apicobasally polarized ones ( chalmers et al . , 2002 ; chenn and mcconnell , 1995 ) .
thus , although the xenopus neuroectoderm shows a much simpler structure , some of the basic principles relating to the cell - cycle control during neurogenesis are highly conserved .
we have used this system to specifically address the role of the apicobasal polarity in controlling the cell cycle via the apically localized key kinase apkc .
apkc is a ubiquitous kinase that gets activated in cell cortex in a pi-3,4,5-trisphosphate ( pip3)-dependent manner ( reviewed by hirai and chida , 2003 ) .
polarized cells are thought to contain a highly active pool of apkc because it is recruited to the apical cortex and/or the junctional complexes ( joberty et al .
, 2000 ; lin et al . , 2000 ) where it should get activated .
therefore membrane targeting of apkc by attaching it to a caax motif makes it constitutively active and mimics the effects of apicobasal polarization on apkc activity ( lee et al . , 2006 ; sabherwal et al . ,
apkc was the first molecule shown to promote the proliferation of polarized neuroblasts in drosophila and neural progenitors in xenopus both by gain- and loss - of - function experiments ( rolls et al .
, 2003 ; chabu and doe , 2008 , 2009 ; sabherwal et al . ,
, the interaction of apicobasal polarity kinase apkc with the cell - cycle machinery in polarized cells was thought to be indirect , via the hippo pathway ( reviewed in genevet and tapon , 2011 ) .
for example , apkc phosphorylates and negatively regulates kibra , an upstream positive regulator of the hippo pathway ( bther et al . , 2004 ;
overexpression of apkc mislocalizes apical hippo to the cytoplasm with its negative regulator rassf , leading to the dampening of the hippo pathway and resulting in enhanced cell proliferation ( grzeschik et al . , 2010 ) .
our current results establish a direct link between apkc and the cell cycle in context of cell polarity . in this study , we show that apkc - caax directly phosphorylates the nuclear cell cycle / cyclin - dependent kinase inhibitor p27xic1 .
phosphorylation primarily takes place in t68 , located in the n - terminal domain , although we can not exclude contribution from other sites .
apkc - caax overexpression leads to a faster cell cycle in the neuroectoderm with shorter g1 and s phases , decreased neuronal differentiation , and enhanced neural proliferation ( this work and sabherwal et al . , 2009 ) .
apkc has also been shown to have a role in cell proliferation of nonpolarized cells .
for example , apkc has been shown to directly phosphorylate and degrade p21cip1 in human colorectal ( hct116 ) cells ( scott et al . , 2002 ) , to upregulate cyd1 transcription , and to reduce p27kip1 s nuclear translocation in a ras - dependent manner in mcf7 cells ( castoria et al . , 2004 ) .
we assume that these nonpolar cells have a basal activity of apkc , which is further enhanced by recruitment to the apical membrane in polarized cells .
how does phosphorylation of p27xic1 by polarity kinase apkc lead to a faster cell cycle with shorter g1 and s phases ?
p27xic1 physically interacts with cdks and cyclins through its cdk / cy interaction domains and reduces the activities of g1 kinase ( cdk4/cyd ) , g1-s transition kinase ( cdk2/cye ) and s progression kinase ( cdk2/cya ) with different half - maximal inhibitory concentration values ( su et al . , 1995 ; finkielstein et al . ,
our data show that phosphomimetic p27xic1 has a reduced ability to bind and inhibit cdk2 . enhanced cdk2 activity through cye overexpression
causes the cells to cycle faster through g1 and enter s phase prematurely ( ohtsubo and roberts , 1993 ) , whereas inhibition of cye / cdk2 complex delays entry into s phase ( van den heuvel and harlow , 1993 ) .
thus , inhibition of cdk2 kinase activity by cdkis , like p27xic1 , is key in inhibiting the cycling of progenitors and promoting their differentiation .
we suggest that in polarized progenitors , unrestricted kinase activities of g1/s - specific cdk / cy complexes due to lower p27xic1 activity would shorten their g1 phase and concomitantly promote s phase entry , thereby promoting their cycling and interfering with the ability to differentiate . the prolongation of total cell - cycle length , and g1 in particular , as cells approach differentiation seems to be a widespread phenomenon ( takahashi et al .
mechanistically , it is thought that elongation of the g1 phase promotes neuronal differentiation by providing sufficient time for the accumulation and posttranscriptional modification of differentiation - promoting factors , such as neurogenin and neurod ( reviewed in lange and calegari , 2010 ; hardwick and philpott , 2014 ) . in support of this idea
, it was recently shown that the activity of neurogenin is controlled by time - dependent , rheostat - like sequential ( de)phosphorylation events in a cell - cycle - dependent manner ( ali et al . , 2011 ) .
in conclusion , our findings provide mechanistic evidence for a direct link between apicobasal polarity and the cell cycle through apkc and p27xic1 , which is used during development to endow polarized neural progenitors with lower propensity for differentiation than their nonpolar counterparts .
p27xic1 has a complex mode of regulation , which includes phosphorylation , ubiquitination , and degradation in the nucleus , and changes in the activity through the cell cycle ( chuang and yew , 2001 ; chuang et al . , 2005 ; lin et al . ,
regulation of apkc is likely to be equally complex , including shuttling between the membrane and the nucleus ( sabherwal et al . , 2009 ) .
one challenge for the future is to understand the dynamic interaction between apkc and p27xic1 during the cell cycle .
this method enables the estimation of the total cell - cycle length ( tc ) and s phase length ( ts ) by injecting two different thymidine analogs ( martynoga et al . , 2005 ) .
embryos collected at open neural plate stage ( nf13 ) were injected with ethynyl deoxyuridine ( edu , 2 10 nl , 10 mm in dmso [ life technologies ] ) in the neural plate midline at two different positions .
after 2 hr incubation , bromodeoxyuridine ( brdu , 2 10 nl , 10 mm in dmso [ roche ] ) was injected in the same place ( figure 1a ) .
embryos were fixed in memfa 30 min later and processed for edu , brdu , and sox3 ( as a marker of both polar and nonpolar neural progenitors ) staining as described elsewhere ( auger et al . , 2012 ) .
this method involves labeling the mitotic ( phosphohistone h3/ph3 + ) cells with a thymidine analog ( edu or brdu ) and gives an estimate of the lengths of total cell cycle , s and g2 + 1/2 m phases , depending on the duration of the experiment ( shackney and ritch , 1987 ; peco et al . , 2012 ) .
embryos at nf13 were injected with edu as described above and collected every 30 min until 240 min as the last point of our experiment ( figure 1b ) .
embryos were fixed , sectioned , and stained for edu , ph3 , and sox3 as described elsewhere ( auger et al . , 2012 ) .
nuclei from nf15 embryos ( 20 per set ) for facs analysis were prepared as described elsewhere ( frederick and andrews , 1994 ) , followed by cell - cycle profiling using a cyan adp flow cytometer from beckman coulter running summit v4.3 software . the modfit analysis on the data was carried out using flojo software .
hela fucci cells were maintained and propagated as described elsewhere ( sakaue - sawano et al . , 2008 ) .
control and experimental cells with inhibitors were imaged on a heated stage at 37c and 5% co2 supply using nikona1 confocal microscope for 23 days . for dual - pulse s phase labeling and percentage of labeled mitoses analyses ,
five to ten sections per embryo from the mid - anterior neural plate were imaged on a nikona1 confocal microscope .
still and time - lapse ( for fucci ) images were processed and analyzed using imagej software .
depending on the outcome of the normality test , appropriate tests for significance ( mentioned in the figure legends ) were chosen from the software and applied to the data .
animal experiments were approved by the university s ethical review panel and were undertaken under uk home office project license ppl 70/7648 .
culturing and overexpression in xenopus laevis embryos were carried out as described elsewhere ( sabherwal et al . , 2009 ) .
staging was made according to the nieuwkoop and faber table of development ( nf stages ; nieuwkoop , 1967 ) .
the following amounts of mrna or dna were injected : apkc - caax 0.250.5 ng , nls - apkc - nt 0.250.5 ng , and p27xic1 0.15 ng ( 0.075ng 2 ) ; 0.5 ng gfp or -galactosidase ( lacz ) mrna was coinjected as lineage
n - tubulin , elrc , and sox3 have been described ( chalmers et al . , 2002
fixation , x - gal staining , in situ hybridization , and sectioning were carried out as described elsewhere ( bourguignon et al . , 1998 ) .
gst - p27xic1 was expressed in bl21 escherichia coli cells and purified as described by the plasmid supplier ( ge healthcare ) .
for in vitro kinase assay , 20 l of eluted gst - p27xic1 was mixed with kinase buffer ( 20 mm tris - hcl [ ph 7.5 ] and 5 mm mgcl2 ) , gst - apkc ( 1 l/0.1 g , calbiochem)/his - apkc ( 1 l/0.1 g , millipore ) , and p32-radiolabelled atp ( 1 l , 0.37 mbq [ perkin elmer ] ) . the final 30 l reaction was incubated at 30c for 30 min , boiled with sample buffer , and loaded on 10%12% polyacrylamide gel .
post - run , dried gel on whatman paper was used to expose x - ray film to get an autoradiogram impression of the kinase reaction . to perform in vitro kinase assays in an embryonic environment ,
the reaction was performed exactly as described above , except that instead of kinase buffer , nf13 - 14 embryonic lysate ( as described below but without edta and egta ) was used as the incubation medium with 10 l ( 3.7 mbq ) of p32-radiolabelled atp . for in vivo kinase assays , hela cells were transfected with flag - p27xic1 alone or with ha - apkc - caax . for inhibitor treatment , flag - p27xic1 transfected cells were incubated with the media containing the inhibitor for 24 hr . p32-radiolabelled orthophosphate ( perkin elmer ) incorporation was performed as described elsewhere ( baril et al . , 2000 ) , followed by immunoprecipitation of flag - p27xic1 and autoradiography as described above . for the immunocomplex kinase assay ,
10 l of protein a / g sepharose beads ( santa cruz biotechnology ) was mixed with 100 l of cleared lysate from hela cells ( overexpressing the protein of interest ) and 25 g of mouse anti - flag antibody ( m2 clone [ sigma ] ) .
for histone h1 in vitro kinase assay , 0.5 g of histone h1 ( sigma ) , 100 ng of active cdk2/cyclina protein ( millipore ) , and 1 l of p32-radiolabelled atp ( 0.37 mbq ) were mixed with immunoprecipitated wild - type or mutant p27xic1 in 1 kinase buffer and incubated at 30c for 30 min . the reaction was stopped and autoradiography was performed as described above .
hela cells were maintained and transfected as described elsewhere ( sabherwal et al . , 2009 ) .
coip assays from hela cells overexpressing proteins of interest were performed as described elsewhere ( roth et al . , 2010 ) .
proteins were immunoprecipitated using 510 g of the following antibodies : mouse anti - flag ( m2 clone [ sigma ] ) , mouse anti - ha ( sigma ) , and mouse anti - myc ( santa cruz biotechnology ) .
coip from embryos was performed using the pierce crosslink ip kit , following the manufacturer s protocol using rabbit anti - apkc ( c20 clone , santa cruz biotechnology ) and rabbit anti - p27xic1 antibodies ( custom made against the antigen as described by shou and dunphy , 1996 ) . for making embryo lysates , embryos were dissociated in lysis buffer ( 50 mm tris ph7.5 + 150 mm nacl + 0.5% np40 + 5 mm edta + 5 mm egta ) . cleared lysates were used for western blot analysis .
the following antibodies were used for detection : rat ha - hrp ( roche ) , mouse flag - hrp ( sigma ) , mouse myc - hrp ( santa cruz biotechnology ) , rabbit apkc ( c20 clone , santa cruz biotechnology ) , rabbit anti - p27xic1 , and mouse anti--tubulin ( dm1a clone [ sigma ] ) .
myristoylated pseudosubstrate inhibitor against apkc ( invitrogen life technologies ; sajan et al . , 1999 ) was used at a working concentration of 12.5 m .
chemical inhibitor against apkc ( g6983 [ calbiochem ] saiz et al . , 2013 ) was used at a working concentration of 5 m .
olomoucine ( calbiochem ; calegari and huttner , 2003 ) was used at a working concentration of 120 m .
antibody staining on sections and whole mounts was performed as described elsewhere ( sabherwal et al . , 2009 ) .
the following primary and secondary antibodies were used : rat anti - ha ( roche ) , rabbit anti - flag ( sigma ) , mouse anti - brdu ( mobu clone [ life technologies ] ) , mouse anti - phosphohistoneh3 ( abcam ) and rabbit anti - sox3 ( custom made ; zhang et al . , 2003 ) , rabbit anti - myt1 ( custom made ; sabherwal et al . , 2009 ) , alexa488-coupled anti - mouse , and alexa647-coupled anti - rabbit ( life technologies ) . for phosphopeptide mapping ,
lc / ms / ms analysis of phosphosites was performed at the taplin biological mass spectrometry facility ( harvard medical school ) as described ( roig et al . , 2005 ) .
please refer to the supplemental experimental procedures for information regarding sample preparation for ms analysis . | summaryduring the development of the nervous system , apicobasally polarized stem cells are characterized by a shorter cell cycle than nonpolar progenitors , leading to a lower differentiation potential of these cells .
however , how polarization might be directly linked to the kinetics of the cell cycle is not understood . here ,
we report that apicobasally polarized neuroepithelial cells in xenopus laevis have a shorter cell cycle than nonpolar progenitors , consistent with mammalian systems .
we show that the apically localized serine / threonine kinase apkc directly phosphorylates an n - terminal site of the cell - cycle inhibitor p27xic1 and reduces its ability to inhibit the cyclin - dependent kinase 2 ( cdk2 ) , leading to shortening of g1 and s phases .
overexpression of activated apkc blocks the neuronal differentiation - promoting activity of p27xic1 .
these findings provide a direct mechanistic link between apicobasal polarity and the cell cycle , which may explain how proliferation is favored over differentiation in polarized neural stem cells . |
extrapulmonary complications of mycoplasma pneumoniae infection involving the central nervous system ( cns ) are not rare . according to previous reports , encephalitis , meningoencephalitis , aseptic meningitis , transverse myelitis , acute disseminated encephalomyelitis , and guillain - barr syndrome
have been associated with m. pneumoniae infection12 ) . meanwhile , there are less than 20 cases of m. pneumoniae - associated cerebral infraction reported in the pediatric population , and only six cases have identified occlusion of the cerebral arteries345678 ) .
we report a case of m. pneumoniae - associated cerebral infraction in a 5-year - old girl in which complete occlusion of the right middle cerebral artery ( mca ) was identified by brain magnetic resonance angiography ( mra ) and transfemoral cerebral angiography ( tfca ) .
a 5-year - old girl was admitted due to left hemiparesis and left facial palsy .
although she had taken oral antibiotics prescribed at a local clinic , her symptoms did not show improvement . on the fourth day of her illness
she was admitted to that hospital and was treated with antibiotics of ceftriaxone and clarithromycin , intravenously . on the sixth day of her illness , her fever subsided . however , she abruptly developed left hemiparesis and left facial palsy , and was referred to inha university hospital .
vital signs showed a blood pressure of 127/60 mmhg , heart rate of 102 beats / min , respiratory rate of 24 breaths / min , and body temperature of 37.6. her heart beat was regular without murmurs , while breath sounds were coarse with inspiratory crackles auscultated on both lung fields . on neurologic examination , pupils were isocoric with prompt light reflexes .
extraocular movements were full in both eyes , while there were defects in the left visual fields .
she was unable to form facial expressions on the lower half of her left face , while she was able to wrinkle her forehead normally , indicating left side central type facial palsy .
motor strengths were decreased to grade iii in both the left upper and lower extremities .
chest radiography conducted on admission showed pneumonic infiltration and pleural effusion in the left lung field ( fig .
1 ) . brain magnetic resonance imaging revealed acute ischemic infarction in the right mca territory ( fig .
2 ) , and brain mra showed complete occlusion of the right mca at the m1 portion ( fig .
tfca revealed complete occlusion of the right mca at the m1 portion , with no evidence of vasculitis or moyamoya disease ( fig .
laboratory exams conducted on the day of admission showed a hemoglobin of 10.5 g / dl , hematocrit 30.1% , white blood cell ( wbc ) count of 5,420/l , comprising 82.8% neutrophils , 9.2% lymphocytes , and 4.2% monocytes , and platelet count of 250,000/l .
c - reactive protein was 7.15 mg / dl while other electrolyte and biochemistry laboratory exams were normal .
coagulation studies revealed normal prothrombin and activated partial thromboplastin time , while fibrinogen level was elevated to 674 mg / dl ( normal range , 200400 mg / dl ) and d - dimer level to 1.7 g / ml ( normal range , 00.4 g / ml ) .
analysis of the patient 's cerebrospinal fluid ( csf ) showed a wbc count of 3/l , glucose concentration of 90 mg / dl , and protein concentration of 15 mg / dl .
initial serum antibody titers to m. pneumoniae detected by enzyme - linked immunosorbent assays ( elisa ) were 5,021 u / ml for igm ( normal range , 0949 u / ml ) and 5.5 au / ml for igg ( normal range , 09.99 au / ml ) . polymerase chain reaction ( pcr ) of m. pneumoniae , influenza viruses , enteroviruses , and herpes simplex viruses were negative in the csf .
the patient was diagnosed as pneumonia , strongly suggestive of m. pneumoniae origin associated with acute cerebral infarction .
antibiotics of ceftriaxone and clarithromycin were administered for 14 days , mannitol for 7 days , and methylprednisolone for 3 days , intravenously .
the results of laboratory exams in order to identify possible predisposing causes of the patient 's stroke were all negative .
serum analysis of prothrombotic agents and factors including protein c , protein s , antithrombin iii , lupus anticoagulant , homocysteine , lipid profiles and plasma lipoprotein levels were normal .
analysis of auto - antibodies associated with autoimmune and rheumatologic diseases , including antiphospholid antibodies , anticardiolipin antibodies , antinuclear antibodies , anticytoplasmic antibodies , anti - dna antibodies , and rheumatoid factors were all negative .
no other bacterial or viral organisms were documented in the serum or csf on culture studies .
serum antibody titers to m. pneumoniae detected by elisa 3 weeks after admission were 7,300 u / ml for igm and 26.65 au / ml for igg .
the patient was discharged on the 32nd hospital day after receiving the inpatient rehabilitation schedule . at discharge , both left hemiparesis and left facial palsy had improved , although mild neurologic deficits regarding fine motor skills were observed .
motor strengths of grade v were checked on all extremities and unaided walking was capable .
although not fully understood , two mechanisms have been proposed in order to explain the pathophysiology of cerebral arterial occlusion associated with m. pneumoniae910 ) .
the direct - type mechanism is due to the vascular transfer of the organism itself , which locally affect the vascular walls of the cerebral circulation by the induction of cytokines and chemokines such as tumor necrosis factor- and interleukin-891011 ) .
this may cause local vasculitis , thrombotic vascular occlusion , or both , even in the absence of a systemic hypercoagulable state .
the indirect - type mechanism is associated with a systemic hypercoagulable state resulting in a generalized thrombotic vascular occlusion , in which the activation of complements are assumed to play a role912 ) .
this postulation is supported by the elevation of fibrinogen and d - dimer levels reported in some cases of m. pneumoniae - associated cerebral arterial occlusion46 ) . in our case ,
negative m. pneumoniae pcr results of the csf and laboratory findings of elevated fibrinogen and d - dimer levels indicate that the pathophysiology of cerebral arterial occlusion might be related to the indirect - type mechanism .
thus , a systemic hypercoagulable state induced by m. pneumoniae infection and resultant inflammation may have contributed to the generation of the cerebral arterial occlusion rather than the direct effects of the organism on the affected cerebral artery .
congenital or acquired heart diseases , sickle cell disease , thrombophilias , and infections are well known etiologies and predisposing factors for acute ischemic cerebral infarction in the pediatric population13 ) . hence , laboratory studies should include examinations regarding coagulopathies , inflammation , autoimmune diseases , and hyperlipidemia .
predisposing diseases or conditions such as sickle cell trait5 ) , hyperhomocysteinemia8 ) , and antiphospholipid and anticardiolipin antibodies6 ) have been identified in cases of m. pneumoniae - associated cerebral arterial occlusion in children , besides the organism itself .
occlusion of cerebral arteries have been documented both in the anterior and posterior circulation in pediatric cases of m. pneumoniae - associated cerebral arterial occlusion , confirmed by conventional angiography or mra .
internal carotid occlusion has been reported in three cases367 ) , posterior cerebral artery occlusion in two cases58 ) , and mca occlusion in one case4 ) among english literature .
our case is the first report of complete arterial occlusion occurring in the right mca with resultant massive ischemic infarct in its territory which was associated with m. pneumoniae infection in a child .
treatment of cns diseases associated with m. pneumoniae infection includes antibiotics , corticosteroids , and intravenous immunoglobulin ( ivig)1 ) .
treatment with steroids and ivig was reported to improve outcome in a pediatric case of m. pneumoniae - associated stroke14 ) .
low dose aspirin has also been administered in some pediatric cases of m. pneumoniae - associated cerebral arterial occlusion58 ) . in general strokes , initial aspirin therapy with doses of 1 to 5 mg / kg
is recommended daily followed by long - term aspirin therapy for a minimum of 2 years in children , except those associated with sickle cell disease15 ) .
although there is no standardized treatment , according to our case we assume that the combination of antibiotics , mannitol , corticosteroids , and aspirin may be effective for initial treatment in pediatric cerebral arterial occlusion associated with m. pneumoniae infection .
the addition of ivig may be considered in cases that do not respond well to initial treatment . in conclusion , we report a case of complete occlusion of the right mca in a child with m. pneumoniae pneumonia .
thorough evaluation of possible predisposing factors for stroke should always be conducted in children and close attention is required on determining the potential systemic hypercoagulable status associated with m. pneumoniae - associated cerebral arterial occlusion .
the administration of corticosteroids and aspirin should also be considered additionally to proper intravenous antibiotics . | we report a case of a 5-year - old girl who developed left hemiparesis and left facial palsy , 6 days after the initiation of fever and respiratory symptoms due to pneumonia .
chest radiography , conducted upon admission , showed pneumonic infiltration and pleural effusion in the left lung field .
brain magnetic resonance imaging showed acute ischemic infarction in the right middle cerebral artery territory .
brain magnetic resonance angiography and transfemoral cerebral angiography revealed complete occlusion of the right middle cerebral artery .
mycoplasma pneumoniae infection was identified by a 4-fold increase in igg antibodies to m. pneumoniae between acute and convalescent sera by enzyme - linked immunosorbent assay .
fibrinogen and d - dimer levels were elevated , while laboratory exams in order to identify other predisposing factors of pediatric stroke were all negative .
this is the first reported pediatric case in english literature of a m. pneumoniae - associated cerebral infarction involving complete occlusion of the right middle cerebral artery . |
the hard tissue of human teeth is composed of enamel , cementum and dentin . multinucleated cells called odontoclasts resorb root dental hard tissues9.the resorption of hard tissue in primary teeth is a normal physiologic phenomenon that leads to their exfoliation and ultimately the transition to permanent dentition .
the hard tissues of permanent teeth , on the other hand , are not resorbed under normal conditions and resorption is a pathologic process .
this pathologic root resorption process has been broadly classified based on the site of occurrence into external or internal resorption2 .
several causes have been attributed to external resorption including inflammation , pressure , and certain systemic disorders .
various methods have been advocated to assess resorption including histology7 , the use of panoramic and periapical radiography6 and scanning electron microscopy ( sem)3 .
the use of traditional methods , such as histologic and radiographic analyses to study this process has inherent shortcomings .
the use of sem has certain advantages over the other traditional methods , as it permits the assessment in multiple planes .
the morphologic image represents the visual expression of functional activity and thus the aim of this study was to examine if there are qualitative differences in the appearance of external root resorption patterns of primary teeth undergoing physiologic resorption and permanent teeth undergoing pathological root resorption in different conditions .
a total of 40 human teeth were collected over a period of 3 months and divided into 4 groups .
all teeth were included in the study after being examined under a stereomicroscope at 10x magnification for the presence of root resorption areas .
group i:10 primary molars that exfoliated due to physiologic root resorption ; group ii : 10 permanent teeth associated with periapical granulomas showing signs of pathologic resorption due to the inflammatory process involved ; group iii : 10 premolars subjected to a short duration of light orthodontic force by conventional mechanics for a period of less than 2 months and therapeutically extracted during the course of therapy ; group iv : 10 permanent teeth associated with odontogenic tumors .
after extraction and removal of the soft tissue remnants , all teeth were immediately immersed in 5% sodium hypochlorite solution to remove the remaining nonmineralized organic components .
the roots were separated from the crown and then dried in a desiccator for 24 h prior to analysis .
the roots were then mounted on the stubs using double - sided adhesive tape and sputter coated with platinum before examination .
the specimens were then analyzed in a scanning electron microscope ( jeol jsm 6380a , jeol , tokyo japan ) at 20 kv beam current .
the specific areas of interest on the teeth were examined under various 20x , 100x , 250x , 500x and 1000x magnifications .
the lowest magnifications were used to identify the areas of resorption and the higher magnifications to study the specific patterns of resorption .
areas of the roots undergoing resorption were compared to that of a normal root and presence of any recurring pattern was noted .
the resorption patterns were described on the basis of extent and the distinct surface appearance .
a total of 40 human teeth were collected over a period of 3 months and divided into 4 groups .
all teeth were included in the study after being examined under a stereomicroscope at 10x magnification for the presence of root resorption areas .
group i:10 primary molars that exfoliated due to physiologic root resorption ; group ii : 10 permanent teeth associated with periapical granulomas showing signs of pathologic resorption due to the inflammatory process involved ; group iii : 10 premolars subjected to a short duration of light orthodontic force by conventional mechanics for a period of less than 2 months and therapeutically extracted during the course of therapy ; group iv : 10 permanent teeth associated with odontogenic tumors .
after extraction and removal of the soft tissue remnants , all teeth were immediately immersed in 5% sodium hypochlorite solution to remove the remaining nonmineralized organic components .
the roots were separated from the crown and then dried in a desiccator for 24 h prior to analysis .
the roots were then mounted on the stubs using double - sided adhesive tape and sputter coated with platinum before examination .
the specimens were then analyzed in a scanning electron microscope ( jeol jsm 6380a , jeol , tokyo japan ) at 20 kv beam current .
the specific areas of interest on the teeth were examined under various 20x , 100x , 250x , 500x and 1000x magnifications .
the lowest magnifications were used to identify the areas of resorption and the higher magnifications to study the specific patterns of resorption .
areas of the roots undergoing resorption were compared to that of a normal root and presence of any recurring pattern was noted .
the resorption patterns were described on the basis of extent and the distinct surface appearance .
figure 1a is the image of a primary molar undergoing resorption examined at 12x magnification .
presence of extensive areas of resorption can be noticed on the lingual aspect of all roots leading to blunting of the apical regions .
figure 1b is a higher magnification ( 100x ) of the apical region that reveals rounding and blunting of this region .
the shiny white area inside the apical foramen ( dark arrow ) is nothing but remnant of pulpal soft tissue .
there is a loss of normal smooth topography of the root surface that now shows the uniform presence of numerous irregularly shaped concavities .
( at 500x ) , the presence of several individual resorption lacunae can be clearly appreciated .
the area looks as if individual adjacent areas of tooth material have been scooped out ( dark arrows ) from the root surface . in figure 1d ( 1000x ) , the dentinal tubule entrance is visible ( dark arrow ) .
figure 2a shows a mandibular permanent molar undergoing root resorption due to an associated periapical granuloma . there is a blunting of the entire apical region of the roots that is evident at 10x magnification . at this magnification
the entire apical root surface has a smooth eggshell like appearance . at the apical region
, however , there is also evidence of resorption occurring near the foramen ( dark arrow ) .
there is clear evidence of resorptive activity seen around the periapical region ( dark arrow ) .
areas of resorption can also be appreciated in the apical third of the root , particularly close to the apex , leading to a funnellike appearance .
there is a widening of the apical foramen diameter due to the loss of cementum around the periapical region .
there is also the presence of a clear border demarcating the affected and unaffected areas at the apical foramen region ( light arrow ) . in figure 2c ( 250x )
, the periphery of the foramen shows areas of normal cementum interspersed with resorption craters ( dark arrows and light arrows ) . differently from the primary teeth ,
the resorptive areas are not uniformly seen . in figure 2d ( 500x ) , the individual craters of resorption are more visible ( dark arrow ) . in figure 3a , the image shows a mandibular first premolar undergoing pressure resorption during the course of orthodontic treatment resorption seen mostly along mesial and buccal aspect .
active resorption is present even though the tooth was subjected to orthodontic force for a relatively short duration of less than two months . here
areas of resorption are ( dark arrows ) seen along most of the entire surface leading to loss of normal smooth appearance of this surface .
a slight tilt of the specimen was carried out so that a more detailed analysis of the mesial aspect could be undertaken . in figure 3b
( 100 x ) , the presence of resorption bays ( dark arrows ) with adjoining areas of cemental repair ( light arrow ) can be appreciated . in figures 3c and 3d ( 250 and 500x ) , the presence of an individual resorption crater of irregular size and shape is specifically visible ( dark arrow ) with adjoining normal cementum ( light arrow ) . in figure 4a ( 13x ) , which shows a permanent molar associated with an ameloblastoma , the most striking feature is the extensive loss of root length as a result of the resorption .
resorptive activity is evident in each of the individual roots and even in between them ( light arrows ) with differing appearance .
however interestingly unlike the smooth blunting of the apex seen in the earlier conditions the roots have a sharp cut appearance ( dark arrows ) . in figure 4b ( 70x ) , the presence of a single very large resorption crater from in between the roots is seen ( dark arrow ) .
higher magnifications ( 250 and 500x ) did not reveal any specific patterns of resorption but loss of normal smooth root surface topography .
figure 4c ( 20x ) shows a haphazard and ragged appearance of the root of an anterior tooth with extensive loss of root length .
figure 1a is the image of a primary molar undergoing resorption examined at 12x magnification .
presence of extensive areas of resorption can be noticed on the lingual aspect of all roots leading to blunting of the apical regions .
figure 1b is a higher magnification ( 100x ) of the apical region that reveals rounding and blunting of this region .
the shiny white area inside the apical foramen ( dark arrow ) is nothing but remnant of pulpal soft tissue .
there is a loss of normal smooth topography of the root surface that now shows the uniform presence of numerous irregularly shaped concavities .
( at 500x ) , the presence of several individual resorption lacunae can be clearly appreciated .
the area looks as if individual adjacent areas of tooth material have been scooped out ( dark arrows ) from the root surface . in figure 1d ( 1000x )
figure 2a shows a mandibular permanent molar undergoing root resorption due to an associated periapical granuloma . there is a blunting of the entire apical region of the roots that is evident at 10x magnification . at this magnification
the entire apical root surface has a smooth eggshell like appearance . at the apical region
, however , there is also evidence of resorption occurring near the foramen ( dark arrow ) .
there is clear evidence of resorptive activity seen around the periapical region ( dark arrow ) .
areas of resorption can also be appreciated in the apical third of the root , particularly close to the apex , leading to a funnellike appearance .
there is a widening of the apical foramen diameter due to the loss of cementum around the periapical region .
there is also the presence of a clear border demarcating the affected and unaffected areas at the apical foramen region ( light arrow ) . in figure 2c ( 250x )
, the periphery of the foramen shows areas of normal cementum interspersed with resorption craters ( dark arrows and light arrows ) .
differently from the primary teeth , the resorptive areas are not uniformly seen . in figure 2d ( 500x ) , the individual craters of resorption are more visible ( dark arrow ) .
in figure 3a , the image shows a mandibular first premolar undergoing pressure resorption during the course of orthodontic treatment resorption seen mostly along mesial and buccal aspect .
active resorption is present even though the tooth was subjected to orthodontic force for a relatively short duration of less than two months . here
areas of resorption are ( dark arrows ) seen along most of the entire surface leading to loss of normal smooth appearance of this surface .
a slight tilt of the specimen was carried out so that a more detailed analysis of the mesial aspect could be undertaken . in figure 3b
( 100 x ) , the presence of resorption bays ( dark arrows ) with adjoining areas of cemental repair ( light arrow ) can be appreciated . in figures 3c and 3d ( 250 and 500x ) , the presence of an individual resorption crater of irregular size and shape is specifically visible ( dark arrow ) with adjoining normal cementum ( light arrow ) .
in figure 4a ( 13x ) , which shows a permanent molar associated with an ameloblastoma , the most striking feature is the extensive loss of root length as a result of the resorption .
resorptive activity is evident in each of the individual roots and even in between them ( light arrows ) with differing appearance .
however interestingly unlike the smooth blunting of the apex seen in the earlier conditions the roots have a sharp cut appearance ( dark arrows ) . in figure 4b ( 70x ) , the presence of a single very large resorption crater from in between the roots is seen ( dark arrow ) .
higher magnifications ( 250 and 500x ) did not reveal any specific patterns of resorption but loss of normal smooth root surface topography .
figure 4c ( 20x ) shows a haphazard and ragged appearance of the root of an anterior tooth with extensive loss of root length .
different causes have been attributed to the root resorption process including pressure , inflammation , neoplastic process and systemic conditions .
the different causes of resorption in physiological and pathological conditions may lead to a diverse resorptive potential by the odontoclasts . as the morphologic image represents functional activity
it was our intention to look at resorption patterns resulting from different pathological and physiological processes applying sem .
the use of histologic sections or radiographs for studying root resorption may lead to missing areas of resorption in certain planes of interest or even small areas of transient resorption may not show up in these methods .
sem on the other hand permits visualization of surface structures and is particularly valuable in recording three - dimensional morphology of mineralized tissues .
this technique provides enhanced visual assessment of root surfaces , unattainable with histological models reconstructed from serial sections . the ability to tilt the mounted sample in all three planes of space to a certain degree while analyzing it permits us to focus on specific areas of interest . in the present study ,
the primary teeth examined with sem were in different stages of root resorption but most of them had lost more than two thirds to three quarters of their root length prior to exfoliation . to this day
it is believed that the emerging permanent tooth applies pressure on the primary predecessor leading to activation of odontoclasts and ultimately exfoliation4 . though the resorption of primary teeth is not solely dependent on the pressure of erupting permanent teeth the site of resorption in all the primary molars studied was in close proximity to the succeeding permanent follicle .
another notable feature was that the resorption was of varying extent in different roots of the same molar .
roots closer to the follicle showed a greater degree of resorption with extent of root divergence and inter root distant affecting the final outcome .
it has been reported that physiologic process is a not a continuous process but an intermittent process with periods of resorption alternating repair activity . however , in all the primary molars examined at various magnifications in the present study the pattern of root resorption in primary teeth had a regular appearance .
this pattern is probably a reflection of the slow ongoing physiologic process of root resorption . on the other hand , the site of the lesion in periapical granuloma accounts for the severity and localization of the resorptive process to the vicinity of the apical region .
this indicates that resorption is actively taking place mainly along the localized areas of pressure from the periapical granuloma .
the areas of resorption were localized at the apex with a clear border demarcating it from the adjoining normal area indicating that pressure from the lesion could be a major contributing factor to the process .
however , root resorption is also seen after the application of light orthodontic force even within a short - duration period10 .
the point of force application with conventional orthodontic mechanics is from the buccal aspect . in the early stages of treatment , this causes crown buccal and root lingual tipping of premolars leading to opposing areas of tension and compression in the crown and root .
the resorption process on examination with sem was however not wholly restricted to any particular area .
based on extent varying degrees of orthodontically induced inflammatory root resorption have been described1 . in the present study , the resorption in all premolars was restricted to mild surface cemental resorption probably because of the short duration for which teeth were subjected to force application .
the extent and site of resorption was not uniform to reach a categorical conclusion as a multitude of local and systemic factors play a vital role in orthodontically induced root resorption .
it is important to know that even in early stages of orthodontic treatment there is evidence of cemental resorption , which may not always be evident on radiographs unless it reaches an advanced stage . in the teeth associated with odontogenic tumors ,
most teeth , however , showed an extensive loss of root length form the resorptive process . previously there have been reports of root resorption in teeth in the proximity of odontogenic tumors , such as ameloblastoma8 .
two teeth associated with ameloblastomas were chosen for closer examination as they had a strikingly different appearance of roots .
a sharp cut appearance of the roots was visible echoing the aggressive nature of the rapidly growing lesion .
this aggressive root resorption pattern reflects the loss of cellular regulatory control synonymous with tumor pathology .
this is an interesting research study for the knowledge of the root resorption pattern under different physiological and pathological conditions .
there were differences in the patterns of external root resorption that resulted from different conditions when studied under sem .
although study is valuable as an additional source of basic information , it has limited clinical application .
further investigations on the quantitative and molecular aspects of root resorption will shed light on several unknown features providing a database for exciting new fields like forensic dentistry . | objective : the aim of this study was to examine if there are qualitative differences in the appearance of external root resorption patterns of primary teeth undergoing physiologic resorption and permanent teeth undergoing pathological root resorption in different conditions.material and methods : a total of 40 teeth undergoing external root resorption in different conditions were divided into 4 groups and prepared for examination under scanning electron microscopy at magnifications ranging from 20x to 1000x .
group i : 10 primary molars exfoliated due to physiologic root resorption ; group ii : 10 permanent teeth with periapical granulomas showing signs of resorption ; group iii:10 permanent teeth therapeutically extracted during the course of orthodontic therapy with evidence of resorption , and group iv : 10 permanent teeth associated with odontogenic tumors that showed evidence of resorption.results:in group i , the primary teeth undergoing resorption showed smooth extensive and predominantly regular areas reflecting the slow ongoing physiologic process . in group ii , the teeth with periapical granulomas showed the resorption was localized to apex with a funnel shaped appearance in most cases .
teeth in group iii , which had been subjected to a short period of light orthodontic force , showed the presence of numerous resorption craters with adjoining areas of cemental repair in some cases .
teeth associated with odontogenic tumors in group iv showed many variations in the patterns of resorption with extensive loss of root length and a sharp cut appearance of the root in most cases.conclusion:differences were observed in the patterns of external root resorption among the studied groups of primary and permanent teeth under physiologic and pathological conditions . |
members of the nuclear factor - kappa b ( nf-baaabbreviations : pkc , protein kinase c , nf-b , nuclear factor - kappa b ; ib , inhibitor of nf-b ; ikk , b- kinase ; carma , caspase recruitment domain ( card ) carrying member of the membrane associated guanylate kinase ( maguk ) family proteins ; bcl-10 , b - cell lymphoma / leukemia 10 protein ; malt , mucosa associated lymphoid tissue translocation protein ; tnf- , tumor necrosis factor alpha ; traf , tnf receptor associated factor ; ubc13 , ubiquitin conjugating enzyme 13 ; c - flip , cellular fas - associated death domain - like il-1-converting enzyme inhibitory protein ; hts , high throughput screening ; mlsmr , nih molecular libraries small molecule repository ; hek293 , human embryonic kidney 293 cells ; il-8 , interleukin-8 ; cd , cluster of differentiation ; pma , phorbol-12-myristate-13-acetate ; cid , compound i d ; sar , structureactivity relationship ; adme / t , absorption , distribution , metabolism , excretion , and toxicology ; pampa , parallel artificial membrane permeability assay ; cyp3a4 , cytochrome p450 3a4 ; nimh - pdsp , national institute of mental health psychoactive drug screening program ; mlpcn , the molecular libraries probe centers network . )
family of transcription factors control many crucial physiological and pathological processes including host - defense , immune response , inflammation , and cancer . a number of intracellular signaling pathways leading to nf-b activation have been elucidated and have been the subject of several excellent reviews .
most nf-b activation pathways converge on ib- kinase ( ikk ) activation , resulting in phosphorylation of ib- , and thereby targeting this protein for ubiquitination with proteasome - dependent destruction
. this cascade releases nf-b heterodimers from the ib- complex in the cytosol and allows for translocation into nucleus where transcription of various pro - inflammatory genes is initiated.(5 ) the various pathways upstream of ikk activation are diverse , with most of them linked to cell surface and intracellular receptors that sense cell damage and pathogens as well as activation in response to cytokines .
accordingly , small molecule pathway selective inhibitors can serve as powerful chemical tools to dissect these molecular networks which are crucial for normal cellular survival but are dysregulated in specific disease states .
abbreviations : pkc , protein kinase c , nf-b , nuclear factor - kappa b ; ib , inhibitor of nf-b ; ikk , b- kinase ; carma , caspase recruitment domain ( card ) carrying member of the membrane associated guanylate kinase ( maguk ) family proteins ; bcl-10 , b - cell lymphoma / leukemia 10 protein ; malt , mucosa associated lymphoid tissue translocation protein ; tnf- , tumor necrosis factor alpha ; traf , tnf receptor associated factor ; ubc13 , ubiquitin conjugating enzyme 13 ; c - flip , cellular fas - associated death domain - like il-1-converting enzyme inhibitory protein ; hts , high throughput screening ; mlsmr , nih molecular libraries small molecule repository ; hek293 , human embryonic kidney 293 cells ; il-8 , interleukin-8 ; cd , cluster of differentiation ; pma , phorbol-12-myristate-13-acetate ; cid , compound i d ; sar , structureactivity relationship ; adme / t , absorption , distribution , metabolism , excretion , and toxicology ; pampa , parallel artificial membrane permeability assay ; cyp3a4 , cytochrome p450 3a4 ; nimh - pdsp , national institute of mental health psychoactive drug screening program ; mlpcn , the molecular libraries probe centers network .
the nf-b pathway activated by antigen receptors is critical for adaptive immunity contributing to t and b lymphocyte activation , proliferation , cell survival , and effector functions .
dysregulated nf-b activation in lymphocytes contributes to development of a variety of autoimmune - based disease states , chronic inflammation , and lymphoid malignancy .
the nf-b activation pathway linked to antigen receptors involves a cascade of adapter and signal transduction proteins that at minimum include a carma family protein , bcl-10 , malt ( paracaspase ) , traf6 , ubc13 , caspase-8 , and c - flip .
formation of this complex is initiated by protein kinase c ( pkc)-mediated phosphorylation of carma proteins . in t and b cells , this pathway is initiated by pkc- and pkc- , respectively , leading ultimately to ikk activation through a mechanism potentially involving lysine 63-linked polyubiquitination of ikk-.(8 ) thus , the antigen receptor pathway for nf-b activation is both initiated and concluded by activation of protein kinases , namely the pkcs and ikks , respectively .
although the ikk complex and its individual subunits represent logical targets for potential drug discovery , small - molecule inhibitors of ikks suppress all known nf-b activation pathways .
this approach lacks the selectivity required to inhibit lymphocyte responses without simultaneously interfering with overall innate immune responses and leads to general immunosuppression attended by risk of pathogenic infection as well as other severe side effects .
a library of approximately 110,000 compounds from the nih molecular libraries small molecule repository ( mlsmr ) compound collection was screened using a hek293 cell - based nf-b luciferase reporter gene strategy .
primary hit compounds with an ic50 10 m were further characterized by 14 additional cell - based counter - screens that helped determine pathway selectivity in the hek293 cell line and other lymphocytes .
ultimately , three hit series emerged and were confirmed through an extensive counter - screen assay platform to be selective for the antigen receptor activation pathway ( figure 1a ) .
of the three primary hit classes identified in the hts campaign , only the 2-aminobenzimidazole series represented by 1-(3,5-di - tert - butyl-4-hydroxyphenyl)-2-(2-((3-hydroxypropyl)amino)-5,6-dimethyl-1h - benzo[d]imidazol-1-yl)ethanone , 1a ( cid-2858522 ) , selectively inhibited the nf-b pathway induced by pkc activators and antigen receptors .
compound 1a did not inhibit activation of nf-b through tnf- stimulation ( figure 1b ) .
details of the comprehensive screening platform and pathway specific cell - based selectivity assessment , as outlined above , are described in our accompanying paper.(13 ) ( a ) three scaffold classes ( with ic50 < 10 m , most active members shown ) identified in the high throughput screening ( hts ) campaign .
( b ) selectivity of 1a for inhibition of the nf-b antigen receptor pathway induced by pma ( a pkc activator ) vs tnf- activation . on the basis of the above information and selectivity profile , the 2-aminobenzimidazole series was selected for chemistry follow - up as it met our initial potency criteria ( < 1 m ) and selective inhibition of antigen receptor - mediated nf-b activation .
we discuss the development of an efficient synthetic route to the 2-aminobenzimidazole scaffold , the design and synthesis of sar analogues , as well as in vitro and in vivo pharmacological profiling of 1a .
these structureactivity relationship studies provide molecular insight for further optimization and synthesis of proteomic probes , which include analogues to be used for the identification of the precise cellular target protein and subsequent mode of action studies .
the initial hts hit compounds of interest ( figure 1 ) were resynthesized so that identity and cellular activity could be confirmed .
additional commercially available dry powder samples for closely related analogues were ordered ( chembridge corporation , san diego , ca ) , and the quality , purity , and identity for each compound was confirmed . in the case
where the commercial analogues were not homogeneous , they were purified by preparative hplc to 98% .
each compound was assessed for bioactivity using a cell - based assay employing hek293 cells containing a stably integrated nf-b - luciferase reporter gene , which was stimulated with phorbol-12-myristate-13-acetate ( pma ) and ionomycin .
approach ultimately provided an initial set of 2-aminobenzimidazoles having nf-b - luciferase ic50 values in the micromolar to nanomolar range as shown in figure 1s ( see supporting information ) .
analysis of the structureactivity relationships ( sar ) pointed to features responsible for optimal activity .
truncation of 2-aminopropanol substituent and removal of both 5- and 6-methyl groups resulted in equipotent inhibitors ( compounds 4 and 5 , figure 1s , see supporting information ) .
most importantly , the di - t - butyl phenol moiety present in the n1-acetophenone substituent of the 2-aminobenzimidazole scaffold seemed to be essential for activity ( compounds 1a and 68 , figure 1s , see supporting information ) .
3,5-di - t - butyl-4-methylphenol and 6,6-methylenebis-(2-tert - butyl-4-methylphenol ) , commercial antioxidants were screened and found to be inactive , thus ruling out any connection between a potential antioxidant pharmacophore ( possibly affecting nitric oxide signaling ) and observed nf-b inhibitory activity of 1a ( compounds 9 and 10 , figure 1s , see supporting information).(14 ) the general synthetic route toward the targeted series is illustrated in scheme 1 .
accordingly , the aryl diamine 11a was condensed with potassium ethyl xanthate in ethanol at reflux to afford 12a , which on bromination furnished the key 2-bromo-5,6-dimethylbenzimidazole intermediate 13a .
amination of 13a with 3-aminopropanol under neat conditions at high temperature afforded the highly polar intermediate 14a , which required extensive chromatography for purification.(18 ) it was found that conducting the amination reaction under solvent - free microwave heating conditions at 180 c/25 min afforded 14a in high yield and purity after simple extraction .
the final alkylation of intermediate 14a with -bromo-3,5-di - tert - butyl-4-hydroxyacetophenone ( 15a ) was conducted in n - butanol at reflux to furnish 1a in moderate yield ( scheme 1 , method a ) .
the low isolated yield was attributed to the formation of tricyclic side product 16 via an intramolecular schiff - base formation.(19 ) purified 16 was found to be inactive ( ic50 > 25 m ) under the assay conditions .
this competing intramolecular cyclization was completely eliminated by conducting the reaction at 23 c in methanol using sodium bicarbonate as a mild base .
although alkylation under these conditions was incomplete even after 3 days , the formation of 16 was attenuated and compound 1a was obtained in 78% yield and in > 98% purity ( scheme 1 , method b ) .
the optimized route was utilized for the synthesis of an analogue library designed to explore , ( a ) monosubstitution at 5- and 6-positions of 2-aminobenzimidazole core , ( b ) substitution at the 2-position with diverse amines , ( c ) substitution of the aryl ring of the n1-acetophenone moiety , and ( d ) replacement of the core 2-aminobenzimidazole with related 2h - benzimidazole and benzimidazolone ring systems .
commercially available 4-monosubstituted - phenyl-1,2-diamines were converted to the corresponding 2-aminobenzimidazoles ( 14b , d , e , scheme 1 ) via the corresponding 2-bromo intermediates ( 13b , d , e ) .
subsequent alkylation with 15a under previously optimized conditions furnished the regioisomeric 5- and 6-substituted benzimidazole analogues ( 1b , d , e ; 17b , d , e ) , which could be separated by chromatography and characterized .
the 5- and 6-bromo analogues ( 1b and 17b ) , when subjected to the suzuki coupling protocol with phenyl boronic acid , afforded 5- and 6-phenyl substituted analogues 1c and 17c , respectively .
the 5- and 6-nitro analogues ( 1e and 17e ) were reduced to the corresponding amino compounds 1f and 17f , respectively , via hydrogenation .
a variety of amines were then reacted with 3a under the previously optimized amination conditions .
subsequent alkylation with 15a generated the corresponding 2-aminoalkyl substituted analogues ( 1g1k , scheme 2a ) .
the analogue library was then evaluated for inhibition of nf-b activation using the cell - based hek293 nf-b luciferase reporter gene assay . to study the effect of variation of substitution on the aryl ring at the n1-position of 1a ,
suitably substituted -bromoacetophenones were synthesized ( 15l15n ) from commercially available starting materials and then treated with intermediate 14a to access analogues 1l1n ( scheme 2b ; see supporting information for details ) . to replace the 2-aminobenzimidazole core of 1a with related heterocycles , commercially available 5,6-dimethyl-1h - benzimidazole and 5,6-dimethyl-1h - benzimidazol-2-one were alkylated with 15a in the presence of sodium hydride to furnish 18 and 19 in 63 and 55% yield , respectively ( scheme 3 ) . through a systematic substitution process
, we probed the individual contribution of the substituents at the 5- and 6-positions of the benzimidazole aryl moiety .
it was found that substituents at the 5-position were better tolerated than at 6-position and activity of analogues was sensitive to both the electronic and steric nature of the substituents ( 1bf and 17bf , table 1a ) .
the results indicate that the 6-position is more sensitive to the steric bulk of the substituent than the 5-position because replacement of either methyl group with a bromine led to a 4080-fold drop in activity ( 1a , 1b , and 17b , table 1a ) .
similarly , the 5-nitro analogue was 20-fold less active than 1a while the 6-nitro analogue was found to be inactive ( 1e and 17e , table 1a ) .
installation of a planar phenyl group resulted in analogues with activity approaching that of 1a , suggesting a positive hydrophobic interaction at the 5-position ( 1c and 17c , table 1a ) .
an unexpected result was that the 5- and 6-amino analogues were the most active analogues in this substitution series , suggesting access to h - bonding interactions ( 1f and 17f , table 1a ) .
it was found that while o - methylation of the 2-aminopropanol side chain retained most of the activity ( 1 g , table 1b ) , removal of the hydroxyl substituent in the 2-butylamino analogue led to a moderate loss of activity ( 1h , table 1b ) , suggesting that the electronegative oxygen atom is not essential for activity .
truncation of the butyl group in the 2-methylamino analogue led to improved activity and was found to be as potent as 1 g ( 1i , table 1b ) .
replacement of 2-aminopropanol side chain with a conformationally restricted 4-hydroxycyclohexylamine did not result in any improvement over 1a ( 1j , table 1b ) .
the presence of a tertiary amine at the benzimidazole 2-position was found to abolish activity ( 1k , table 1b ) .
these results , taken together , suggest that the entire 2-aminopropanol substituent is likely solvent exposed and may not be completely required ( except the nh moiety ) for binding to the cellular target protein .
replacement of the n-3,5-dimethyl-4-hydroxyacetophenone with either simple acetophenones or 3,5-dimethyl-4-hydroxyacetophenone resulted in complete loss of activity ( 610 , figure 1s , see supporting information ) .
it was found that the des-4-phenoxy analogue retained complete activity ( 1l , table 1c ) , and hence the 4-phenoxy substituent is not essential for activity , further confirming that nf-b inhibition is not due to antioxidant activity .
elimination or replacement of one of the tert - butyl groups with a tert - butoxy group resulted in completely inactive analogues , suggesting that the di - tert - butyl phenyl group might represent a conformational lock and is extremely important for binding / activity ( 1 m and 1n , table 1 ) .
replacement of the 2-alkylamino moiety with hydrogen led to a significant loss of activity , however , the 2-benzimidazolone analogue was essentially equipotent to 1a ( 18 and 19 , figure 2 ) .
these results indicate that a polar heteroatom is needed at the 2-position for activity , yet the 2-amino group is completely replaceable .
this observation has important bearing on future probe sar because it allows further derivatization at the n position allowing exploration of additional chemical space via alkylation or acylation .
bioactivity of 2h - benzimidazole 18 , 2-benzimidazolone 19 , azide 1o , and alkynyl ester 1p .
because the precise cellular protein target is unknown , we capitalized on this information to synthesize two analogues that have an azide and a terminal alkyne incorporated in the 2-aminoalkyl side chain ( 1o , 1p , figure 2 ) .
the objective was to utilize these as building blocks for the synthesis of pull down probes for protein target identification .
we were gratified that both analogues retain sufficient bioactivity ( within 6-fold ) and are currently being utilized in ongoing studies involving bioconjugation via click chemistry to furnish biotinylated derivatives for affinity studies .
compound 1a was subjected to in vitro adme / t assays to profile its general pharmacological properties ( table 2 ) .
compound 1a exhibits good to moderate solubility across ph ranges and moderate to high effective cell permeability ( pampa ) .
although plasma stability is good , the probe exhibits high affinity toward plasma protein binding , possibly limiting available in vivo free fraction concentration .
the compound was found to be nontoxic to human hepatocytes ( fa2n-4 cells ; ld50 = 47 m ) and in the hek293 cell line ( ld50 > 50 m ) used in the primary activity assay .
compound 1a is moderately stable to human hepatic microsomes at 1 and 10 m but was rapidly metabolized by murine microsomes .
pampa pe : low 5 10 , moderate 250 10 , high 1000 10 .
plasma / pbs ; compound at 40 m , 3 h. in vivo dose - exposure profiling of 1a was conducted using a small cohort of three male mice was subjected to single ip doses at 10 , 30 , and 50 mg / kg .
blood was drawn at 0.5 and 3 h , and subsequent lc / ms analysis of pooled samples was performed to determine the overall blood levels of compound ( table 3 ) .
probe compound 1a exhibited nonlinear pharmacokinetics , showing higher serum levels at the 0.5 h measurement time for the 30 mg / kg dose compared to 50 mg / kg but displaying typical dose - dependent behavior when measured at t = 3 h. the increasing accumulation seen at a dose of 50 mg / kg may be due to a depot effect created by cyp3a4 inhibition .
the cohort exhibited clear signs of morbidity at t = 3 h at the 50 mg / kg dose .
on the basis of the above data , blood levels can be expected to reach maximum concentrations approximately 10-fold higher than ic50 at a dose of 30 mg / kg , thus defining a promising dose for future applications in short - term , acute biomarker studies such as in vivo measurements of cytokine production .
pkcs are proximal kinases in the nf-b pathways activated by pma / ionomycin and by t - cell and b - cell antigen receptors , while the ikks are distal kinases operating in the terminal segments of these and other nf-b activation pathways.(2 ) at concentrations up to 8 m , 1a failed to suppress pkc- and pkc- ( the pkc family members implicated in tcr / bcr signaling ) , and ikk- ( a component of the ikk complex ) , while known pkc and ikk inhibitors and the broad - spectrum kinase inhibitor staurosporine afforded potent inhibition .
in addition , 1a did not inhibit endogenous pkc activity or phosphorylation of downstream substrates.(13 ) probe 1a did not directly inhibit pkc- , pkc- , or ikk-. a broad kinome screen was then conducted using kinomescan ( ambit biosciences ) platform
. of 353 protein kinases surveyed , 1a inhibited only three by more than 50% at 10 m ; raf ( 57% inhibition ) , tlk1 ( 70% inhibition ) , and jak2 ( 53% inhibition ) , none of which are clearly implicated in nf-b regulation .
compound 1a was screened for general functional activity to determine effects on second messenger systems using a subset of cloned human or rodent receptors , ion channels , and transporters ( nimh - pdsp , university of north carolina chapel hill ) .
of the 92 targets screened , 1a showed greater than 50% inhibition at 10 m for only eight targets ( see supporting information for details ) .
for all active targets assessed , the subsequent ic50 values were greater than 5 m , highlighting that 1a does not exhibit significant off - target activity.(20 )
a series of substituted 2-aminobenzimidazoles has been identified that serve as promising research tools as pathway - selective chemical inhibitors of nf-b activation induced by activators of pkc signaling through b and t cell antigen receptors .
interestingly , the mlpcn probe candidate ( 1a ) was identified directly from the screening hit set and only lesser or equipotent analogues have been prepared , indicating a limited sar for the series .
compound 1a was found to be noncytotoxic and inhibited il-8 production induced by pkc activators in hek293 cells ( ic50 < 0.1 m ) , while failing to inhibit nf-b reporter gene activation by agonists of the other nf-b activation pathways .
compound 1a attenuated cd3/cd28 and pma / ionomycin - induced production of il-2 by jurkat t - cells ( ic50 < 5 m ) and anti - igm - stimulated proliferation of murine b - lymphocytes ( ic50 2 m ) .
we have used the sar data to generate the azide and alkynyl containing derivatives 1o and 1p .
these have been utilized in the synthesis of biotinylated conjugates directed toward affinity - based target identification and future mode of action studies that will be reported separately .
in addition , the equipotent 2-benzimidazolone analogues ( 19 ) provide a modified scaffold with an additional n3-site for future exploration .
compound 1a and its analogues represent new chemical tools that may be useful toward further development of therapeutically useful pathway selective modulators of nf-b activity that may find application in disease models of inflammation , cancer and other autoimmune diseases .
to a 20 dram vial equipped with a stir bar was added 2-aminobenzimidazole 14a ( 0.37 g , 1.6 mmol ) followed by corresponding 2-bromoacetophenone 15a ( 0.62 g , 1.9 mmol ) .
to the mixture was added 10 ml of methanol and solid nahco3 ( 3 equiv ) .
the mixture was stirred at 23 c for 3 days , after which the solvent was evaporated to a suitable volume and subsequent purification by silica gel flash column chromatography ( hexanes to 1:4 hexanes / ethyl acetate ) resulted in a yellow film , which on lyophilization from a 2:8 acetone / water mixture afforded 1a as white solid ( 0.61 g , 78% ) .
h nmr ( 400 mhz , acetone - d6 ) 7.87 ( s , 2h ) , 6.95 ( s , 1h ) , 6.77 ( s , 1h ) , 5.94 ( s , 1h ) , 5.52 ( s , 2h ) , 3.553.47 ( m , 2h ) , 3.42 ( t , j = 5.6 hz , 2h ) , 2.14 ( s , 2h ) , 2.12 ( s , 3h ) , 1.56 ( p , j = 6.0 hz , 2h ) , 1.40 ( s , 18h ) .
c nmr ( 100 mhz , acetone - d6 ) 191.58 , 159.66 , 156.73 , 141.17 , 137.58 , 134.43 , 128.85 , 127.29 , 127.19 , 126.16 , 116.74 , 108.72 , 57.65 , 48.47 , 39.19 , 34.90 , 34.67 , 19.77 , 19.74 .
lrms ( esi ) : calcd for c28h39n3o3 [ m + h ] = 466.3 , obsd [ m + h ] = 466.1 .
hrms ( esi ) : calcd for c28h39n3o3 [ m + h ] = 466.3070 , obsd [ m + h ] = 466.3091 . | a unique series of biologically active chemical probes that selectively inhibit nf-b activation induced by protein kinase c ( pkc ) pathway activators have been identified through a cell - based phenotypic reporter gene assay .
these 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by b and t cell antigen receptors . starting from the founding member of this chemical series 1a ( notated in pubchem as cid-2858522 ) , we report the chemical synthesis , sar studies , and pharmacological profiling of this pathway - selective inhibitor of nf-b activation . |
mild cognitive impairment ( mci ) is considered to be the transitional phase between normal
aging and dementia and is recognized as the prodromal stage of alzheimer s disease ( ad ) ,
which is the most common form of dementia1 .
evidence from neuropsychological and neuroimaging studies have
suggested that mild cognitive impairment ( mci ) represents a clinical prodromal status for
degenerative dementias such as ad2 .
for
example , a population - based study in sweden reported that the relative risks of progression
to dementia in a 3 year follow - up in subjects with mild , moderate , and severe cognitive
impairment without dementia were 3.6 , 5.4 , and 7.0 , respectively3 .
older people with mci show not only cognitive decline but
also frailty , which is one of the most crucial health issues among older adults4 .
the concept of frailty comprises five general factors : shrinking , weakness , exhaustion ,
slowness , and a low activity level5 , 6 .
older adults with cognitive impairment ,
including mci , are at higher risk of becoming frail than older adults who are cognitive
healthy .
in addition , older non - frail individuals with lower cognitive scores are
significantly more likely to acquire one or more components of frailty over 10 years
compared with those with higher cognitive scores7 . in turn , older adults with cognitive decline , such as mci , are more
vulnerable , so it is not enough to approach them from the standpoint of cognitive function
only . among the five factors of the frailty concept ,
exhaustion in particular contributes to a
more rapid onset of frailty and affects 80% of frail older adults8 .
based on self - reported symptoms , exhaustion was defined as
a condition characterized by unusual fatigue or a general loss of energy .
exhaustion in the
older population results from lower physiological capacity , as reflected in less total
energy expenditure and a low physiological reserve compared with younger people9 . these effects lead to an inactive lifestyle
that is accompanied by behavioral changes to save energy , thus preventing exhaustion from
daily activities9 , and a subsequent
decline in physical or cognitive function .
in fact , self - reported exhaustion negatively
affects functions among older people : gait speed10 , gait endurance11 , physical activity12 , and cognitive function13 .
so , self - reported exhaustion in older people is one of the health
problems to prevent . in older adults with mci , maintaining physical performance through a physically active
lifestyle may play an important role in delaying or preventing dementia14 .
it remains unclear whether or not older people with mci
have the association with self - reported exhaustion , although self - reported exhaustion was
found to be negatively associated with physical function and physical activity among healthy
older adults .
thus , it is important to clarify the relationship between exhaustion and
physical function , including physical activity , in older adults with mci .
the first aim of
this study was to investigate the prevalence of exhaustion among older adults with mci and
to compare it with the prevalence of exhaustion in the elderly population , which is
estimated to be about 20%15 .
the second
aim of this study was to investigate the prevalence of self - reported exhaustion among older
adults with mci and whether exhaustion is related to physical ability and physical activity
levels .
the study population and data in this study were obtained at baseline in a larger cohort
study , the obu study of health promotion for the elderly ( oshpe)16 . the classification of an individual as having mci was
based on the consensus previously published by petersen1 .
these criteria included ( 1 ) a subjective memory complaint , ( 2 )
objective cognitive decline , ( 3 ) intact general cognitive function ( mini - mental state
examination ( mmse ) score > 23)17 , ( 4 )
ability to perform activities of daily living ( adl ) independently , and ( 5 ) absence of
clinical criteria indicating dementia .
objective cognitive decline was defined as being
indicated by a score of more than 1.5 standard deviations from the score for healthy in the
oshpe database after adjustment for age16 .
we assessed objective cognitive function using the national center
for geriatrics and gerontology functional assessment tool .
this software is used on a tablet
pc and contains a battery of cognitive tests ; the details of these tests have been described
in detail elsewhere18 .
the tests comprise
eight tasks to assess memory , attention , executive memory , processing speed , and
visuospatial skills .
six hundred forty - nine older adults with mci were selected from the
larger cohort study as the potential study population for this study . after applying the
exclusion criteria , 356 people with mci
the exclusion criteria were : ( 1 ) history of cerebrovascular disease , parkinson s
disease , depression , or connective tissue disease ; ( 2 ) presence of a heart pacemaker ; ( 3 )
severe auditory or visual deficit ; ( 4 ) depression symptoms ( geriatric depression scale-15
score > 5)19 ; or ( 5 ) prior commitment
to other studies .
informed consent was obtained from all participants before inclusion in
the study , and the ethics committee of the national center for gerontology and geriatrics
approved the study protocol .
demographic data were collected including age , sex , number of medications , and body mass
index ( bmi ) .
self - reported exhaustion was identified using a questionnaire do you feel full
of energy ? from the study of osteoporotic fractures ( sof ) index5 .
participants who responded no to the question were placed
in the exhausted group ( eg ) , and those who answered yes were placed in the nonexhausted
group ( non - eg ) .
the sof index has been found to be as reliable and valid as the
cardiovascular health study ( chs ) index which is used most in the world , and this is
reliability .
in fact , ensrud et al . showed that in the sof index predicted disability ,
fracture , and death as well as the more complex chs index even though the sof index is a
simple index5 , 20 . to measure gait speed ,
participants were asked to walk on a straight walkway ( flat floor ,
11 m long ) at a comfortable and consistent gait speed .
gait speed was measured over a 5 m
distance from the midpoint of the walkway . the capacity for gait endurance
was assessed
using the 6 min walk test ( 6mwt ) , which was performed as described previously21 .
participants were instructed to walk as
far as possible in 6 min along a 10 m course . the distance ( in meters )
walked during the
6 min was recorded as the gait endurance . licensed and well - trained physical therapists
assessed these physical performance tests .
the life space of older adults was assessed using a japanese translation of life - space
assessment ( lsa)22 , 23 .
scores on the lsa range from 0 to 120 , with higher scores
reflecting a larger life space .
the frequency of movement ( how many days per week ) for the
five different life space levels was reported by the participants for four weeks before the
assessment . these levels are ( 1 ) rooms of the home besides where one sleeps ; ( 2 ) an area
directly outside the home ( e.g. , porch ) ; ( 3 ) places in one s neighborhood , other than one s
yard or apartment building ; ( 4 ) places outside one s neighborhood but within one s town ; and
( 5 ) places outside one s town .
participants were also asked whether they had assistance from
another person or devices to reach each level of life space .
the overall life space score
was computed by summing the products of the each life space level ( 15 ) multiplied by : ( a )
degree of independence ( 2 = independent , 1.5 = used equipment , 1 = had personal assistance )
and by ( b ) frequency of attainment ( 1 = less than once per week , 2 = 13 times per week , 3 =
46 times per week , 4 = daily ) . differences between groups were examined using the wilcoxon signed - rank test for
nonparametric data or student s t test for parametric data . only sex ( dichotomous outcome )
then , multivariate logistic regression analysis was
used to investigate whether the potential determinants were associated independently with
self - reported exhaustion , and odds ratios ( or ) and 95% confidence intervals ( 95% ci ) were
calculated . in the adjusted model , we examined the association of each variable ( gait speed ,
gait endurance , and life space ) with self - reported exhaustion status while accounting for
all covariates ( sex , age , bmi , number of medications , and mmse score ) . in the fully adjusted
model
, we then identified which variables were independently related to self - reported
exhaustion status when adjusted for gait speed , 6mwt result , lsa score , and all covariates .
all analyses were performed using the ibm spss ( version 20 ; ibm corp . ,
the characteristics of the participants in the non - eg and eg are summarized in table 1table 1.characteristics of participants in eg and non - egall participants(n = 356)non - eg(n = 294)eg(n = 62)p valuesex ( number of female ) [ n ( % ) ] 181 ( 50.8)145 ( 49.3)36 ( 58.1)0.26 age ( years ) [ mean se]71.6 0.371.5 0.371.9 0.70.61bmi ( kg / m2 ) [ mean se]23.4 0.223.4 0.223.4 0.20.80medications ( number of medication ) [ mean se]2.13 0.112.05 0.112.50 0.290.11mmse ( score ) [ mean se]26.7 0.126.7 0.127.1 0.20.10lsa ( score)90.4 0.891.8 0.983.9 2.2 < 0.016mwt ( m)449.5 4.1453.2 4.6432.1 9.1
< 0.05normal gait speed ( m / sec)1.36 0.011.37 0.011.33 0.030.16eg , exhaustion group ; se , standard error ; bmi , body mass index ; mmse , mini - mental
state examination ; lsa , life space assessment ; 6mwt , 6 minutes walking test .
significance set at
p < 0.05 .. sixty - two participants were classified into the eg , giving a 17.4% prevalence
of self - reported exhaustion .
the participants characteristics , including the mmse score ,
did not differ significantly between the groups .
the lsa scores and the 6mwt results ( but
not normal gait speed ) were significantly lower ( p < 0.01 , p < 0.05 , respectively ) in
the eg compared with the non - eg ( table 1 ) .
eg , exhaustion group ; se , standard error ; bmi , body mass index ; mmse , mini - mental
state examination ; lsa , life space assessment ; 6mwt , 6 minutes walking test . a was
test and the other variables were student t test .
significance set at
p < 0.05 . the results of the logistic regression analyses are shown in table 2table 2.univariate and multiple logistic regression analyses showing the cross - sectional
associations between exhaustion ( dependent variable ) and life - space assessment
( independent variable ) adjusted for the participants characteristicsadjusted model fully adjusted model or95% cip - valueor95% cip - valuelsa
( score)0.970.950.99 < 0.010.970.950.99 < 0.016mwt ( m)0.990.990.990.040.990.991.000.31normal gait speed ( m / sec)0.330.081.450.140.740.124.680.75all analyses were adjusted for age , gender , bmi , number of medications , and
mini - mental state examination score . or : odds ratio , ci : confidence interval , lsa :
life - space assessment , 6mwt : 6 min walking test . :
significance set at p < 0.05 . in the adjusted model , the lsa score ( or = 0.97 , 95% ci ; 0.950.99 ) and 6mwt
results ( or = 0.99 , 95% ci ; 0.990.99 ) were independently associated with exhaustion status .
however , in the fully adjusted model ( model 3 ) , only the lsa score was independently
associated with exhaustion status ( or = 0.97 , 95% ci ; 0.950.99 ) .
all analyses were adjusted for age , gender , bmi , number of medications , and
mini - mental state examination score . or : odds ratio , ci : confidence interval , lsa :
life - space assessment , 6mwt : 6 min walking test . :
the results of this study showed that the prevalence of self - reported exhaustion was 17.4% .
a previous study reported that the prevalence of self - reported exhaustion was 14.3% among
older males and 20.4% among older females15 .
the prevalence among older adults with mci in our study was
similar to that of healthy older adults .
people with mci who self - reported exhaustion
exhibited lower gait endurance performance and a lower lsa score compared with those without
exhaustion . in the logistic analyses adjusted for covariates ,
the lsa score was
significantly negatively associated with self - reported exhaustion , although there was no
significant relationship between exhaustion and gait speed or gait endurance .
with respect in the
frequency of going outdoors , frequency going outdoors was positively associated with health
status in older adults including psychological health , such as self - reported exhaustion ,
compared with older people who were housebound .
going outdoors frequently is positively
associated with psychological health but not with physical function or ability to perform
adl24 , 25 . on the other hand , infrequently going outdoors is negatively
associated with health problems among older people26 ,
27 .
going outdoors less than once a
week is linked to poorer psychological status , physical function , and adl ability compared
with going outdoors 23 times per week24 .
in the present study ,
our
current findings complement those of previous studies because we found an association
between exhaustion status and lsa score even after adjusting for physical function and other
covariates . in the fully adjusted multivariate regression analysis ,
self - reported exhaustion was
independently associated with decreased life space but not with gait speed or gait
endurance .
considering the effect of exhaustion on the capacity of energy in the physical
activity cycle , older adults cease to perform activities that are not necessary for daily
life to prevent exhaustion9 . in the
present study ,
expanding the life space may not be as important as maintaining gait function
for adl ; thus , older adults with self - reported exhaustion would have incorporated a smaller
life space . on the other hand , maintaining gait speed or gait endurance may be considered to
be more necessary for daily living for older adults than expanding life space and thus were
maintained despite self - reported exhaustion . in the present study , self - reported exhaustion was not associated with physical function
including normal gait speed or the results of the 6wmt . the discrepancy between our results
and those of other studies may reflect differences in methodology , although previous studies
showed an association between self - reported exhaustion and physical function .
other studies
have assessed gait speed using maximum speed10 and gait endurance using the 400 m walking test11 , whereas our study included a comfortable
speed test and the 6mwt , respectively .
another explanation for the discrepancies may relate
to the differences in the populations characteristics .
for example , previous studies have
sampled older adults between 65 and 102 years of age ( mean 75 years)10 or have targeted only persons of 75 years of age11 , whereas the mean age was 71.7 years in
our study . the influence of these variables should be investigated using a similar
methodology in future studies . to our knowledge
, our study is the first to report on the association between exhaustion
status and life space among older adults with mci .
lsa was developed to evaluate mobility
status by measuring the life space for elderly individuals living in a community22 .
however , the lsa score is influenced by
physical activity as well as physical performance , adl , and sociodemographic factors28 .
for example , the life space is sensitive
to marginal limitations before an older person experiences difficulties in performing adl or
instrumental adl22 .
life space may be a
useful identifier of older adults at risk for cognitive decline29 . in a previous study
, life space was related more closely
to cognitive function than to physical performance among older adults with amnestic mci30 .
self - reported exhaustion has also been
reported to be associated with cognitive function31 .
we found that self - reported exhaustion was negatively related to
life space among older adults with mci .
because both a small life space and self - reported
exhaustion adversely affect cognitive function , the combination of a small life space and
exhaustion may indicate a high risk for cognitive decline .
thus , it may be important to
assess not only cognitive function but also self - reported exhaustion for older adults with
mci .
first , the analysis was based on a
cross - sectional design , and we were therefore able to examine only the relationships with
exhaustion .
second , we did not collect data on other factors that may influence one s
feeling of exhaustion such as the cortisol level , which is a biomarker for the history of
stressful life events32 .
this factor and
others should be examined in future studies of exhaustion in older people . in summary ,
older adults with mci who have a restricted life space had self - reported
exhaustion .
less life space may lead to several health problems such as disability or
dementia in older adults with mci .
evaluation and preventive strategies to target
self - reported exhaustion are recommended to help maintain health status in this population .
future research is needed to identify ways for older people with mci to improve their
exhaustion status . | [ purpose ] older adults experience exhaustion - induced health problems , such as poor
physical function and low physical activity levels .
the associations between self - reported
exhaustion and physical function and activity are not clear in older adults with mild
cognitive impairment ( mci ) .
the aim of this study was to investigate the relationships
between self - reported exhaustion and physical function and activity in older adults with
mild cognitive impairment .
[ subjects ] a total of 356 older adults with mild cognitive
impairment ( mean age = 71.6 0.3 years , 50.8% women ) were included in this study .
[ methods ] self - reported exhaustion was identified by one item from the study of
osteoporotic fractures index .
gait speed , gait endurance , and life space were also
assessed .
[ results ] sixty - two participants reported having exhaustion , giving a 17.4%
prevalence of self - reported exhaustion among these individuals .
logistic regression
analysis showed that the life - space assessment score was the only parameter significantly
independently associated with exhaustion status ( adjusted odds ratio 0.97 , 95% confidence
interval 0.950.99 ) .
[ conclusion ] these results suggest that self - reported exhaustion is
associated with life space .
future research is needed to identify ways for older people
with mci to improve their exhaustion status . |
septic complications of central venous catheters ( cvcs ) remain a significant cause of patient morbidity and mortality both in the intensive care unit ( icu ) and on general hospital wards .
approximately 25% of cvcs inserted have been reported to become colonised , with rates of catheter - related blood stream infection ( crbsi ) varying between 0% and 11% [ 28 ] . on the basis of varied items of data [ 1 , 9 , 10 ] ,
there is preliminary evidence that colonisation of cvcs may play a role as a pathogenic mechanism for the development of crbsi .
there is limited data on cvc colonisation and crbsi rates outside the icu , in part due to the perceived high usage of cvcs within the icu setting .
however , because of the larger population of patients managed at ward level , the majority of cvcs may in fact be inserted and maintained in patients located outside of the icu on general wards , as high as 70% in one survey .
this suggests that hospital wide surveillance of cvc complications should be a priority and focus is now turning toward the measurement of crbsi incidence and epidemiology in cvcs inserted in patients at ward level .
it has also been contended , but with limited evidential support [ 1315 ] , that cvcs inserted in icu patients may have different incidence of infectious complications relative to cvcs inserted in ward patients , with different patterns of causative organisms .
this has obvious implications for best cvc insertion practice and empiric antimicrobial therapy in the event of cvc - related sepsis .
the aims of this 12-year study were to document the proportion of cvcs in a hospital - wide tpn population that became tip culture positive ( incorporating colonised and infected cvcs ) , and to examine the consistency of any relationship between culture positivity of cvc tips and development of crbsi
. we also aimed to define the microbiological pattern of causative organisms of culture positive and crbsi cvcs and to compare the pattern of organisms from cvcs inserted in the icu with cvcs inserted in the operating theatre ( ot ) for general hospital ward patients .
the tpn service is run from the icu under the clinical direction of the intensive care consultant staff and receives referrals from all disciplines .
all patients commenced on tpn are subject to a hospital audit process and a monitoring database was established in 1990 .
data were entered prospectively into tpn audit records by the dedicated tpn surveillance nurse practitioner .
all patients referred to the hospital tpn service for placement of a standard cvc for administration of tpn between january 1997 and january 2009 were included .
tunneled cvcs , peripherally inserted central venous catheters ( picc ) , peripheral intravenous cannulae , and cvcs that had been inserted in other hospitals were excluded , as were patients receiving home tpn .
a tpn audit committee , comprising the intensive care consultant and senior registrar , tpn surveillance nurse , and microbiology consultant , met quarterly throughout the study period .
data regarding all complications of tpn cvcs , including septic complications , were collected by the tpn surveillance nurse and these records were reviewed by the tpn audit committee .
the committee assigned a diagnostic category ( noninfected , colonised , or crbsi ) to all catheters potentially responsible for episodes of sepsis occurring in the hospital tpn population , using cdc compatible definitions .
all cvc tips from tpn patients were cultured , regardless of whether removal was because the cvc was no longer required or because of suspected cvc - related sepsis .
catheter tip culture positivity ( cp ) was defined as growth of > 15 colony forming units ( cfu ) per ml from the inside of a distal catheter segment using semiquantitative microbiological techniques as described by maki et al . .
culture positive cvcs were deemed to be colonised when catheter tip was culture positive but not associated with clinical sepsis [ 17 , 19 ] .
crbsi episodes were diagnosed in patients with culture - positive cvcs meeting the cdc criteria for crbsi current at the time , outlined as follow .
isolation of an organism by semiquantitative culture from a catheter segment in a patient with clinical sepsis , and isolation of the same organism from the patient 's blood , or defervescence of sepsis following removal of the catheter .
the subclavian vein was the recommended anatomical approach provided the operator was comfortable with this in the individual patient , and there were no contraindications to this approach .
the cvcs were placed by predetermined registrars in intensive care / anaesthesia under tpn service consultant supervision .
all cvcs were inserted either in the icu ( intensive care and high dependency units ) or in the ot for tpn administration in general ward patients ( ot / ward cvcs ) .
cvcs were carefully removed by nurses using written protocols involving initial antiseptic cleaning , the tip being removed under aseptic conditions and sent for culture in all situations .
organisms isolated from cvc tips were categorised as follows : methicillin - sensitive staphylococcus aureus ( mssa ) coagulase - negative staphylococcus ( cns ) methicillin - resistant staphylococcus aureus ( mrsa ) gram - negative bacilli ( gnb ) polymicrobial causes .
all cvcs that became culture positive , incorporating colonised cvcs and those responsible for crbsi , were examined .
statistical analysis was by means of paired t tests , comparing the incidence of each causative organism .
incidence rates for crbsi and culture positivity were also compared using paired t tests . in order to examine whether there were any changes in infection rates over the course of the study period , ordinary least squares regressions were conducted . in each regression ,
the incidence rate in each group ( crbsi , culture positive ) for a particular year was the dependent variable , with the sole independent variable being the year .
the difference in rates between the two groups was also used as a dependent variable .
these three regressions allow us to identify whether , for each outcome ( viz . the infection rate in the crbsi , the incidence rate in the culture - positive group , and the difference in incidence rates between crbsi and culture - positive groups ) , whether there was any statistical reduction or increase over time .
throughout the twelve - year study period , spanning january 1997 to january 2009 , a total of 1392 patients received tpn via 2,565 cvcs over 15,397 cvc days .
25.4% ( 651/2565 ) of cvcs inserted for tpn administration throughout the study period became culture positive . of these culture
positive cvcs , 68% ( 441/651 ) were colonised and 32% ( 210/651 ) were associated with the development of crbsi .
figure 1 depicts the annual incidence of culture - positive cvcs subdivided into colonised cvcs and those responsible for crbsi expressed as episodes per 1000 cvc days .
table 1 shows that the proportion of culture positive cvcs that were associated with crbsi averaged 32% ( derived from mean annual number of episodes = 17.5/54.25 ) , the difference between culture positive and crbsi was statistically significant at 36.75 ( se 1.83 ) , ( p < 0.01 ) .
table 2 shows the decline in the proportion of cvcs that became culture positive in the study period .
in contrast to the previously reported significant fall in crbsi rate ( the regression analysis estimates a significant fall of 1.182 on average for each year in the study period , ( p < 0.05 ) ) ; the decline in culture - positive cvcs ( 0.465 ) was not statistically significant ( p > 0.05 ) .
table 3 depicts the pattern of organisms isolated from cvcs that became culture positive and those responsible for development of crbsi .
it demonstrates that a similar pattern of organisms was isolated from cvc tips of both groups for all categories of causative organisms with the exception of enterococci , which were statistically more likely to be isolated in culture positive than crbsi cvc tips ( p = 0.04 ) .
table 4 displays causative organisms isolated from cuture positive cvcs in icu and ot / ward cvcs , and shows that the proportion of culture positive cvcs that grew staphylococci ( incorporating mssa , cns and mrsa ) was significantly higher in ot / ward cvcs ( 74.7% ) compared to icu ( 64% ) , ( p = 0.01 ) .
more specifically , mssa isolation was significantly higher in the ot / ward cvcs compared to icu ( p < 0.01 ) .
there were no other significant differences in the pattern of organism isolated between icu and ot / ward cvcs .
in this large study of hospital - wide tpn patients , we examine the prevalence of cvc tip culture positivity , incorporating both those cvcs that became culture positive in the absence of systemic sepsis ( colonised cvcs ) and culture - positive cvcs causative of systemic infection ( crbsi cvcs ) , in a hospital - wide tpn service .
we do not recommend that routine culture of cvc tips be carried out , however , given that the policy and practice was to culture all cvc tips used for tpn administration throughout the study period in such a large , hospital - wide tpn population , we were afforded the unique opportunity to carry out the current analysis allowing us to fully define the relationship between culture positive cvc tips and subsequent development of crbsi using standard cdc diagnostic criterion .
our results demonstrate that 25% of standard , nontunnelled cvcs inserted for tpn administration became culture positive throughout the study period , and that there was a consistent relationship between culture positivity and the development of crbsi ( table 1 ) .
the terminology utilized in much of the literature to describe colonised and infected cvcs is inconsistent as many studies appear to use the word
colonised to include all catheters that are tip positive ( by local / standard criteria ) [ 1 , 2 , 6 , 2224 ] .
however , these studies then describe the proportion of these colonised " cvcs that resulted in systemic infection ( crbsi ) thus creating an inherent contradiction as cvcs responsible for crbsi episodes are thus being included in the colonised group .
we concur with the clarifying terminology utilized in a recent study examining crbsi / colonisation in arterial cannulae which utilises the term culture positive to cover both categories ( colonised cvcs and those cvcs that resulted in crbsi ) , as it is descriptively precise and we have used this terminology here . in a review by marik
however , this estimate was based on evidence using varying terminology , from a variety of clinical settings including arterial catheters , cvcs , and antibiotic - impregnated cvc studies [ 2527 ] and there was considerable variability in colonisation and infection rates .
the current study , of a large population of uniform , nontunneled , standard , nonantibiotic impregnated cvcs , confirms a culture positive , not colonization , rate of 25.4% for all cvcs .
there is limited published evidence to support the relationship between culture - positive cvcs and the subsequent development of crbsi . in the review
, the authors demonstrate a linear correlation between catheter tip colonisation and crbsi .
this correlates with the evidence of the current study where 32% of tpn cvcs that became culture positive resulted in the development of crbsi , a relationship that was consistent throughout the study period ( table 1 ) .
this study provides further circumstantial evidential support for cvc tip colonisation as an important pathogenic mechanism for crbsi in that it demonstrates a similar pattern of organism growth from both colonised and crbsi cvcs ( table 3 ) .
the decline in cvc culture positive rates seen ( table 2 , an estimated reduction of 0.465 on average per year during the study period , p > 0.05 ) did not reach statistical significance in this study but is likely related to the known significant decline in crbsi rate in this population ( table 2 , an estimated reduction of 1.182 on average per year during the study period , p < 0.05 ) and reflective of the value of a surveillance nurse implementing hygiene and other preventative measures as a probable means of reducing crbsi .
this decline in crbsi correlates with data presented in the literature describing falling crbsi rates , resulting in overall crbsi incidences tending to be generally low , in the region of 5 episodes per 1000 cvc days .
with such a low prevalence of crbsi , it is difficult to detect differences in outcome measures such that extremely large numbers of patients and cvcs would need to be recruited in future studies to demonstrate significant differences between groups , and others have alluded to this difficulty .
this has led to the suggestion of use of cvc tip colonisation as a representation , or surrogate marker , for crbsi in many clinical studies [ 3034 ] .
this use of colonisation data for cvc tips has been demonstrated to be a practicable endpoint in future studies by rijnders et al . in a large review of the current evidence .
given that the current study provides further evidential support for the relationship between culture positivity and crbsi and presuming the consistency of this relationship is confirmed in further studies , it supports the auditing of cvc tip culture positivity as a surrogate surveillance marker for crbsi which may provide a more dynamic measurement of trends than crbsi measurement alone especially where numbers of cvcs inserted are low .
however , this would entail culturing all removed cvcs , regardless of whether or not catheter - related infection was suspected , and the issue of cost versus benefit would require analysis in any hospital / laboratory considering such a regular process .
it has been proposed that tpn , being a potential culture medium , is an independent risk factor for crbsi [ 3538 ] .
however , there is a paucity of studies related to cvc colonisation and crbsi and in patients receiving tpn via short - term cvcs . of the limited prospective studies of crbsi among tpn patients , levels reported are diverse with crbsi rates ranging from 55.2% in one study and 11.46% in another .
insofar as one can judge the current evidence base , the proportion of culture - positive cvcs that resulted in crbsi was little different from those quoted for cvcs for general use .
suggestions that tpn is an independent risk factor for crbsi ( and perhaps culture positivity ) are therefore questioned by data from this study . in a cdc survey published in 2003 , 55% of patients in the icu had a cvc in situ , compared with 24% of ward patients . however , because of the much larger population of patients located on general hospital wards , the majority of cvcs in use were in fact inserted in patients who were located outside of the icu ( 70% ) , and this is probably the case for most institutions .
one study reports higher risks of breaches in cvc care to occur on wards . in another study
, non - icu patients tended to have cvcs in situ for longer periods of time .
both of these are factors that place patients at higher risk of crbsi and focus is now turning toward the measurement of crbsi incidence and epidemiology in cvcs inserted in patients at ward level . despite this , there are very limited studies examining cvc colonisation and crbsi among non - icu populations , and less is still that compare these in a homogenous population .
the current study is unique in that it provides comprehensive data on the microbiology of cvc tip colonisation and crbsi in cvcs inserted in patients both in the icu and at ward level . in a recent study by zingg et al .
, similar rates of crbsi were determined among icu and non - icu patients , with most crbsi being due to gram - positive organisms ( 60% ) , followed by gram - negative organisms ( 13% ) .
however , they do not separate out causative organisms in icu and non - icu cvcs . in fact
, we could find no studies comparing causative organisms for colonised or crbsi cvcs in icu and non - icu patients within the same institution .
causative organisms for crbsi among icu patients tend to most commonly be due to gram - positive organisms between 55.488% , followed by gram - negative organisms 8.530.1% and candida 2.811.7% [ 10 , 4345 ] .
other studies examining crbsi in non - icu populations determined gram - positive organisms to be the most common causative organisms responsible for between 4057% of crbsi episodes [ 13 , 46 , 47 ] .
gram - negative organisms were causative for 17% of crbsi in these studies of non - icu patients followed by candida in 1214% [ 13 , 46 ] .
these data correlate with the results of the current study , with gram - positive organisms being the most common causative organism in both culture - positive and crbsi cvcs ( table 4 ) in both the icu and at ward level , followed by gram - negative organisms with a trend ( although nonsignificant ) toward increased incidence among icu cvcs .
the use of vancomycin as empiric antibiotic therapy to treat suspected crbsi is further supported by the current study .
however , table 4 shows that this consideration appears to be even more appropriate in ot / ward cvcs where there was a significantly higher ( p < 0.01 ) staphylococcal isolation rate ( 74% of cvcs ) than in icu ( 64% ) and the rate of mssa isolation specifically was also significantly higher ( p < 0.01 ) in ot / ward patients ( 4.6% ) than in icu ( 0.3% ) .
this may reflect the slightly ( but not significantly ) higher gnb , enterococcal , and polymicrobial isolates from icu cvcs .
strengths of this study include its large size , the systematic tracking of prospectively collected data on 1392 patients over 15,397 cvc days in patients receiving tpn , and the use of the same diagnostic microbiological criteria ( for catheter tip positivity ) and the same multidisciplinary process using cdc criteria for crbsi diagnosis over the 13 years of the study .
weaknesses are the observational nature of the study design , the questionable relevance of cvc colonisation as a clinical entity , and the possibility of cvc culture positivity being merely representative of contamination at the time of cvc removal . however , the standard procedure for cvc removal outlined in the methods and the tpn surveillance nurse may have provided protection against this . furthermore , although the cvc tip culture methodology used has been arguably superseded , its 13 years of consistent use lent validity to this study . in conclusion , this large study of tpn cvcs demonstrates a consistent relationship between cvc culture positivity and crbsi , and that the organism pattern for culture - positivity and crbsi is similar thus supporting the contention that colonisation is related to crbsi and that cvc culture positivity may be a useful surrogate marker for crbsi rates .
staphylococci are the primary organism causing culture positivity in all cvcs and are significantly more likely to be cultured in ot / ward patients than icu patients .
this study also highlights , that as 25% of cvcs became culture positive and 32% of these were responsible for development of crbsi , continued vigilance to reduce infection through prevention methods remains important . | a relationship between central venous catheter ( cvc ) tip colonisation and catheter - related blood - stream infection ( crbsi ) has been suggested .
we examined culture positivity of cvc tips ( colonised and infected cvcs ) in a total parenteral nutrition ( tpn ) population .
our aims were to define the relationship between culture positivity and crbsi , and to compare causative organisms between culture positive and crbsi cvcs , and between ward and icu cvcs .
all patients receiving tpn via non - tunnelled cvcs during the study ( 19972009 ) were included .
all cvc tips were analysed .
data were collated contemporaneously .
a tpn audit committee determined whether cvc tip culture positivity reflected colonisation / crbsi using cdc criteria .
1,392 patients received tpn via 2,565 cvcs over 15,397 cvc days .
25.4% of cvcs tips were culture positive , of these 32% developed crbsi .
there was a nonsignificant trend of higher gram negative bacilli isolation in icu cvcs ( p = 0.1 ) , ward cvcs were associated with higher rates of staphylococcal isolation ( p = 0.01 ) .
a similar pattern of organisms were cultured from crbsi and culture positive cvcs .
the consistent relationship between crbsi and culture positive cvcs , and similar pattern of causative organisms further supports an aetiological relationship between culture positive cvc tips and crbsi , supporting the contention that cvc culture - positivity may be a useful surrogate marker for crbsi rates . |
the initial treatment of differentiated thyroid cancer ( dtc ) includes surgery and selective administration of radioactive iodine ( rai ) .
rai has shown to decrease mortality and recurrence in high risk patients , and it is considered an important component of the treatment in invasive or metastatic disease [ 1 , 2 ] . however , the benefit of rai in low risk patients has not been fully supported by evidence [ 36 ] and current guidelines recommend an individualized approach for its indication [ 1 , 7 , 8 ] .
several cohorts of patients treated with rai have shown an increased risk of secondary malignancies even when rai was used to treat low risk patients [ 9 , 10 ] .
more frequently , rai can impact nonthyroidal tissues that express sodium iodide symporter ( nis ) , including the salivary glands and lachrymal system .
dysfunction may appear as salivary gland pain , taste impairment such as hypogeusia , also xerostomia , sialolithiasis , dental caries , stomatitis , salivary gland or oral infections , facial nerve damage , and even salivary gland neoplasia .
these side effects may be persistent in nearly 7% of patients and profoundly impair patient quality of life ( qol ) [ 13 , 14 ] .
lachrymal system side effects can be manifested as epiphora , xerophthalmia , and recurrent or chronic conjunctivitis . the time course and resolution of lacrimal effects are not completely known .
although tissues in the nasal cavity do not express nis , rai may be transported nonspecifically from the lachrymal system into the nasal ducts .
several nasal complaints have been documented including nasal pain , nasal tenderness , bloody discharge , and dry nose , with a frequency up to 10.5% . as for lachrymal complaints ,
little information has been published regarding the duration of nasal symptoms . despite the uncertainty regarding the benefit of rai and the potential adverse effects related to its use , recent evidence showed an increase in the use of rai in patients with dtc in the last two decades , including those with low risk of recurrence [ 17 , 18 ] .
the objective of this study was to evaluate the frequency and intensity of early and late rai related symptoms in patients with dtc .
we designed an observational , prospective study to evaluate and compare the frequency and intensity of early and late adverse effects associated with rai administration in patients with dtc treated with total thyroidectomy .
our study protocol was approved by the research ethics committee of our institution , and all patients accepted to participate providing their informed consent .
patients over 18 years of age with dtc , surgically treated in our institution between january and august 2013 , were consecutively invited to answer 2 online surveys on 12 adverse effects associated with rai administration .
these surveys were created by our team based on information published in literature regarding rai related symptoms [ 12 , 13 , 15 , 16 ] .
early rai related symptoms were assessed within 6 months after initial treatment ( considering rai administration date in patients who received rai or surgery date in those who did not receive rai ) .
intensity of symptoms was evaluated with a likert - like scale from 0 ( none ) to 5 ( severe ) . for the purpose of data analysis
, we determined four categories of intensity : 0 ( inexistent ) , 1 - 2 ( mild ) , 3 - 4 ( moderate ) , and 5 ( severe ) .
the detail of the survey is shown in table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/2586512 .
lymph node dissection was performed in those in whom preoperative ultrasound , fine needle aspiration biopsy , or intraoperative findings suggested lymph node metastases .
the administration of rai and its dose were decided by the attending physician following the ata and local guidelines , considering postoperative stimulated thyroglobulin [ 7 , 19 ] .
a low - iodine diet was prescribed 2 weeks before rai administration and all patients received rai at least 3 weeks after thyroid hormone withdrawal .
a tsh above 30 miu / l was certified before rai administration as well as a negative pregnancy test in fertile women .
thyroid hormone replacement therapy was started 48 hours after rai administration and all patients had tsh levels below 1.0 ui / ml six weeks after starting treatment . a posttherapy whole body scan ( wbs )
we defined rai doses as low and high when 50 mci ( 1850 mbq ) and 100 mci ( 3700 mbq ) were administered , respectively . considering the results of preoperative ultrasound , intraoperative findings , postsurgical histology , and wbs
when rai was administered , patients were risk - stratified using the 7th edition of the ajcc / uicc staging system ( stage i , ii , iii , or iv ) and the ata risk of recurrence stratification system ( low , intermediate , or high risk of recurrence ) .
categorical variables are expressed as number and frequencies ; continuous variables are expressed as mean sd or median ( range ) , when appropriate .
categorical comparisons were made using chi - square testing with fischer 's exact test when appropriate .
the comparison between frequencies of early and late rai related symptoms was tested with mcnemar 's test .
chicago , il ) and stata 12 ( statacorp , collage station , tx ) .
one hundred and ten patients answered the first survey and 61 the second one regarding early and late rai related symptoms , respectively .
the demographics , clinical features , risk stratification , and final outcomes included in this study are presented in table 1 . as expected , most patients were female ( 91.8% ) and had low or intermediate dtc risk of recurrence based on the ata 2009 risk stratification system .
all patients underwent total thyroidectomy , and 26 ( 23.6% ) also underwent lymph node dissection .
of the total cohort , 86 ( 78.2% ) patients received rai : 38 ( 63.3% ) in the low and 48 ( 96% ) in the intermediate risk of recurrence patients .
patients received a median dose of 75 mci ( 30150 mci ) ( median 2775 mbq ; range 11105550 mbq ) .
patients who received rai were similar to those not receiving rai regarding age , gender , type of surgery , and time to surveys .
the first survey was answered at a median of 2.5 ( 0.55.8 ) months after initial treatment .
the following rai related symptoms were more frequent among patients who received rai : periorbital edema ( 36% versus 12.5% ; p = 0.027 ) , excessive tearing ( 24.4 versus 4.2 : p = 0.028 ) , salivary gland swelling ( 55.8 versus 16.7 ; p = 0.001 ) , salivary gland pain ( 58.1 versus 12.5 ; p < 0.01 ) , dry mouth ( 60.5 versus 29.2 ; p = 0.007 ) , taste disorders ( hypogeusia [ 65.1 versus 16.7 : p < 0.01 ] and dysgeusia [ 65.1 versus 12.5 : p < 0.01 ] ) , smell impairment ( 30.2 versus 4.2 : p = 0.009 ) , and nausea ( 59.3 versus 29.2 : p = 0.009 ) ( figure 1 )
. intensity of symptoms was also significantly higher in patients receiving rai treatment compared with those who did not ( figure 2 ) .
also , excessive tearing , salivary gland swelling and pain , dry mouth , and taste and smell disorders were more frequent in patients receiving low rai doses ( 50 mci ) ( 1850 mbq ) than in those who did not ( figure 3 ) . when low and high rai doses were compared , we found no differences neither in frequency nor in intensity of any of the studied rai related symptoms .
the second survey was answered at a median of 11.5 ( 618.6 ) months after initial treatment .
the following rai related symptoms were more frequent and intense among patients who received rai : periorbital edema ( 36.7 versus 0 : p = 0.012 ) , salivary gland pain ( 63.3 versus 8.3 : p = 0.001 ) , salivary gland swelling ( 49 versus 16.7 : p = 0.042 ) , and dry mouth ( 71.4 versus 33.3 : p = 0.014 ) ( figure 2 ) .
the same differences were found between low dose and no rai administration ( figure 3 ) . when low and high rai doses were compared , we found no differences regarding the frequency or intensity of any of the studied symptoms . among those patients who received rai
, we found that epiphora and nausea were significantly less frequent in the second survey than in the first one . in terms of intensity , periorbital edema , salivary gland pain and swelling , taste disorders , and nausea were significantly less intense after six months of follow - up ( figure 1 ) .
in this study , we found a higher frequency of some early rai related symptoms ( periorbital edema , excessive tearing , salivary gland swelling and pain , dry mouth , taste and smell disorders , and nausea ) among patients with dtc who received rai than in those who did not .
it is remarkable that , even after 6 months of initial treatment , periorbital edema , salivary gland pain and swelling , and dry mouth were persistently more frequent among patients who received rai .
it is also important to note that most of these rai related symptoms were more intense among patients who received rai . in our series , most rai related side effects and their intensity decreased 6 months after initial treatment .
however , symptoms like excessive tearing , salivary gland pain , and dry mouth were more frequent , though less intense , 6 months after initial therapy .
although side effects usually decline in a longer follow - up period , the higher frequency and intensity of symptoms during the first year after rai administration force us to take this information into account when considering rai use in low risk dtc patients .
we are aware that the latest evidence does not support rai administration in this group of patients , but guidelines followed to treat these patients were more permissive regarding this issue [ 7 , 19 ] .
. also found comparable frequency of rai related symptoms between low and high rai doses administered after thyroid hormone withdrawal ( thw ) .
found a direct relationship between rai related symptoms frequencies and rai dose [ 13 , 21 ] .
after a thorough review of the literature regarding rai related symptoms , we could not find a validated instrument to evaluate their frequency and intensity .
the lack of this tool motivated us to develop a web - based survey to evaluate the presence of early and late rai related symptoms , including the 12 most frequently published in previous studies [ 12 , 13 , 15 , 16 ] .
the aim of these surveys was to study both the frequency and intensity of these rai related symptoms .
the frequency of rai related symptoms reported by patients who did not receive rai was unexpectedly high . one plausible explanation could be the effect of hypothyroidism required before rai administration . as reported by mallick et al . and schlumberger et al .
in hilo and estimabl trials , respectively , patients who received rai with thw had a higher frequency of rai related symptoms compared to those with rhtsh administration [ 20 , 21 ] .
some of the rai related symptoms they found , such as puffy face or weight gain , could be considered as equivalent to some of the symptoms reported by our cohort .
this could also explain , to some extent , the higher frequency of early rai related symptoms in those who received rai ( with thw ) compared to those who did not . however , this higher frequency persisted in late rai related symptoms when all patients were under thyroid hormone replacement .
this fact verifies that the influence of rai administration in the frequency of rai related symptoms is not entirely dependent on the hypothyroid state .
another explanation for the high frequency of rai related symptoms reported by patients who did not receive rai is potential selection bias of the study , considering that nearly half of patients answered both surveys .
further , these patients may have been more prone to report symptoms than those who did not accept our invitation and may be especially willing to interpret any discomfort as a positive outcome .
nonetheless , there were significant differences between those who received rai and those who did not , confirming the validity of our survey .
currently , our group is prospectively surveying all patients who receive rai in our center .
this will allow us to increase the number of analyzed patients , avoid bias , and validate our results .
other relevant adverse effects have been reported in the literature for the use of rai .
there is an increased risk of secondary malignancies , including solid tumors and leukemia , even in patients with low risk thyroid cancer that have received rai [ 9 , 10 ] .
these issues are beyond the scope of our study , but they should also be considered when defining the need of rai ablation in patients with ptc , thus restricting its use only in patients who will get a real benefit .
recent publications have shown important effects of dtc on qol [ 22 , 23 ] . a recent publication by applewhite et al .
has shown that qol scores among thyroid cancer patients were the lowest at initial diagnosis , surgery , thw , and , interestingly , rai ablation .
when compared to patients affected by other neoplastic diseases with poorer survival , thyroid cancer patients showed similar qol scores .
this fact reveals that qol impairment is defined not only by prognosis but , probably , by the intrinsic nature of the disease and some treatment related factors .
hence , in order to reduce distress associated with dtc diagnosis and treatment , we should be cautious when deciding therapeutic approaches , weighting benefits and physical and psychological side effects . probably , rai is a paradigm of a therapy with questionable benefit and evident side effects in low risk dtc patients . in conclusion
, this study shows a high frequency of early and late rai related symptoms in patients with dtc , even when low doses are used .
this information should be carefully considered when defining what dose to administer , especially among patients with low risk of recurrence , since an improvement on prognosis is questionable . | context . radioiodine ( rai ) administration has adverse effects in patients treated for thyroid cancer ( dtc ) , but there is scarce information regarding their intensity and duration .
objective . to evaluate frequency and intensity of early and late rai - related symptoms in patients with dtc
. design .
observational prospective study
. patients . dtc patients who underwent thyroidectomy , with or without rai . measurements .
patients answered 2 surveys : ( 1 ) from 0 to 6 months and ( 2 ) between 6 and 18 months after initial treatment .
results .
110 patients answered the first survey and 61 both .
nearly 80 percent received rai . among early symptoms , periorbital edema , excessive tearing , salivary gland disturbances , dry mouth , taste disorders , and nausea were more frequent and intense among rai patients .
regarding late symptoms , periorbital edema , salivary gland pain and swelling , and dry mouth were more frequent and intense in rai patients .
frequency and intensity of adverse effects were not different between low and high rai doses ( 50 versus 100 mci ) . conclusion .
rai - related symptoms are frequent and usually persist after 6 months of administration , even when low doses are given .
this finding must be considered when deciding rai administration , especially in low risk patients , among whom rai benefit is controversial . |
osteoporosis is a disorder of low bone mass and damaged skeletal architecture resulting in impaired bone strength and an increased risk of fragility fracture . during the past 20 years
, several classes of drugs with different mechanisms of action have been shown to protect patients with osteoporosis from fragility fractures .
most of our current treatment options are anti - remodeling agents that reduce both bone resorption and formation , bringing the balance of bone metabolism back toward or to normal .
these drugs strengthen trabecular bone by reducing the number and depth of stress risers in thin trabeculae .
the most effective of these drugs , potent bisphosphonates and the receptor activator of nuclear factor kappa - b ( rank ) ligand inhibitor denosumab , reduce the risk of vertebral fracture by about 70% , of hip fracture by 40% to 50% and of all non - vertebral fractures by 20% to 30% .
the only bone building or anabolic agents now available are parathyroid hormone ( pth ) analogues , pth 1 - 84 and teriparatide .
these drugs stimulate both bone formation and bone resorption . in the early months of treatment , bone formation
is activated more than is resorption , resulting in a positive bone balance , especially in the trabecular skeleton .
while teriparatide therapy may thicken cortical bone , it also causes , at least temporarily , an increase in the porosity of cortical bone . both teriparatide and pth 1 - 84 reduced the risk of vertebral fracture by 65% and 61% , respectively .
some drugs such as oral bisphosphonates require complex dosing regimens that are inconvenient and may result in poor compliance with the dosing rules .
overall there is very poor adherence to recommended treatment regimens ; more than half of patients discontinue their treatment within 12 months of beginning therapy .
also , concerns about long - term safety with bisphosphonates and perhaps denosumab limit the acceptance of these drugs and cause concerns about the benefit : risk ratio of long - term treatment .
thus , opportunities exist for new therapeutic agents to fill the unmet needs of having a drug that reduces the risk of non - vertebral fracture more effectively than current treatments , that has a good safety profile and that can be given conveniently .
the other two are drugs with unique mechanisms of action that have the potential to substantially strengthen cortical bone and to become important new treatment options to reduce fracture risk in patients with osteoporosis .
parathyroid hormone related peptide ( pthrp ) shares modest structural homology with pth 1 - 84 and teriparatide .
both pth and pthrp bind to the same pth receptor , but the kinetics of binding differ , and the duration of the cellular activation of cyclic amp with pthrp is shorter than with pth .
preclinical studies suggested that , compared to pth , pthrp could achieve an anabolic skeletal effect with less activation of bone resorption and less calcium mobilization causing hypercalcemia , thereby broadening the therapeutic window . in a phase 2 clinical trial , 600 g pthrp 1 - 36 administered daily for 2 months resulted in similar gains in bone mineral density ( bmd ) as did teriparatide 20 g daily .
a more promising candidate molecule is abaloparatide , a synthetic analog of pthrp . in a phase 2 study ,
the stimulation of resorption and formation markers was less with abaloparatide 80 g daily than with teriparatide 20 g . however , the bmd response to abaloparatide was greater in both the spine and especially in the hip than with teriparatide .
results of the phase 3 pivotal fracture trial with abaloparatide were recently presented at the annual meeting of the endocrine society . over 18 months of treatment with abaloparatide and teriparatide ,
the incidence of vertebral fracture was decreased by 86% and 80% , respectively , compared to placebo .
teriparatide reduced non - vertebral fracture by 28% , a difference that was not statistically significant compared to placebo .
the difference in non - vertebral risk reduction between abaloparatide and teriparatide was also not significant .
the study is continuing for another 6 months during which time , all patients will take alendronate 70 mg once weekly .
a more detailed analysis of the data from this study will be necessary to determine whether and how abaloparatide would provide meaningful clinical advantages over teriparatide , other than the convenience of not having to refrigerate the drug .
cathepsin k ( catk ) is the major osteoclast - derived proteolytic enzyme in bone .
genetic deficiency of catk ( pyknodysostosis ) is characterized by an osteoclast - rich form of osteopetrosis with high bone mass , reduced bone resorption but with preservation of bone formation . targeted disruption of the catk gene in mice results in a phenotype with an osteosclerotic skeleton with increased trabecular and cortical bone mass of good quality .
histomorphometry demonstrates a high rate of bone formation , normal bone mineralization and normal or increased osteoclast numbers , but a decrease in the osteoclasts ' ability to resorb bone matrix .
compared to normal animals , catk deficient mice have increased bone strength at the vertebral body and femoral mid - shaft .
several small molecular weight inhibitors of human catk have been evaluated in pre - clinical studies and clinical trials .
balicatib ( novartis , basel , switzerland ) is a basic , highly selective nitrile - based catk inhibitor in enzyme - based assays , but the selectivity is largely lost in whole cell assays due its accumulation in lysosomes ( a property called lysosomotropism ) where high concentrations exert effects on non - k cathepsins .
odanacatib ( merck , kenilworth , nj , usa ) is a non - basic , non - lysosomotropic , nitrile based molecule that retains its high enzyme selectivity in cell - based assay systems .
ono-5334 ( ono pharmaceutical , trenton , nj , usa ) is a potent hydrazine - based non - lysosomotropic inhibitor of catk with high selectivity compared to other cathepsins . in preclinical studies in ovariectomized rabbits and monkeys ,
catk inhibitors caused dose - dependent decreases bone resorption at both trabecular and cortical sites and preservation of areal bmd .
odanacatib treatment increased osteoclast numbers when assessed by histomorphometry , and increased serum levels of tartrate resistant acid phosphatase 5b ( trap5b ) , a marker of osteoclast number .
the effects of catk inhibition on bone formation are more complex . like other resorption inhibitors ,
balicatib and odanacatib inhibit trabecular bone formation in ovariectomized monkeys . in ovariectomized rabbits , both odanacatib and alendronate
however , the reduction in bone formation rate was less with catk inhibition than with alendronate . unlike what occurs with other anti - resorptive agents , catk inhibition is associated with maintenance of endocortical bone formation and increased periosteal bone formation in the femur , resulting in increased cortical bone thickness and volume in the hip and improved bending strength of long bones .
these effects in preclinical studies demonstrated the unique effects that catk inhibition has on bone remodeling .
inhibiting catk results in decreased capacity of viable osteoclasts to resorb bone . by inhibiting the extracellular activity of catk in the resorption space , the depth of resorption cavities made by osteoclasts
however , the number and other functions of osteoclasts is preserved , unlike the effects of bisphosphonates or rank ligand inhibitors which result in decreased numbers and function of osteoclasts . as a result ,
communication between these functioning osteoclasts and osteoblasts remains intact in the presence of catk inhibition .
preservation of osteoblast function might also be due to reduced degradation by catk of matrix - derived growth proteins such as transforming growth factor and insulin - like growth factor 1 .
the favorable preclinical studies led to clinical trials with the three catk inhibitors . in a phase 2 study ,
treatment with balicatib led to reduced markers of bone resorption and increased bmd . however , because several cases of morphea - like skin lesions were observed , further development of this compound was halted .
the dermatologic toxicities of balicatib might be due to inhibition of catk in skin or to the lysosomotropism described above , causing off - target inhibition of other cathepsins expressed in skin fibroblasts .
the effects of oral ono-5334 ( 50 mg twice daily , 100 mg once daily , or 300 mg once daily ) were compared with placebo and alendronate 70 mg weekly in postmenopausal women with osteoporosis in a phase 2 trial .
after 12 months of therapy , the 300 mg daily dose of ono-5334 reduced bone resorption markers similarly to alendronate , but the effects on bone formation markers bone specific alkaline phosphatase ( bsap ) and procollagen type i n - terminal peptide ( p1np ) were less than that observed with alendronate .
the effects of ono-5334 300 mg daily and alendronate on bmd of the lumbar spine were comparable .
no clinically relevant safety concerns were noted , and no difference was detected in the rate of skin adverse events among the study groups .
odanacatib is the most extensively studied catk inhibitor in clinical trials and is the only agent remaining in clinical development . in a placebo - controlled phase 2 trial ,
postmenopausal women with low bmd received odanacatib in once weekly doses ranging from 3 to 50 mg . over 24 months of treatment ,
a dose dependent increase in bmd at the lumbar spine and femoral neck was observed . at the highest dose of odanacatib ( 50 mg weekly ) ,
bmd at the lumbar spine and total hip increased by 5.7% and 4.1% , respectively , versus placebo .
urinary n - terminal telopeptide ( ntx ) was quickly reduced , with persistent decreases observed at 12 and 24 months ( 58% and 52% versus placebo , respectively ) .
markers of bone formation were initially reduced ( 20% for bsap and 30% for p1np ) , but levels then gradually returned toward baseline by 24 months . in the extension of this study , continued therapy with odanacatib 50 mg once weekly for 5 years resulted in almost linear increases in bmd from baseline at the lumbar spine ( 11.9% ) and total hip ( 8.5% ) ( fig .
urinary ntx values remained well below baseline ( 50% at 3 years ; 55.6% at 5 years ) , while after year 2 , bone formation markers remained close to baseline levels .
after 2 years , therapy was withdrawn in a group of participants that had received odanacatib 50 mg weekly .
markers of both bone resorption and formation quickly rose above baseline values , returning to baseline 12 to 24 months after therapy was discontinued .
bmd values decreased to levels seen in the placebo group 12 months after withdrawing treatment .
the incidence of skin and pulmonary events were similar in the treatment and placebo groups , and morphea - like skin lesions were not reported during the 5 years of therapy .
the effects of treatment with odanacatib 50 mg weekly on the incidence of hip and spine fracture is being evaluated in an international , placebo - controlled phase 3 event - driven study in more than 16,000 postmenopausal women with osteoporosis . based upon the results of a pre - planned interim analysis ,
this study was recently discontinued early because of " robust effectiveness . " after a median duration of therapy of about 3 years , it was recently reported that odanacatib significantly reduced the incidence of vertebral , hip and non - vertebral fractures .
more detailed analyses of adverse events of special interest are being made . to collect more safety information
, most patients will continue on their blinded treatment allocation for 5 years with additional follow - up on open label odanacatib therapy .
secretion is increased by unloading or immobilization and is inhibited by pth and by skeletal loading .
both diseases are characterized by very high bone mass but , unlike the brittle bones of patients with osteopetrosis , the bone tissue is normal and of good quality . as a result , these patients with sclerostin deficiency are at very low risk for fracture .
homozygous patients experience cranial and facial distortion during growth and , as adults , bony overgrowth such as cranial basilar stenosis .
heterozygotes , who have intermediate levels of sclerostin , have a normal phenotype except for high bone mass , suggesting that inhibiting sclerostin has promise as a treatment strategy to activate bone formation and to improve bone mass as a treatment for osteoporosis and other bone disorders .
sclerostin - deficient mice also exhibit high bone mass with increased rates of bone formation in both trabecular and cortical bone .
inhibiting sclerostin activity with an anti - sclerostin antibody in animals demonstrated the potential of sclerostin inhibition to improve bone mass and structure . in aged ,
ovariectomized rats given a sclerostin - inhibiting antibody , increased bone formation was observed on trabecular , endocortical , intracortical and periosteal bone surfaces .
treatment for 5 weeks resulted in bone mass and bone strength that exceeded the sham - operated control animals and restored the skeletal abnormalities induced by ovariectomy . in gonad - intact female cynomolgus monkeys , treatment with a humanized sclerostin - neutralizing antibody
once monthly for 2 months transiently increased markers of bone formation , increased bmd in the lumbar spine , femoral neck , proximal tibia , and distal radius . increased modeling - based bone formation
substantial increase in bone strength occurred , with a strong correlation between bone mineral content ( measured by quantitative computed tomography ) and peak load observed in both the lumbar spine and femoral diaphysis . in clinical studies ,
single doses of humanized anti - sclerostin antibodies , administered subcutaneously , induced rapid and substantial increases in markers of bone formation accompanied by a moderate decrease in bone resorption markers , resulting in a very positive bone balance . in phase 2 studies in which patients received anti - sclerostin antibody injections every 2 to 4 weeks , does - dependent increases in bmd were observed ( fig .
the bmd responses to the larger doses of romosozumab were substantially greater than those observed in patients randomly assigned to receive open label alendronate or teriparatide therapy . despite ongoing treatment
, markers of bone formation returned to baseline levels within 6 to 12 months of beginning treatment .
the mechanism(s ) of this resistance to the anabolic effect of anti - sclerostin therapy is not known .
anti - sclerostin therapy has been well tolerated , complicated only by injection site reactions that were generally mild and transient .
phase 3 studies with romosozumab , currently underway , will evaluate the efficacy of treatment to reduce fractures in women with postmenopausal osteoporosis and will provide much more information about the tolerability and safety of therapy with anti - sclerostin antibodies .
new forms of treatment , based on new or refined knowledge about the molecular mechanisms regulating bone metabolism , hold the promise of providing large gains in bone mass and reconstructing damaged skeletal architecture
. the clinical challenge will be to most appropriately use these new medications to treat our patients most effectively . | although several effective therapies are available for the treatment of osteoporosis in postmenopausal women and older men , there remains a need for the development of even more effective and acceptable drugs .
several new drugs that are in late - stage clinical development will be discussed .
abaloparatide ( recombinant parathyroid hormone related peptide [ pthrp ] analogue ) has anabolic activity like teriparatide .
recent data from the phase 3 fracture prevention trial demonstrate that this agent is effective in reducing fracture risk .
inhibiting cathepsin k reduces bone resorption without decreasing the numbers or activity of osteoclasts , thereby preserving or promoting osteoblast function .
progressive increases in bone mineral density ( bmd ) have been observed over 5 years .
early data suggest that odanacatib effectively reduces fracture risk .
lastly , inhibiting sclerostin with humanized antibodies promotes rapid , substantial but transient increases in bone formation while inhibiting bone resorption .
marked increases in bmd have been observed in phase 2 studies .
fracture prevention studies are underway .
the new therapies with novel and unique mechanisms of action may , alone or in combination , provide more effective treatment options for our patients . |
the study was performed between september 2004 and december 2009 at three study sites in sweden , finland , and the netherlands .
, 150 patients were screened of which 69 patients were randomized using a permutated block randomization scheme stratified by site and screenings for hba1c to receive exe or glar in addition to ongoing metformin treatment .
inclusion criteria were as follows : age 3075 years , hba1c 6.59.5% , bmi 2540 kg / m , and metformin treatment at a stable dose for at least 2 months .
no other blood glucose lowering agents were allowed within 3 months prior to screening .
the study protocol was approved by each site s ethics review committee and was in accordance with the principles described in the declaration of helsinki .
patients randomized to exe ( n = 36 ) initiated treatment at a dose of 5 g b.i.d . , injected 15 min before breakfast and dinner for a period of 4 weeks , followed by dose increase to 10 g b.i.d .
exe was titrated to a maximum dose of 20 g t.i.d . or the maximum tolerated dose when hba1c ranged 7.17.5% at two consecutive visits or when hba1c was 7.6% at any given visit .
patients randomized to glar ( n = 33 ) started at an initial dose of 10 iu once daily ( q.d . ) , which was injected at bedtime .
patients were instructed to increase the daily dose based on their fasting self - monitored blood glucose ( smbg ) levels ( < 5.6 mmol / l ) according to a prespecified treat - to - target algorithm ( 8) . when necessary , the importance of proper titration of insulin was emphasized .
after completing the 1-year main treatment period of the study , patients were asked to return to their randomly assigned study medication for an additional 104-week treatment , which was followed by a 12-week off - drug period ( supplementary fig .
forty - six patients gave their written consent to participate in the extension phase and continued their allocated treatment with exe ( n = 21 ) or glar ( n = 25 ) . during the extension phase , patients visited the study centers at 12-week intervals until the end of the treatment period ( week 168 ) . at that point
the patients stopped the exe or glar treatment and continued their ongoing metformin treatment , which they had been using in an unchanged dose during the total 168-week treatment period .
after a 4-week off - drug period , patients returned to the center for their final combined euglycemic hyperinsulinemic and hyperglycemic clamp with arginine stimulation .
the final study visit was at week 180 ( approximately 3.5 years after randomization ) .
insulin sensitivity and c - peptide secretion measures were measured during a combined euglycemic hyperinsulinemic and hyperglycemic clamp procedure as previously described ( supplementary fig .
clamps were performed prior to randomization , following 52 weeks of treatment , after a 4-week off - drug period ( week 56 ) , and finally after a 4-week off - drug period following the total 3 years of treatment ( week 172 ) as previously described ( 7 ) . during the clamp at week 52 , patients randomized to exe were given the study drug 15 min prior to the onset of the hyperglycemic clamp , and patients randomized to glar received their last insulin dose the night before at bedtime . during the clamp at week 56 and week 172 , patients did not receive either exe or glar .
arginine was administered at t = 260 min during the hyperglycemic clamp to estimate maximum insulin secretory capacity at a steady - state glucose concentration of 15 mmol / l ( 11 ) .
whole - body insulin mediated glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 30 min of the euglycemic hyperinsulinemic clamp between 90120 min ( 9 ) .
first- and second - phase c - peptide secretion was calculated as area under the curve ( auc ) 180190 min and auc 190260 min , respectively .
arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc 260270 min above the c - peptide concentration prior to the start of the hyperglycemic clamp ( t = 175 min ) .
the disposition index ( di ) , a measure of -cell function and adjusted for insulin sensitivity , was calculated by multiplying the first - phase incremental c - peptide secretion with the m value ( airgluc*m ) ( 12 ) .
hba1c ( normal range : 4.36.1% , diabetes control and complications trial [ dcct]-standardized bio - rad assay ) , fasting plasma glucose ( fpg ) , and safety parameters were measured by a central laboratory ( quintiles , livingston , u.k . ) prior to randomization and during each follow - up visit until the end of the 12-week off - drug period .
plasma glucose concentrations during the clamp were measured using a ysi 2300 stat plus ( ysi , yellow springs , oh ) in sweden and the netherlands , and a beckman - coulter glucose analyzer 2 ( beckman - coulter , fullerton , ca ) in finland .
c - peptide samples were analyzed at the vu university medical center using a single batch immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , the netherlands ) .
the extension study s primary efficacy end point was the treatment effect on the -cell function measured as the first - phase glucose - stimulated c - peptide secretion adjusted for the m value .
the dependent variable used in the model was the change from pretreatment for the -cell function parameters ( airarg , first phase , second phase ) .
for all other end points , the dependent value used was the mean at the corresponding visit .
the model included factors for treatment group ( exe / glar ) , site ( the netherlands / sweden / finland ) , and baseline hba1c stratum ( 8.5%/>8.5% ) , and the pretreatment variable of the corresponding dependent variable as a covariate . if the parameter did not approximate the normal distribution after base - e transformation , a nonparametric test was used ( di statistics using mann - whitney test ) .
statistical analysis was performed using spss 16.0 for mac os x ( spss , chicago , il ) .
all inferential statistical tests were conducted at a significance level of 0.05 ( two - sided ) .
patients randomized to exe ( n = 36 ) initiated treatment at a dose of 5 g b.i.d . , injected 15 min before breakfast and dinner for a period of 4 weeks , followed by dose increase to 10 g b.i.d .
exe was titrated to a maximum dose of 20 g t.i.d . or the maximum tolerated dose when hba1c ranged 7.17.5% at two consecutive visits or when hba1c was 7.6% at any given visit .
patients randomized to glar ( n = 33 ) started at an initial dose of 10 iu once daily ( q.d . ) , which was injected at bedtime .
patients were instructed to increase the daily dose based on their fasting self - monitored blood glucose ( smbg ) levels ( < 5.6 mmol / l ) according to a prespecified treat - to - target algorithm ( 8) . when necessary , the importance of proper titration of insulin was emphasized . after completing the 1-year main treatment period of the study ,
patients were asked to return to their randomly assigned study medication for an additional 104-week treatment , which was followed by a 12-week off - drug period ( supplementary fig .
forty - six patients gave their written consent to participate in the extension phase and continued their allocated treatment with exe ( n = 21 ) or glar ( n = 25 ) . during the extension phase
, patients visited the study centers at 12-week intervals until the end of the treatment period ( week 168 ) . at that point
the patients stopped the exe or glar treatment and continued their ongoing metformin treatment , which they had been using in an unchanged dose during the total 168-week treatment period .
after a 4-week off - drug period , patients returned to the center for their final combined euglycemic hyperinsulinemic and hyperglycemic clamp with arginine stimulation .
the final study visit was at week 180 ( approximately 3.5 years after randomization ) .
insulin sensitivity and c - peptide secretion measures were measured during a combined euglycemic hyperinsulinemic and hyperglycemic clamp procedure as previously described ( supplementary fig .
clamps were performed prior to randomization , following 52 weeks of treatment , after a 4-week off - drug period ( week 56 ) , and finally after a 4-week off - drug period following the total 3 years of treatment ( week 172 ) as previously described ( 7 ) . during the clamp at week 52 , patients randomized to exe were given the study drug 15 min prior to the onset of the hyperglycemic clamp , and patients randomized to glar received their last insulin dose the night before at bedtime . during the clamp at week 56 and week 172
arginine was administered at t = 260 min during the hyperglycemic clamp to estimate maximum insulin secretory capacity at a steady - state glucose concentration of 15 mmol / l ( 11 ) .
whole - body insulin mediated glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 30 min of the euglycemic hyperinsulinemic clamp between 90120 min ( 9 ) .
first- and second - phase c - peptide secretion was calculated as area under the curve ( auc ) 180190 min and auc 190260 min , respectively .
arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc 260270 min above the c - peptide concentration prior to the start of the hyperglycemic clamp ( t = 175 min ) . the disposition index ( di ) , a measure of -cell function and adjusted for insulin sensitivity ,
was calculated by multiplying the first - phase incremental c - peptide secretion with the m value ( airgluc*m ) ( 12 ) .
hba1c ( normal range : 4.36.1% , diabetes control and complications trial [ dcct]-standardized bio - rad assay ) , fasting plasma glucose ( fpg ) , and safety parameters were measured by a central laboratory ( quintiles , livingston , u.k . )
prior to randomization and during each follow - up visit until the end of the 12-week off - drug period .
plasma glucose concentrations during the clamp were measured using a ysi 2300 stat plus ( ysi , yellow springs , oh ) in sweden and the netherlands , and a beckman - coulter glucose analyzer 2 ( beckman - coulter , fullerton , ca ) in finland .
c - peptide samples were analyzed at the vu university medical center using a single batch immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , the netherlands ) .
the extension study s primary efficacy end point was the treatment effect on the -cell function measured as the first - phase glucose - stimulated c - peptide secretion adjusted for the m value .
the dependent variable used in the model was the change from pretreatment for the -cell function parameters ( airarg , first phase , second phase ) .
for all other end points , the dependent value used was the mean at the corresponding visit .
the model included factors for treatment group ( exe / glar ) , site ( the netherlands / sweden / finland ) , and baseline hba1c stratum ( 8.5%/>8.5% ) , and the pretreatment variable of the corresponding dependent variable as a covariate . if the parameter did not approximate the normal distribution after base - e transformation , a nonparametric test was used ( di statistics using mann - whitney test ) .
statistical analysis was performed using spss 16.0 for mac os x ( spss , chicago , il ) .
all inferential statistical tests were conducted at a significance level of 0.05 ( two - sided ) .
no significant between - treatment group differences were present at baseline . at the end of the 1-year main study phase , five patients withdrew their consent and did not participate in the extension phase ( one in the glar group and four in the exe group ) because of the demanding study procedures .
of the patients randomized to exe , 69% ( n = 11 ) were treated with exe 10 g b.i.d . at 168 weeks of treatment .
, one a combination of 4 q.d./8 b.i.d . , and one a combination of 6 q.d./8 q.d .. the daily exe dose was reduced to 5 g b.i.d . in one ( 6.25% )
data are mean sd . at 3 years , glycemic control was still comparable for exe and glar treatment : the hba1c values were 6.6 0.2% , and 6.9 0.2% at 168 weeks ( between - group difference : p = 0.186 ) ( fig .
because of the treat - to - target titration , the glar group showed a significantly greater reduction in fpg as compared with exe ( 2.0 0.4 vs. 0.2 0.5
after 12 weeks off - drug , both hba1c and fpg increased in both groups to pretreatment values ( fig .
time course for hba1c ( a ) , fasting plasma glucose ( b ) , and change in body weight ( c ) .
data are mean ( sem ) . = exe ; = glar . vertical hatched lines at weeks 52 , 64 , and 168 represent cessation and restart of study medication .
after 168 weeks of treatment , exe reduced body weight by 5.7 1.3 kg , while treatment with glar resulted in a body weight increase of 2.1 1.3 kg ( between - group least squares mean difference , 7.9 1.8 kg ; p < 0.001 ) ( fig .
2c ) . during the 12-week off - drug period , body weight slightly trended back toward baseline values in both treatment groups , leaving a statistical significant difference at the end of the study in favor of exe ( between - group difference at week 180 : 5.5 1.7 kg ;
, whole - body insulin mediated glucose uptake did not differ between the two treatment groups .
three - year treatment with exe improved the m value by 39% ( p = 0.006 ) while glar had no effect ( p = 0.647 ) . at week 0 , both glucose- and arginine - stimulated c - peptide secretion did not significantly differ between the two treatment groups ( fig .
exe significantly improved -cell function during 52 weeks of active treatment compared with titrated glar ( fig .
after 4-week cessation of both exe and glar therapy , -cell function returned to pretreatment values at week 56 ( fig .
-cell function parameters during 3 years of exe ( n = 16 ) and glar ( n = 20 ) treatment .
serum c - peptide concentrations during hyperglycemic clamp are shown at week 0 ( a ) , week 52 ( b ) , week 56 ( c ) , and week 172 ( d ) .
-cell secretory capacity ratio - to - pretreatment is shown in the exe- ( e ) and glar - treated ( f ) groups .
= week 0 ( pretreatment ) ; = week 52 ( on - drug ) ; = week 56 ( off - drug ) ; = week 172 ( off - drug ) .
= exe week 0 ( pretreatment ) ; = exe week 172 ( off - drug ) ; = glar week 0 ( pretreatment ) ; = glar week 172 ( off - drug ) .
data are mean ( sem ) in a d and g h ; geometric mean ( sem ) in e f .
airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; airgluc and 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose .
following 3-year treatment with either exe or glar and a 4-week off - treatment period , the glucose- and arginine - stimulated c - peptide secretion , as compared with baseline , remained similar : 1.02 0.11 and 1.06 0.10 for exe and glar , relative to baseline , respectively ( fig .
interestingly , first- and second - phase glucose - stimulated c - peptide responses were significantly lower in the exe - treated patients after 3 years of treatment when compared with glar ( fig .
3d ) : first phase relative to pretreatment exe 0.88 0.09 , glar 1.08 0.10 , p = 0.038 , and second phase relative to pretreatment exe 0.97 0.08 , glar 1.17 0.08 , p = 0.017 . however , the di change from pretreatment showed a sustained effect on -cell function 4 weeks after cessation of treatment in the exe - treated patients , whereas a reduction was observed in the glar - treated patients ( fig .
3 g and h : 1.43 0.78 and 0.99 0.65 , respectively ; between - group difference p = 0.028 ) .
this is in contrast to the 1-year data , where no sustained effect on the di was observed after cessation of treatment .
no statistical significant between - treatment group difference was observed in the di calculated over the second - phase c - peptide secretion ( 36.89 17.51 and 25.02 14.24 for exe and glar , respectively ; between - group difference p = 0.763 ) . during the extension phase ,
most common adverse events with exe treatment were gastrointestinal in nature ( 42.9% ) and mild to moderate in intensity : nausea 38.1% , vomiting 9.5% , abdominal distention 4.8% , and diarrhea 4.8% . in the exe - treated group ,
two patients ( 9.5% ) withdrew their consent as a result of nausea or vomiting .
nineteen percent of exe - treated subjects experienced treatment - emergent minor hypoglycemia , defined as a self - measured blood glucose concentration < 3.0 mmol / l . at week 168 , 31% of exe - treated subjects ( 5/16 ) had detectable anti - exe antibody titers with the majority ( 4/5 ) of titers in the low range ( < 1/25 titer ) .
the anti - exe antibodies had no predictive effect on the magnitude of an individual s glycemic response or the incidence of adverse events .
most reported adverse events with glar treatment were treatment - related minor hypoglycemia , 28% ; gastrointestinal disorders , 16% ; and vomiting , 8.0% .
no major hypoglycemia and no treatment - related withdrawal because of hypoglycemia were observed in both the exe and glar groups . in the glar - treated group , one patient ( 4.0% )
no significant between - treatment group differences were present at baseline . at the end of the 1-year main study phase , five patients withdrew their consent and did not participate in the extension phase ( one in the glar group and four in the exe group ) because of the demanding study procedures .
of the patients randomized to exe , 69% ( n = 11 ) were treated with exe 10 g b.i.d . at 168 weeks of treatment .
, one a combination of 4 q.d./8 b.i.d . , and one a combination of 6 q.d./8 q.d .. the daily exe dose was reduced to 5 g b.i.d . in one ( 6.25% )
at 3 years , glycemic control was still comparable for exe and glar treatment : the hba1c values were 6.6 0.2% , and 6.9 0.2% at 168 weeks ( between - group difference : p = 0.186 ) ( fig .
because of the treat - to - target titration , the glar group showed a significantly greater reduction in fpg as compared with exe ( 2.0 0.4 vs. 0.2 0.5
after 12 weeks off - drug , both hba1c and fpg increased in both groups to pretreatment values ( fig .
time course for hba1c ( a ) , fasting plasma glucose ( b ) , and change in body weight ( c ) .
data are mean ( sem ) . = exe ; = glar . vertical hatched lines at weeks 52 , 64 , and 168 represent cessation and restart of study medication .
after 168 weeks of treatment , exe reduced body weight by 5.7 1.3 kg , while treatment with glar resulted in a body weight increase of 2.1 1.3 kg ( between - group least squares mean difference , 7.9 1.8 kg ; p < 0.001 ) ( fig .
body weight slightly trended back toward baseline values in both treatment groups , leaving a statistical significant difference at the end of the study in favor of exe ( between - group difference at week 180 : 5.5 1.7 kg ;
p = 0.004 ) ( fig . 2c ) . before randomization , whole - body insulin
three - year treatment with exe improved the m value by 39% ( p = 0.006 ) while glar had no effect ( p = 0.647 ) .
at week 0 , both glucose- and arginine - stimulated c - peptide secretion did not significantly differ between the two treatment groups ( fig .
exe significantly improved -cell function during 52 weeks of active treatment compared with titrated glar ( fig .
after 4-week cessation of both exe and glar therapy , -cell function returned to pretreatment values at week 56 ( fig .
-cell function parameters during 3 years of exe ( n = 16 ) and glar ( n = 20 ) treatment .
serum c - peptide concentrations during hyperglycemic clamp are shown at week 0 ( a ) , week 52 ( b ) , week 56 ( c ) , and week 172 ( d ) .
-cell secretory capacity ratio - to - pretreatment is shown in the exe- ( e ) and glar - treated ( f ) groups .
= week 0 ( pretreatment ) ; = week 52 ( on - drug ) ; = week 56 ( off - drug ) ; = week 172 ( off - drug ) .
= exe week 0 ( pretreatment ) ; = exe week 172 ( off - drug ) ; = glar week 0 ( pretreatment ) ; = glar week 172 ( off - drug ) .
data are mean ( sem ) in a d and g h ; geometric mean ( sem ) in e f .
airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; airgluc and 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose .
following 3-year treatment with either exe or glar and a 4-week off - treatment period , the glucose- and arginine - stimulated c - peptide secretion , as compared with baseline , remained similar : 1.02 0.11 and 1.06 0.10 for exe and glar , relative to baseline , respectively ( fig .
interestingly , first- and second - phase glucose - stimulated c - peptide responses were significantly lower in the exe - treated patients after 3 years of treatment when compared with glar ( fig .
3d ) : first phase relative to pretreatment exe 0.88 0.09 , glar 1.08 0.10 , p = 0.038 , and second phase relative to pretreatment exe 0.97 0.08 , glar 1.17 0.08 , p = 0.017 .
however , the di change from pretreatment showed a sustained effect on -cell function 4 weeks after cessation of treatment in the exe - treated patients , whereas a reduction was observed in the glar - treated patients ( fig .
3 g and h : 1.43 0.78 and 0.99 0.65 , respectively ; between - group difference p = 0.028 ) .
this is in contrast to the 1-year data , where no sustained effect on the di was observed after cessation of treatment .
no statistical significant between - treatment group difference was observed in the di calculated over the second - phase c - peptide secretion ( 36.89 17.51 and 25.02 14.24 for exe and glar , respectively ; between - group difference p = 0.763 ) .
during the extension phase , most common adverse events with exe treatment were gastrointestinal in nature ( 42.9% ) and mild to moderate in intensity : nausea 38.1% , vomiting 9.5% , abdominal distention 4.8% , and diarrhea 4.8% . in the exe - treated group ,
two patients ( 9.5% ) withdrew their consent as a result of nausea or vomiting .
nineteen percent of exe - treated subjects experienced treatment - emergent minor hypoglycemia , defined as a self - measured blood glucose concentration < 3.0 mmol / l . at week 168 , 31% of exe - treated subjects ( 5/16 ) had detectable anti - exe antibody titers with the majority ( 4/5 ) of titers in the low range ( < 1/25 titer ) .
the anti - exe antibodies had no predictive effect on the magnitude of an individual s glycemic response or the incidence of adverse events .
most reported adverse events with glar treatment were treatment - related minor hypoglycemia , 28% ; gastrointestinal disorders , 16% ; and vomiting , 8.0% .
no major hypoglycemia and no treatment - related withdrawal because of hypoglycemia were observed in both the exe and glar groups . in the glar - treated group , one patient ( 4.0% )
the main result of this 3-year follow - up study in patients treated with exe is the sustained improvement in first - phase glucose - stimulated c - peptide secretion , adjusted for prevailing insulin sensitivity ( the di ) 4 weeks after discontinuation of treatment .
no significant effect was seen in the glar - treated patients despite achievement of similar glycemic control .
additionally , exe treatment was associated with continued weight loss and improvement in whole - body insulin sensitivity . both exe and glar
were generally well tolerated with nausea and minor hypoglycemia being the most frequently reported adverse event in the exe and glar groups , respectively . although human data are not available , exposure to glp-1 and glp-1ra in the preclinical setting results in -cell proliferation , islet neogenesis , and inhibition of -cell apoptosis in ( human ) cell lines , primary rodent islets , and in vivo in different rodent species ( 13 ) .
it has therefore been hypothesized , although human islet - cell biology differs widely from that in rodents , that long - term glp-1ra treatment may enhance -cell mass or health in humans , thereby potentially modifying the progressive course of type 2 diabetes ( 14 ) . the current study , particularly the 3-year data presented herein , reports that exe treatment may lend some support to this idea , whereas following 1-year exe exposure , the treatment - related improvement of -cell function was lost after 4-week drug cessation ( 7 ) .
the current 3-year treatment data show a small but statistically significant effect on the di following a 4-week off - therapy period .
our results therefore suggest that a 3-year treatment with a glp-1ra ( such as exe ) is necessary to delineate an effect on -cell function .
additional factors , such as duration of type 2 diabetes and achieved glycemic control and body weight reduction , may play a role in the ultimate efficacy of the glp-1ra . an even longer treatment or intervention at an earlier stage of the disease
additionally , our results confirm findings in diabetic fatty zucker rats that prolonged exe treatment in humans does not result in tachyphylaxis ( 15 ) .
prolonged exposure to elevated glucose and lipid concentrations is detrimental to -cell function ( 16 ) .
these combined glucolipotoxic effects result in impaired insulin secretion and -cell apoptosis , and may contribute to the loss of -cell function in the pathogenesis of type 2 diabetes ( 17 ) .
our results show a similar reduction in hyperglycemia , i.e. , glucose toxicity , in the exe and glar groups after 3-year treatment .
although we did find a 0.2% greater hba1c reduction in the exe - treated group , this finding did not reach statistical significance because of the decreased number of participants left in the study .
since glycemic control was similar for both treatments , this improvement can not be solely attributed to an improvement of glycemic control ; therefore , a glp-1ra related factor should be considered . in as much as -cell function integrity
is determined by the combined effects of variables related to -cell stress versus -cell health , it is important to note that 3-year exe versus glar treatment resulted in body weight reduction of approximately 8 kg with concomitant improvement of insulin sensitivity .
body weight reduction per se has been shown to improve -cell function in subjects with and without type 2 diabetes ( 18,19 ) .
interestingly , the improvement in -cell function reported in our article can not be explained fully by the reduction in body weight alone .
however , in about half of the patients treated with exe , a combined improvement in body weight and di was observed .
additionally , there appeared to be no statistical correlation between the body weight reduction and the di improvement in both exe- and glar - treated patients .
unfortunately , additional post hoc analysis is not possible because of the small sample size .
we recently demonstrated that exe predominantly reduces trunkal fat mass , whereas lean body mass was not affected ( 20 ) .
the observed 39% improvement in m value may at least partly be because of a lowering of the ( trunkal ) body fat mass .
obesity - related insulin resistance is a key feature of type 2 diabetes and is associated with metabolic and cardiovascular complications ( 16 ) .
( 21 ) was the first to report a beneficial effect of continuous glp-1 infusion on clamp - measured -cell function as well as insulin sensitivity in the presence of concomitant weight loss of 1.9 kg in spite of the mere 6-week duration .
subsequent clinical studies using the euglycemic hyperinsulinemic clamp , insulin - modified frequently sampled intravenous glucose tolerance test , or homeostasis model assessment of insulin resistance do not provide a clear view of the effects of glp-1ra on insulin sensitivity and suggest that such an effect may be secondary to the weight reduction ( 22,23 ) . because insulin sensitivity and -cell secretory function are closely interrelated , it is essential to measure both when studying ( long - term ) therapeutic interventions that may affect insulin secretion and body weight ( 1 ) .
the di adjusts for the interaction between changes in insulin sensitivity and insulin secretion as differences in insulin sensitivity must be balanced by reciprocal changes in insulin release in order to maintain glucose tolerance ( and prevent hypoglycemia ) . in the case of exe ,
given the observed weight loss related improvement in insulin sensitivity ( + 39% ) , one may have expected that if -cell secretory function had remained unaltered , the c - peptide response would rather decline . however , inasmuch as the c - peptide remained substantially unaltered ( compared with pretreatment ) , one may conclude that -cell function , following 3 years of exe exposure , was improved ( 1 ) .
additionally , no correlation was observed between treatment - related changes in di and body weight ( supplementary fig .
recently , defronzo et al . ( 22 ) demonstrated a similar phenomenon following 20-week exe monotherapy as compared with the combination of exe and rosiglitazone ( rosi ) .
the di was similar in the exe alone and exe / rosi groups , although the amount of insulin secreted in response to glucose alone or with arginine during the hyperglycemic clamp was markedly reduced in the group receiving exe / rosi combination therapy .
interestingly , defronzo et al . also showed an improvement in the di calculated from the second - phase c - peptide secretion , a finding we did not observe in our study .
in contrast to the former study , we did not administer exe prior to the hyperglycemic clamp .
additionally , a di that is not derived from the rapid c - peptide response to intravenous glucose may not present correct physiology ( 1,12 ) .
the strength of our study is the long - term follow - up and the use of state - of - the - art gold standard methodology to quantify of insulin sensitivity and -cell function in the study population .
additionally , as patients did not receive study medication prior to the hyperglycemic clamp at the end of the extension phase , the effects of a 3-year treatment period on -cell health were measured rather than the acute effects of exe administration .
one limitation of our study is the relatively modest proportion of randomized patients who completed the entire study .
fifty - two percent ( 36/69 ) of all randomized patients completed the entire 3-year treatment period .
of the original 51 patients who completed the 64-week main study , 46 agreed to participate in the extension phase . from these
, 78% completed the additional 2-year treatment period , with similar numbers remaining in both treatment arms .
most patients withdrew their consent and did not participate in the 2-year extension phase of the study because of the demanding nature of the study protocol including long - term follow - up , which included a total of 30 visits ( during 3.5 years ) to the study center .
the proportion of patients who did enter the additional 2-year treatment period show characteristics comparable to those participating in large intervention studies in patients with type 2 diabetes ( 24,25 ) .
only three patients discontinued the 2-year extension phase because of loss of glycemic control ( exe : n = 2 ; glar : n = 1 ) .
finally , three patients dropped out as a result of an adverse event : one patient randomized to glar experienced an ischemic stroke , and two patients randomized to exe dropped out as a result of nausea following reinitiating of exe treatment during the first weeks of the extension period .
generally , both exe and glar were well tolerated during the 3-year study period , and reported adverse events were mainly mild to moderate in intensity , confirming previous long - term study tolerability results ( 6 ) .
interestingly , no patients randomized to exe withdrew their consent during the 3-month off - drug period , whereas four patients in the glar group did .
of the patients randomized to glar , 30.6% did not want to continue with the off - drug period of the study as they observed an increase in smbg , confirming the important role smbg plays in the treatment of patients with type 2 diabetes . in conclusion ,
3-year exe treatment in metformin - treated patients with type 2 diabetes resulted in sustained improvement in -cell function and progressive weight reduction .
long - term follow - up in a wide variety of patients at earlier stages of type 2 diabetes is needed to study possible disease modifying effects of glp-1ra .
| objectivewe previously showed that exenatide ( exe ) enhanced insulin secretion after 1 year of treatment , relative to insulin glargine ( glar ) , with a similar glucose - lowering action .
these effects were not sustained after a 4-week off - drug period .
this article reports the results after additional 2 years of exposure.research design and methodssixty - nine metformin - treated patients with type 2 diabetes were randomized to exe or glar .
forty - six patients entered the 2-year extension study in which they continued their allocated therapy .
thirty - six completed ( exe : n = 16 ; glar : n = 20 ) the 3-year exposure period .
insulin sensitivity ( m value ) and -cell function were measured by euglycemic hyperinsulinemic clamp followed by hyperglycemic clamp with arginine stimulation at pretreatment ( week 52 ) and 4 weeks after discontinuation of study medication ( week 56 and week 172 ) .
first - phase glucose stimulated c - peptide secretion was adjusted for m value and calculated as the disposition index ( di).resultsat 3 years , exe and glar resulted in similar levels of glycemic control : 6.6 0.2% and 6.9 0.2% ,
respectively ( p = 0.186 ) .
exe compared with glar significantly reduced body weight ( 7.9 1.8 kg ; p < 0.001 ) .
after the 4-week off - drug period , exe increased the m value by 39% ( p = 0.006 ) while glar had no effect ( p = 0.647 ) . following the 4-week off - drug period ,
the di , compared with pretreatment , increased with exe , but decreased with glar ( 1.43 0.78 and 0.99 0.65 , respectively ; p = 0.028).conclusionsexe and glar sustained hba1c over the 3-year treatment period , while exe reduced body weight and glar increased body weight .
following the 3-year treatment with exe , the di was sustained after a 4-week off - drug period .
these findings suggest a beneficial effect on -cell health . |
ca release from intracellular stores is mediated by two major classes of intracellular channels , ryanodine and ins(1,4,5)p3 receptors .
mammalian genomes encode three ins(1,4,5)p3 receptor genes , type 13 , with disparate tissue distribution and molecular regulation .
the ins(1,4,5)p3 receptor is a complex signal integrator that consolidates input from multiple signaling cascades into ca release from intracellular stores .
the ins(1,4,5)p3 receptor ( ~2700 amino acids ) is functionally divided into three domains : the ins(1,4,5)p3 binding domain , the coupling domain and the channel domain ( fig .
the pore forming domain localizes to the c - terminal end of the protein and is composed of six membrane spanning regions and a pore forming loop between trans - membrane domains 5 and 6 , followed by a short cytoplasmic tail . the ins(1,4,5)p3 binding domain in the n - terminus of the protein binds the ligand ins(1,4,5)p3 , and the coupling region spans the region between the ins(1,4,5)p3 binding region and the channel domain .
the coupling domain transmits ins(1,4,5)p3 binding into gating of the channel to release ca from the endoplasmic reticulum ( er ) .
in addition to ins(1,4,5)p3 , ca functions as a co - agonist of the ins(1,4,5)p3 receptor regulating it s gating in a biphasic fashion . the bulk of the ins(1,4,5)p3 receptor protein is cytoplasmic and offers multiple sites for regulation by interacting proteins , small molecules and post - translational modifications .
figure 1 . generation and characterization of stable dt40 cell lines expressing ins(1,4,5)p3 receptor phosphorylation mutants .
( a ) cartoon representation of ins(1,4,5)p3 receptor structure with sequence alignment around t-930 , for the rat ( rip3r ) , human ( hip3r ) and xenopus ( xip3r ) ins(1,4,5)p3 receptors .
different cell lines were generated using the dt40 cell line where all three ins(1,4,5)p3r isoform are deleted ( ko ) .
the 3ko cells were electroporated with the linearized plasmid for either the t930a or t930e mutants .
wt : dt40 cell line expressing only the wild - type ins(1,4,5)p3 receptor ( type 1 ) ; t930a and t930e : expressing the respective mutant ins(1,4,5)p3rs .
the same number of cells was plated at time zero and cells were counted at different time points under equivalent culturing conditions to determine whether the expression of the different mutants affects cell growth .
although the t930e mutant grew at a slightly slower rate the difference were not significant .
( d ) resting ca levels and store ca content were measured in dt40 cells loaded with fura-2 and incubated in ca - free solution .
the ins(1,4,5)p3 receptor requires both ins(1,4,5)p3 and ca as co - agonists to gate open and release ca from stores .
phosphorylation increases the probability of opening ( po ) of the ins(1,4,5)p3 receptor resulting in bursting activity without affecting its ca dependence , arguing for an increase in the apparent sensitivity to ins(1,4,5)p3 .
although pka phosphorylation is best understood , the ins(1,4,5)p3 receptor has been shown to be phosphorylated and modulated by other kinases , including pkc , camkii and non - receptor tyrosine kinases .
pkc phosphorylation of the ins(1,4,5)p3 receptor leads to enhanced ins(1,4,5)p3-dependent ca release ; and camkii phosphorylation modulates ca oscillations and inhibition of ins(1,4,5)p3-dependent ca release .
ins(1,4,5)p3-dependent ca release is also modulated during the cell cycle with the most dramatic example at fertilization .
ca release through ins(1,4,5)p3 receptors is essential for egg activation and for mediating the egg to embryo transition in vertebrates . in xenopus oocytes , ins(1,4,5)p3-dependent ca release is sensitized during meiosis , and this sensitization is essential to generate the fertilization - specific ca transient that is required for egg activation at fertilization . in addition , we have recently shown that remodeling of the endoplasmic reticulum during oocyte meiosis contributes to ins(1,4,5)p3 receptor sensitization through a process termed geometric sensitization .
geometric sensitization is due to er remodeling during meiosis resulting in the formation of large er patches enriched in ins(1,4,5)p3 receptors .
ins(1,4,5)p3 receptors that localize to these er patches display increased sensitivity to ins(1,4,5)p3 as compared with their counterparts in the reticular er .
we have also argued that phosphorylation by cell cycle kinases is involved in ins(1,4,5)p3 receptor sensitization during meiosis .
xenopus oocytes express the sii- variant of the type 1 ins(1,4,5)p3 receptor , and the receptor is phosphorylated at both pka sites throughout oocyte maturation . during meiosis
the ins(1,4,5)p3 receptor is specifically phosphorylated on three additional residues t931 , t1136 and t1145 .
both t931 and t1136 localize to consensus sites for proline - directed kinases such as mapk and cdk1 . furthermore , activation of the mapk cascade or cdk1 sensitizes ins(1,4,5)p3-dependent ca release in xenopus oocytes .
all three residues , t-931 , t-1136 and s-1145 , localize within the coupling domain and correspond to t-930 , t-1140 and s-1152 of the rat si , sii , siii- isoform . the ins(1,4,5)p3 receptor is also phosphorylated during mitosis . here
we test the role of phosphorylation at t-930 of the rat ins(1,4,5)p3 receptor ( si , sii , siii- splice variant ) . we focus on t-930 because it is specifically phosphorylated during meiosis and because the sequence surrounding this residue is conserved among vertebrates ( fig .
we show that a phosphomimetic mutation at t-930 ( t930e ) inhibits ca release through the ins(1,4,5)p3 receptor . furthermore ,
ins(1,4,5)p3-binding affinity of the t930e mutant is significantly decreased compared with the wild - type or t930a mutant .
these data show that ins(1,4,5)p3 receptor phosphorylation at t-930 , a site within the coupling domain , decreases ins(1,4,5)p3 binding affinity of the receptor resulting in decreased ins(1,4,5)p3-dependent ca release .
to investigate the role of phosphorylation at t-930 on ins(1,4,5)p3 receptor function , we engineered two mutants one with an alanine substitution at t-930 ( t930a ) , and the other with a phosphomimetic mutation where the threonine is replaced by a glutamic acid ( t930e ) .
we chose glutamic acid as its side chain closely resembles that of a phosphorylated negatively charged threonine side chain .
as such the t930e mutant is expected to mimic the behavior of the ins(1,4,5)p3 receptor phosphorylated at t-930 .
in contrast , the alanine substitution at t930 serves as a control and provides a mutant that is not phosphorylatable at this residue .
furthermore , it also controls for the structural need for a threonine at this position .
the ubiquitous distribution of the ins(1,4,5)p3 receptor makes it challenging to study the effect of different mutants on ins(1,4,5)p3 receptor function because of the background signal due to endogenous ins(1,4,5)p3 receptors in most cells .
therefore to study the functional consequences of phosphorylation at t-930 , we used the dt40 chicken lymphocyte cell line because of the existence of a dt40 line ( 3ko ) , where all three ins(1,4,5)p3 receptor genes ( type 1 , 2 , and 3 ) have been knocked out .
this provides a clean background to analyze the function of different expressed ins(1,4,5)p3 receptor mutants . using the 3ko line as the parental line , we generated stable dt40 cell lines expressing the wild - type rat ins(1,4,5)p3 receptor , the t930a or the t930e mutants .
following the establishment of the cell lines we amplified and sequence confirmed that they are in fact expressing the relevant ins(1,4,5)p3 receptor mutant .
western blot analysis shows that the three different cell lines express the ins(1,4,5)p3 receptor at equivalent levels , whereas no immuno - reactivity is detected in the 3ko line ( fig .
the growth rate of the different dt40 cell lines were equivalent , arguing that expression of the different mutants did not have a dramatic effect on cell survival or ability to replicate ( fig .
finally , both resting ca levels and ca store content were similar among the different cell lines ( fig .
1d ) , showing that expression of the t930e or t930a mutant does not alter ca homeostasis significantly at rest . to test the sensitivity of the different ins(1,4,5)p3 receptor mutants , we developed an assay to measure ca release from intracellular stores in situ in response to an ins(1,4,5)p3 dose response .
for these experiments dt40 cells were loaded with caged - ins(1,4,5)p3 and ins(1,4,5)p3 was uncaged for different durations as indicated in figure 2 .
cells expressing wild - type ( wt ) ins(1,4,5)p3 receptor respond with an exponential increase in ca release along the ins(1,4,5)p3 uncaging dose response ( fig . 2 ) .
this assay provides a direct test of ins(1,4,5)p3 receptor function because ins(1,4,5)p3 produced following uncaging binds to and gates the ins(1,4,5)p3 receptor without invoking additional intermediaries .
dt40 cells were loaded with a ca dye and caged - ins(1,4,5)p3 and exposed to different uv uncaging pulse durations to produce a dose response of ins(1,4,5)p3 intracellulary .
the level of ca release following the uncaging pulses was used as a measure of the sensitivity of the ins(1,4,5)p3 receptor mutants as compared with the wild - type receptor .
surprisingly in cells expressing the t930a mutant ins(1,4,5)p3-dependent ca release is sensitized compared with the wild - type receptor ( fig . 2 ) .
the t930a mutant receptor responds with increased ca release at lower ins(1,4,5)p3 concentrations although the maximum amount of ca release is comparable to the wild - type receptor ( fig .
this sensitization is unlikely to be due to higher expression levels of the t930a mutant as compared with wt , because the different cell lines express equivalent levels of ins(1,4,5)p3 receptors ( fig . 1 ) and because the maximal ca release is similar between the two cell lines ( fig . 2 ) .
in contrast , cells expressing the t930e mutant exhibit the opposite phenotype of decreased ca release in response to ins(1,4,5)p3 uncaging ( fig . 2 ) .
interestingly , even at high levels of ins(1,4,5)p3 these cells release significantly smaller levels of ca ( fig . 2 ) .
this is not due to decreased store ca content , since t930e expressing cells have a similar store ca load as cells expressing the wild - type receptor or the t930a mutant ( fig .
in contrast cells expressing the t930a mutant or the wild - type receptor produce significantly higher ca release levels at long uncaging durations ( fig .
because the t930e cell line expresses equivalent amount of ins(1,4,5)p3 receptors , these results argue that either the affinity of the t930e mutant to ins(1,4,5)p3 is low or that gating / permeation or ca dependence of the receptor are affected by the t930e mutation leading to short lived or small ca release . to further investigate which of these mechanisms is affected in the t930e mutant , we directly measured the ins(1,4,5)p3 binding affinity of the different mutants in microsomes derived from the stable dt40 cell lines ( fig .
microsomes from brain , which is enriched in ins(1,4,5)p3 receptors , were used as a positive control and showed significant ins(1,4,5)p3-binding that was competed effectively by cold ins(1,4,5)p3 ( fig .
ins(1,4,5)p3 binding to microsomes isolated for the dt40 cell lines expressing the wild - type ins(1,4,5)p3 receptor showed lower binding than brain microsomes , which was also effectively competed away using cold ins(1,4,5)p3 ( fig . 3 ) .
2 ) , microsomes from cells expressing the t930a mutant exhibited higher ins(1,4,5)p3 binding affinity than their wild - type counterparts , whereas ins(1,4,5)p3 binding to t930e microsomes was hardly detectable and only slightly above the background binding observed in the 3ko cells ( fig . 3 ) .
these data show that the behavior of the t930a and t930e mutants in situ can be explained solely based on their ins(1,4,5)p3 binding affinity without the need to invoke alterations to ca dependence , gating or permeation of the receptor .
our results further show that phosphorylation at t930 leads to a significant decrease in ins(1,4,5)p3 binding affinity .
microsomal preparations from brain as a positive control and from the different dt40 cell lines as indicated were subjected to the ins(1,4,5)p3 binding assay to measure the binding affinity of ins(1,4,5)p3 receptor mutants .
briefly lysates were allowed to bind radioactively ( h ) labeled ins(1,4,5)p3 and the label competed with cold ins(1,4,5)p3 .
the competitive assay provides a relative comparative measure ins(1,4,5)p3r affinity in the different mutants as compared with the wild type receptor ( wt ) .
the negative control was the 3ko cell line which does not express any of the three ins(1,4,5)p3 receptor isoforms and as such provides non - specific background binding .
using stable dt40 cell lines expressing different ins(1,4,5)p3 receptor mutants we show that phosphorylation of the type 1 receptor at t930 is likely to decrease ins(1,4,5)p3-binding affinity . as discussed above the ins(1,4,5)p3 receptor
is targeted by various kinases and in most cases phosphorylation results in increased sensitization of ins(1,4,5)p3-dependent ca release . in contrast , a phosphomimetic mutation at t930 results in decreased ins(1,4,5)p3 binding affinity and reduced ins(1,4,5)p3-dependent ca release .
the t930 residue localizes within the coupling domain and is distant from the ins(1,4,5)p3 binding domain in the linear sequence of the ins(1,4,5)p3 receptor .
this brings into question the ability of phosphorylation at this site to modulate ins(1,4,5)p3 binding affinity .
however , the available structure of the ins(1,4,5)p3 receptor obtained from cryo - em studies shows that the coupling domain lies below the ins(1,4,5)p3 binding domain in the 3-d structure of the receptor .
hence phosphorylation at t-930 could modulate ins(1,4,5)p3 binding if this residue interacts with the ins(1,4,5)p3-binding and/or suppressor domains .
alternatively , phosphorylation at t930 could alter the conformation of the coupling domain in close proximity of the ins(1,4,5)p3-binding domain thus modulating its ability to bind ins(1,4,5)p3 .
one argument supporting this possibility is the sensitization of ins(1,4,5)p3-dependent ca release observed in the cells expressing the t930a mutant .
this sensitization argues that structural / sequence modifications within this region of the coupling domain affect ins(1,4,5)p3 receptor sensitivity .
furthermore , ins(1,4,5)p3-dependent ca release is highly sensitized during meiosis and this sensitization is critical for egg activation and the egg - to - embryo transition at fertilization .
this creates a conundrum , since the phosphomimetic t930e mutation decreases ins(1,4,5)p3 binding affinity and as such reduces the sensitivity of ins(1,4,5)p3-dependent ca release .
this is quite interesting as it implicates cross talk among different phosphorylation sites within the ins(1,4,5)p3 receptor to define its biological function . during meiosis
the xenopus ins(1,4,5)p3 receptor is also phosphorylated at two additional residues that match the consensus for proline - directed kinases , t-1136 and s-1145 .
so it is possible that the combinatorial phosphorylation at all three residues sensitizes ins(1,4,5)p3-dependent ca release .
another formal possibility is that the rat receptor used in this study behaves differently in response to phosphorylation at t-930 as compared with the xenopus receptor .
however , given the high degree of sequence conservation between the two receptors ( 89% identity ) this possibility seems remote .
in addition , because of the high levels of endogenous pka activity in oocytes , the ins(1,4,5)p3 receptor is also phosphorylated at the consensus pka sites during maturation .
interestingly , all these residues localize within the coupling domain , arguing that combinatorial phosphorylation within this domain may differentially affect ins(1,4,5)p3 binding affinity and the function of the ins(1,4,5)p3 receptor .
plasmid pcdna3 containing the rat ins(1,4,5)p3 receptor ( pcdna3 rip3r ) was a kind gift from suresh joseph .
mutations in the coding region of the ins(1,4,5)p3 receptor were generated using quick change ii site - directed mutagenesis kits ( strategene ) to change the bases act to gct in the coding dna strand resulting in an amino acid substitution of threonine ( t930 ) with alanine ( t930a ) .
likewise , act was replaced with gaa resulting in the substitution of amino acid threonine ( t930 ) with glutamic acid ( t930e ) .
to generate dt40 cell lines expressing the mutated ip3r , linearized pcdna3 t930a and pcdna3 t930e plasmids were electroporated into a dt403ko cell line where all three ins(1,4,5)p3 receptor isoforms are deleted .
lysates from ~5 10 dt40 cells expressing wild - type rip3r , t930a , t930e , or from the 3ko cell line were separated on denaturing nupage 38% tris - acetate gradient gels ( invitrogen ) .
primary rabbit anti - ip3r ( t443 ) antibody and secondary goat anti - rabbit - hrp antibody ( jackson immunoresearch ) at 1:1000 and 1:7500 dilution , respectively , in 1% hammerstein casein , 2% bsa were used for western analysis .
ins(1,4,5)p3 receptor protein bands were detected using ecl - plus ( amersham ) detection reagent .
microsomes were prepared from rabbit cerebellum by homogenizing 1 g of tissue in 12 ml of e buffer ( 20 mm tris - hcl ph 8.3 , 10 mm kcl , 1 mm edta , 1 mm dtt , cocktail inhibitor iii ( calbiochem ) and 1 mm pmsf ) using a polytron homogenizer .
the lysate was centrifuged at 1000 xg for 15 min at 4c and the resulting supernatant was transferred to a microfuge tube and placed on ice .
the pellet was suspended in 3 ml of e buffer and centrifuged again at 1000x g. the supernatants were combined and centrifuged at 2000 xg for 15 min .
the pellet containing the microsomes was suspended in e buffer and the protein concentration was determined using a biorad protein assay .
the cell pellets were washed once in e buffer then centrifuged at 1,500 xg for 15 min .
the cells were lysed with 20 strokes in a glass dounce homogenizer ( pyrex 7727 - 15 ) in 5 ml of e buffer .
the supernatants were transferred to polyallomer ultracentrifuge tubes ( beckman # 326814 ) and centrifuged at 100,000 xg for 30 min at 4c .
the pellets were suspended in e buffer and protein concentrations were determined using a biorad protein assay .
ins(1,4,5)p3 binding assays were performed using microsome preparations from rabbit cerebellum and dt40 cells expressing wild - type ins(1,4,5)p3 receptor , t930a , t930e , and from the 3ko dt40 cells .
the reaction mix contained 15 nm [ h]ins(1,4,5)p3 ( 5 nm [ h]ins(1,4,5)p3/l net911 , 0.005mci , 21.4ci / mmol ) ; various concentrations ranging from 02,000 m of cold ins(1,4,5)p3 ( 10 mm ins(1,4,5)p3 , invitrogen ) , ins(1,4,5)p3 binding buffer ( 50 mmtris - hcl , ph 8.3 , 1 mm edta , 1 mm dtt , 0.1 m kcl ) , eds buffer ( 20 mm tris - hcl , ph 8.3 , 20 mm nacl , 10 mm kcl , 1 mm edta , 1 mm dtt , 10% sucrose plus protease inhibitors ) in a final volume of 500 l .
the reaction mixtures were incubated on ice for 10 min and then 5 l of -globulin and 5 l of glycogen were added and incubated for additional 5 min .
the mixtures were centrifuge at 20,000 xg for 15 min , the supernatants were aspirated , and the pellets were solubilized in solubilization buffer ( 0.625
m tris - hcl , ph 7.5 , 5% 2-mercaptoethanol , 2% sds ) and the amount of h was determined using a scintillation counter .
measurements of intracellular free calcium concentration ( [ ca]i ) were made using the fluorescent indicator ca - green - am .
cells ( approximately 2.5 10 ) were loaded for 45 min in growth medium at 40 containing 5 m ci - ip3 , 2 m ca - green - am and 0.0025% pluronic f-127 .
cells were washed three times and re - suspended at 4c in hbss ( in mm : 137 nacl , 5.4 kcl , 0.25 na2hpo4 , 0.44 kh2po4 , 1.3 cacl2 , 1.0 mgso4 , 4.2 nahco3 , 5.5 d - glucose , 10 hepes ph 7.4 ) to a final density of 10 cells ml and kept on ice for up to 1 h. cells were then pre - warmed to 40 for 15 min .
fluorescence of the stirred cell suspension was then measured by emission at 520 nm while uncaging of ci - ins(1,4,5)p3 was performed at 330 40 nm .
removal of all residual extracellular calcium was achieved by addition of 3 mm egta at the beginning of fluorescence measurements .
plasmid pcdna3 containing the rat ins(1,4,5)p3 receptor ( pcdna3 rip3r ) was a kind gift from suresh joseph .
mutations in the coding region of the ins(1,4,5)p3 receptor were generated using quick change ii site - directed mutagenesis kits ( strategene ) to change the bases act to gct in the coding dna strand resulting in an amino acid substitution of threonine ( t930 ) with alanine ( t930a ) .
likewise , act was replaced with gaa resulting in the substitution of amino acid threonine ( t930 ) with glutamic acid ( t930e ) .
to generate dt40 cell lines expressing the mutated ip3r , linearized pcdna3 t930a and pcdna3 t930e plasmids were electroporated into a dt403ko cell line where all three ins(1,4,5)p3 receptor isoforms are deleted .
lysates from ~5 10 dt40 cells expressing wild - type rip3r , t930a , t930e , or from the 3ko cell line were separated on denaturing nupage 38% tris - acetate gradient gels ( invitrogen ) .
primary rabbit anti - ip3r ( t443 ) antibody and secondary goat anti - rabbit - hrp antibody ( jackson immunoresearch ) at 1:1000 and 1:7500 dilution , respectively , in 1% hammerstein casein , 2% bsa were used for western analysis .
ins(1,4,5)p3 receptor protein bands were detected using ecl - plus ( amersham ) detection reagent .
microsomes were prepared from rabbit cerebellum by homogenizing 1 g of tissue in 12 ml of e buffer ( 20 mm tris - hcl ph 8.3 , 10 mm kcl , 1 mm edta , 1 mm dtt , cocktail inhibitor iii ( calbiochem ) and 1 mm pmsf ) using a polytron homogenizer .
the lysate was centrifuged at 1000 xg for 15 min at 4c and the resulting supernatant was transferred to a microfuge tube and placed on ice .
the pellet was suspended in 3 ml of e buffer and centrifuged again at 1000x g. the supernatants were combined and centrifuged at 2000 xg for 15 min .
the pellet containing the microsomes was suspended in e buffer and the protein concentration was determined using a biorad protein assay .
the cell pellets were washed once in e buffer then centrifuged at 1,500 xg for 15 min .
the cells were lysed with 20 strokes in a glass dounce homogenizer ( pyrex 7727 - 15 ) in 5 ml of e buffer .
the supernatants were transferred to polyallomer ultracentrifuge tubes ( beckman # 326814 ) and centrifuged at 100,000 xg for 30 min at 4c .
the pellets were suspended in e buffer and protein concentrations were determined using a biorad protein assay .
ins(1,4,5)p3 binding assays were performed using microsome preparations from rabbit cerebellum and dt40 cells expressing wild - type ins(1,4,5)p3 receptor , t930a , t930e , and from the 3ko dt40 cells .
the reaction mix contained 15 nm [ h]ins(1,4,5)p3 ( 5 nm [ h]ins(1,4,5)p3/l net911 , 0.005mci , 21.4ci / mmol ) ; various concentrations ranging from 02,000 m of cold ins(1,4,5)p3 ( 10 mm ins(1,4,5)p3 , invitrogen ) , ins(1,4,5)p3 binding buffer ( 50 mmtris - hcl , ph 8.3 , 1 mm edta , 1 mm dtt , 0.1 m kcl ) , eds buffer ( 20 mm tris - hcl , ph 8.3 , 20 mm nacl , 10 mm kcl , 1 mm edta , 1 mm dtt , 10% sucrose plus protease inhibitors ) in a final volume of 500 l .
the reaction mixtures were incubated on ice for 10 min and then 5 l of -globulin and 5 l of glycogen were added and incubated for additional 5 min .
the mixtures were centrifuge at 20,000 xg for 15 min , the supernatants were aspirated , and the pellets were solubilized in solubilization buffer ( 0.625 m tris - hcl , ph 7.5 , 5% 2-mercaptoethanol , 2% sds ) and the amount of h was determined using a scintillation counter .
measurements of intracellular free calcium concentration ( [ ca]i ) were made using the fluorescent indicator ca - green - am .
cells ( approximately 2.5 10 ) were loaded for 45 min in growth medium at 40 containing 5 m ci - ip3 , 2 m ca - green - am and 0.0025% pluronic f-127 .
cells were washed three times and re - suspended at 4c in hbss ( in mm : 137 nacl , 5.4 kcl , 0.25 na2hpo4 , 0.44 kh2po4 , 1.3 cacl2 , 1.0 mgso4 , 4.2 nahco3 , 5.5 d - glucose , 10 hepes ph 7.4 ) to a final density of 10 cells ml and kept on ice for up to 1 h. cells were then pre - warmed to 40 for 15 min .
fluorescence of the stirred cell suspension was then measured by emission at 520 nm while uncaging of ci - ins(1,4,5)p3 was performed at 330 40 nm .
removal of all residual extracellular calcium was achieved by addition of 3 mm egta at the beginning of fluorescence measurements . | the ins(1,4,5)p3 receptor acts as a central hub for ca2 + signaling by integrating multiple signaling modalities into ca2 + release from intracellular stores downstream of g - protein and tyrosine kinase - coupled receptor stimulation . as such ,
the ins(1,4,5)p3 receptor plays fundamental roles in cellular physiology .
the regulation of the ins(1,4,5)p3 receptor is complex and involves protein - protein interactions , post - translational modifications , allosteric modulation , and regulation of its sub - cellular distribution .
phosphorylation has been implicated in the sensitization of ins(1,4,5)p3-dependent ca2 + release observed during oocyte maturation . here
we investigate the role of phosphorylation at t-930 , a residue phosphorylated specifically during meiosis .
we show that a phosphomimetic mutation at t-930 of the rat ins(1,4,5)p3 receptor results in decreased ins(1,4,5)p3-dependent ca2 + release and lowers the ins(1,4,5)p3 binding affinity of the receptor .
these data , coupled to the sensitization of ins(1,4,5)p3-dependent ca2 + release during meiosis , argue that phosphorylation within the coupling domain of the ins(1,4,5)p3 receptor acts in a combinatorial fashion to regulate ins(1,4,5)p3 receptor function . |
diseases of the myocardium associated with cardiac disfunction by world health organization ( who ) , and it is an important cause of chronic congestive cardiac failure in children .
the reported incidence for cardiomyopathies is 1,13 - 1,24 per 100,000 children 1 , 2 .
although the pathogenesis of disease is not fully understood , disturbances of the cellular and humoral immune system are frequently observed in cmps , and myocardial inflammation is one of the commonest mechanisms in cardiomyopathy 3 .
mannose binding lectin ( mbl ) and macrophage migration inhibitory factor ( mif ) play substantial roles in the pathogenesis of several inflammatory and autoimmune disorders 4 , 5 .
mannose binding lectin is a key molecule in innate immunity with the capacity to bind to microorganisms and kill them by initiating the lectin pathway of complement activation 5 .
furthermore , mbl has a major role in the modulation of inflammation but the mechanisms responsible for mbl interactions with inflammatory pathways is remain unclear 6 .
several studies suggest that , there is a modulatory role of mbl in autoimmune disease such as rheumatoid arthritis and systemic lupus erythematosus 6 - 8 .
previous studies show that the absence of mbl may affect occurrence of cardiovascular complications and myocardial ischemia / reperfussion injury , and cmp in mbl null animal models ( 9 - 11 ) .
mannose binding lectin deficiency has been reported by three single nucleotide polymorphisms ( snps ) in codon 52 , 54 and 57 of exon 1 in the mbl2 gene 6 .
these snps are frequently referred to as variants b , c , and d ( b , c , and d , denoting the substitution of aspartic acid for glysine codon 54 , the substitution of gutamic acid for glycine codon 57 , and the substitution of cysteine for arginine codon 52 , respectively ) .
each of these variant alleles affect the stability of the final protein product , resulting in decreased serum levels and a dysfunctional mbl variant with a lower molecular weight than the normal mbl 6 , 12 .
macrophage migration inhibitory factor is a constitutive element of the host antimicrobial defences and stress response that promotes proinflammatory function of the innate and acquired immune system .
macrophage migration inhibitory factor plays a regulator role in the immune response system and promotes proinflammatory biological activities .
mif is constitutively expressed in variety types of tissue and cells , including innate immune cells such as monocytes and macrophages 4 .
recently , it has been shown that mif gene expression is higher in the heart with impaired glucose tolerance with cardiac dysfunction in rats , and elevated levels of mif were associated with cardiac dysfunction in diabetic patients 13 .
mutations of the human mif gene would predispose affected hosts to altered to sensibility or severity of inflammatory diseases such as juvenile idiopathic rheumatoid arthritis , and glomerulonephritis 4 , 14 , 15 . to our knowledge
, no studies have investigated the possible roles of mbl2 and mif gene polymorphisms in children with cmp .
the aim of the present study is to investigate any possible association between polymorphisms of mbl2 and mif genes and cmp in a group of turkish children , and to investigate the association between the identified genotypes and their clinical features .
patients and controls : twenty unrelated turkish children with cmp , followed up in the paediatric cardiology clinic of the gaziantep university , medical faculty , were compared with 30 age - and sex - matched healthy controls .
the diagnosis of cmp were made by signs and symptoms ( irritability , feeding difficulties , weakness , fatigue , dizziness , syncope , tachypnea , tachycardia , hepatomegaly , and evidence of fluid retension ) , chest x ray ( cardiomegaly , pulmonary venous congestion , pulmonary oedema ) , electrocardiography ( hypertrophy of left ventricle with strain , low voltage complexes ) and echocardiographic signs . cardiomyopathies were classified according to their structural and functional abnormalities such as dilated , in the setting of reduced left ventricular systolic function ; hypertrophic , in the presence of unexplained septal hypertrophy of the left ventricule ; restrictive , when impaired diastolic filling with preserved systolic function and normal ventricular wall thickness 1.the study was approved by the local ethics committee of the faculty of medicine , and informed consents were obtained from the parents of children .
the medical records of all children with cmp were reviewed for information about age , sex , and to document clinical presentation including symptoms , family history , laboratory and echocardiographic findings .
genotyping : all patients and controls were analyzed for codon 54 a / b ( gly54asp ) variation in exon 1 of mbl2 gene and -173 g / c polymorphism in mif gene .
genotyping of mbl2 gene codon 54 a / b : polymerase chain reaction ( pcr ) was performed using a forward ( 5'-taggacagagggcatgctc-3 ' ) and a reverse ( 5'-caggcagtttcctctggaagg-3 ' ) primers in a 25 l volume containing 50 ng dna , 2 mm dntps , 2 nmol of each primer , 1.5 mm mgcl2 and 3u taq polymerase .
the product 349 bp was digested with restriction enzyme bani ( fermentas ) identify codon 54 polymorphism , respectively .
the bani restriction site is present on wild type allele a and absent on variant allele b 17 .
g / c : pcr was performed using a forward ( 5'-actaagaaagacccgaggc-3 ' ) and reverse ( 5'-ggggcacgttggtgtttac-3 ' ) primers . for mif ( -173 ) ,
a 330 bp fragment was amplified , which was then digested with alui restriction enzyme ( fermentas ) , overnight at 37 c. the products were then separated on 3% agarose gel .
the pcr product contains two restriction site for allele c and one of these sites is destroyed when the presence of allele g 18 . statistical analysis : all statistical analyses were performed with the statistical package for the social science for windows ( version 18.0 ; spss inc , chicago , il , u.s.a.).results are given as meansd , while allele frequencies and the distribution of genotype are given as % .
clinical features and mbl / mif gene polymorphisms were compared using the chi - square and the fisher 's exact tests .
differences between groups were compared by kruskal - wallis variant analysis and the mann - whitney u - test .
hardy - weinberg equilibrium ( hwe ) was calculated using de - finetti program 19 .
differences in allele and genotype distributions were assessed using odds ratios ( ors ) and 95% confidence intervals .
the minimum sample size was determined as 44 person in each group at the 80% power level with an error of 5% .
clinical features : the age ranged from 3 months to 13 years ( mean : 3.47 3.38 years ; median : 2.00-ir:3.00 ) in patients with cmp ( n=20 , 10 females/10 males ) . according to the echocardiographic evaluation , 80% ( n=16 ) of the patients had dilated cardiomyopathy , 15% ( n=3 ) hypertrophic cardiomyopathy and 5% ( n=1 ) restrictive cardiomyopathy .
the distribution of aa , ab , and bb genotypes for mbl codon 54 were 65 % ( 13 ) , 25 % ( 5 ) and 10 % ( 2 ) in cmp compared with 76.7 % ( 23 ) , 23.3 % ( 7 ) and 0 % ( 0 ) in the controls .
the allele frequency of a / b in mbl was 77.5 % ( 31 ) , and 22.5 % ( 9 ) in cmp compared with 88.3 % ( 53 ) , and 11.7 % ( 7 ) in the controls .
no significant difference was found between genotypes and alleles of mbl2 gene in patients and controls ( p>0.05 ) .
the distribution of gg , gc , and cc genotypes for mif ( -173 ) were 50 % , 30 % , and 20 % in cmp compared with 56.7 % , 43.3 % and 0 % in the controls ( table 2 ) .
the allele frequency of g / c in mif was 65 % , and 35 % in cmp compared with 78.3 % , and 21.7 % in the controls .
the observed genotype counts were not deviated significantly from those expected according to the hardy - weinberg equilibrium for mbl and mif gene polymorphisms ( p>0.05 ) .
the distribution of aa , ab , and bb genotypes for mbl codon 54 were 65 % ( 13 ) , 25 % ( 5 ) and 10 % ( 2 ) in cmp compared with 76.7 % ( 23 ) , 23.3 % ( 7 ) and 0 % ( 0 ) in the controls .
the allele frequency of a / b in mbl was 77.5 % ( 31 ) , and 22.5 % ( 9 ) in cmp compared with 88.3 % ( 53 ) , and 11.7 % ( 7 ) in the controls .
no significant difference was found between genotypes and alleles of mbl2 gene in patients and controls ( p>0.05 ) .
the distribution of gg , gc , and cc genotypes for mif ( -173 ) were 50 % , 30 % , and 20 % in cmp compared with 56.7 % , 43.3 % and 0 % in the controls ( table 2 ) .
the allele frequency of g / c in mif was 65 % , and 35 % in cmp compared with 78.3 % , and 21.7 % in the controls .
the observed genotype counts were not deviated significantly from those expected according to the hardy - weinberg equilibrium for mbl and mif gene polymorphisms ( p>0.05 ) .
association between the identified genotypes and patients clinical / laboratory characteristics : we investigated correlations of mbl / mif genotypes with clinical and laboratory findings of patients such as duration of symptoms , ejection fraction ( ef ) , fractional shortening ( fs ) , left ventricle end diastolic diameter ( lvedd ) , left ventricle end systolic diameter ( lvesd ) , left ventricle end diastolic volume ( lvedv ) , and left ventricle end systolic volume ( lvesv ) .
no relationship was found between mbl / mif genotypes and these parameters ( data not shown ) . in children with dilated cmp ( n=16 ) ,
serum uric acid levels were higher in patients with mbl aa genotype ( p=0.033 , table 3 ) , while plasma sodium ( na ) levels were higher in patients with mif cc genotype ( p=0.042 , table 4 ) .
the distribution of aa , ab , and bb genotypes for mbl codon 54 were 65 % ( 13 ) , 25 % ( 5 ) and 10 % ( 2 ) in cmp compared with 76.7 % ( 23 ) , 23.3 % ( 7 ) and 0 % ( 0 ) in the controls .
the allele frequency of a / b in mbl was 77.5 % ( 31 ) , and 22.5 % ( 9 ) in cmp compared with 88.3 % ( 53 ) , and 11.7 % ( 7 ) in the controls .
no significant difference was found between genotypes and alleles of mbl2 gene in patients and controls ( p>0.05 ) .
the distribution of gg , gc , and cc genotypes for mif ( -173 ) were 50 % , 30 % , and 20 % in cmp compared with 56.7 % , 43.3 % and 0 % in the controls ( table 2 ) .
the allele frequency of g / c in mif was 65 % , and 35 % in cmp compared with 78.3 % , and 21.7 % in the controls .
the observed genotype counts were not deviated significantly from those expected according to the hardy - weinberg equilibrium for mbl and mif gene polymorphisms ( p>0.05 ) .
the distribution of aa , ab , and bb genotypes for mbl codon 54 were 65 % ( 13 ) , 25 % ( 5 ) and 10 % ( 2 ) in cmp compared with 76.7 % ( 23 ) , 23.3 % ( 7 ) and 0 % ( 0 ) in the controls .
the allele frequency of a / b in mbl was 77.5 % ( 31 ) , and 22.5 % ( 9 ) in cmp compared with 88.3 % ( 53 ) , and 11.7 % ( 7 ) in the controls .
no significant difference was found between genotypes and alleles of mbl2 gene in patients and controls ( p>0.05 ) .
the distribution of gg , gc , and cc genotypes for mif ( -173 ) were 50 % , 30 % , and 20 % in cmp compared with 56.7 % , 43.3 % and 0 % in the controls ( table 2 ) .
the allele frequency of g / c in mif was 65 % , and 35 % in cmp compared with 78.3 % , and 21.7 % in the controls .
the observed genotype counts were not deviated significantly from those expected according to the hardy - weinberg equilibrium for mbl and mif gene polymorphisms ( p>0.05 ) .
association between the identified genotypes and patients clinical / laboratory characteristics : we investigated correlations of mbl / mif genotypes with clinical and laboratory findings of patients such as duration of symptoms , ejection fraction ( ef ) , fractional shortening ( fs ) , left ventricle end diastolic diameter ( lvedd ) , left ventricle end systolic diameter ( lvesd ) , left ventricle end diastolic volume ( lvedv ) , and left ventricle end systolic volume ( lvesv ) .
no relationship was found between mbl / mif genotypes and these parameters ( data not shown ) . in children with dilated cmp ( n=16 ) ,
serum uric acid levels were higher in patients with mbl aa genotype ( p=0.033 , table 3 ) , while plasma sodium ( na ) levels were higher in patients with mif cc genotype ( p=0.042 , table 4 ) .
although the pathogenesis of cmp is not fully understood , cellular as well as humoral autoimmun responses are critically associated with the pathogenesis and progression of the disease .
furthermore , disturbances of the cellular and humoral immune system are frequently observed , and myocardial inflammation is one of the commonest mechanisms in cardiomyopathy 3 .
two of the postulated factors are ; firstly , myocardial inflammation mediated by the effector cells of the immune system ; and secondly locoregional effect of inflammatory mediators , released by the infiltrating lymphocytes , macrophages or endothelial cells 21 . both mif and mbl play several roles in innate and adaptive immune responses , and changes in levels of mbl and mif
are implicated as playing causative role in many disease states 4 , 6 , 22 .
better understanding of the molecular genetics underlying cmp may provide a means of early diagnosis , genotype - based therapy , and even prevention of the disease .
mannose binding lectin deficiency is associated with susceptibility to infectious and autoimmun diseases and serum mbl levels vary substantially because of the variant alleles in exon 1 of the mbl2 gene , located on chromosome 10 in the humans 22 . in the present study , homozygosity for mbl
variant allele was observed only two patients with dilated cmp and the a / a genotype was higher than the variant alleles in cmp patients , but these results were not statistically significant .
messias - reason et al , suggested that wild type variants of mbl2 gene was significantly higher in rheumatic hearth diseases 23 .
reported that subjects homozygous for the wild type allele had a higher concentration of mbl than heterozygous subjects and than homozygous for the variant mbl2 alleles 24 .
schafranski et al showed that , genotypes associated with a higher level of mbl seem to represent a risk factor for the evolution of rheumatic carditis , and mbl play a substantial role in the progression of the disease to chronic form 25 .
however , some studies indicated that there was either no association of mbl gene polymorphisms and systemic lupus erythematosus ( sle ) and rheumatoid arthritis ( ra ) , or even an increased risk for ra has been demonstrated in mbl insufficiency 8 , 26 . in the present study , homozygosity for mbl
variant allele was observed only two patients with dilated cmp and the a / a genotype was higher than the variant alleles in cmp patients , but these results were not statistically significant .
these different results in literature highlight the need for further detailed studies to understand the exact role of mbl2 gene polymorphism in several diseases .
we investigated correlations of mbl genotypes with clinical and laboratory findings of the disease , and found that uric acid levels were higher in patients with mbl aa genotype than the other genotypes .
uric acid is a useful marker for the decompensation trigger in chronic heart failure , and might be related to inflammatory responses 27 .
it has been shown that the left ventricular hypertrophy has an important potential to increase uric acid level 28 .
measured uric acid levels in idiopathic dilated cardiomyopthy , and they observed that serum uric acid levels are significantly higher in the lower coronary flow reverse group than in the higher coronary flow reverse group 29 .
interestingly , garred et al reported that homozygous for wild type alleles in exon 1 of mbl2 gene were more likely to show evidence of persistent inflammation 30 .
we suggest that the reason of increased uric acid level in our patients may be both inflammation and chronic heart failure .
macrophage migration inhibitory factor plays an important role in the control of innate immun responses and promotes proinflammatory biological activities .
four polymorphisms of the human mif gene ( -794 , -173 , + 254 , + 656 ) have been reported , and this polymorphisms would predispose affected hosts to altered susceptibility to or severity of inflammatory or infectious disease 4 .
patients with -173 c allele ( that is , guanine - to - cytosine transition at position -173 ) had increased levels of mif , and increased mif concentrations had been associated with severe clinical manifestations , high severity scores , and poor outcome of inflammatory disease 4 , 31 , 32 . in the other studies , no association were found in genotype distributions of mif -173 g / c polymorphism between ulcerative colitis , juvenile rheumatoid arthritis and healthy controls 14 , 33 .
however , donn et al showed that mif -173 c allele was associated with juvenile idiopathic arthritis 31 , 32 .
moreover , mif-173 c allele had a significantly greater number of joints with active arthritis and was associated with a poor response to glucocorticoids in patients with juvenile idiopathic arthritis 31 .
berdeli et al showed that , the mif -173 c allele was a poor outcome predictor in jra 14 .
miller et al . demonstrated that mif released from ischemic cardiomyocytes stimulates adenosine monophosphate - activated protein kinase ( ampk ) activation and promotes glucose uptake , and thereby protects the hearth against ischaemia reperfusion injury 34 .
jian et al found that mif protein is constitutively expressed by cardiomyocytes in vivo and is increased in the myocardium of infants with cyanotic cardiac defects in myocardial biopsy materials 35 .
tereshchenko et al did not reveal an association of the myocardial infarction with the mif-173 c allele polymorphism 36 . in the present study , homozygosity for mif-173
recently , it has been shown that presence of -173c allele indicates higher mif levels 37 , and cardiac inflammation ( autoimmune , viral or post viral ) has an important component in the pathogenesis of dilated cmp 38 .
therefore we suggest that , cc genotype in our patients may be partially responsible from inflammation in dilated cmp , and mif polymorphism may contribute to mif release from cardiomyocytes in children with cmp .
however , we could not find any relationship between mif genotypes and cardiac functions . considering the limited number of our patients
it has been shown that , plasma brain natriuretic peptide ( bnp ) concentrations were increased in various forms of heart disease with impaired left ventricular systolic function including cardiomyopathy 39 .
natriuretic peptides inhibit the transport of sodium and water in proximal tubules and block reabsorption of sodium 40 . in this study ,
plasma sodium levels were higher in patients with mif cc genotype than the other genotypes .
however , we could not conclude whether cc genotype of mif has any effect on bnp with this study .
this study is the first to investigate the mbl and mif gene polymorphisms in turkish children with cmp .
we conclude that cc genotype of mif ( -173 ) gene may be a risk factor for cmp patients .
however , further studies with larger samples are needed to address the exact role of this polymorphism in cmp . | myocardial inflammation is one of the commonest mechanisms in cardiomyopathy ( cmp ) .
mannose binding lectin ( mbl ) is a key molecule in innate immunity , while macrophage migration inhibitory factor ( mif ) is a constitutive element of the host defenses .
we investigated the possible association between polymorphisms of mbl2 and mif genes and cmp in turkish children .
twenty - children with cmp and 30 healthy controls were analyzed for codon 54 a / b polymorphism in mbl , and -173 g / c polymorphism in mif genes by using pcr - rflp methods .
no significant difference was found between genotypes and alleles of mbl2 gene codon 54 a / b polymorphism in patients and controls ( p>0.05 ) .
however , serum uric acid levels was found higher in dilated cmp patients with aa genotype .
frequency of mif -173 cc genotype was significantly higher in patients ( p<0.05 ) , and sodium levels were higher in patients with mif -173 cc genotype .
this study is the first to investigate the mbl and mif gene polymorphisms in turkish children with cmp .
we conclude that cc genotype of mif ( -173 ) polymorphism may be a risk factor for cmp patients .
however , further studies with larger samples are needed to address the exact role of this polymorphism in cmp . |
the sporadic ( i.e. , non - familial ) form of the alzheimer 's disease ( ad ) syndrome ( sads , where the first s is for sporadic , and the second s is for syndrome ) is by far the most common form of this disorder , and appears to result from a more or less individually unique set of converging risk factors , inadequately counterbalanced by a set of hypothetical protective factors that is also relatively unique to the individual affected ( fernndez et al . , 2008 ; kulji , 2009a , 2010b ) .
here we postulate that these sets of opposing factors operate over decades primarily ( i.e. , pathogenically ) or secondarily ( i.e. , as a common final pathway of prior events ) to target the so - called polydendrocytes , a.k.a .
beta astrocytes and the fourth neuroglial type , a relatively recently discovered ( reyners et al . , 1982 , 1986 ) and subsequently confirmed type of neural cell ( peters , 2004 ; nishiyama et al .
we designate them here as the fourth element cell ( 4ec ) following the tradition established by santiago ramn y cajal when he made a distinction between neurons , macroglia , and all other types of cells in the brain , and lumped the latter types of cell into a third element .
po del ro hortega subsequently discovered and named what we know today as the microglia from among the third element , employing a battery of silver carbonate impregnation methods .
modern technology has confirmed del ro hortega 's discoveries , and demonstrated that altered forms of microglia participate in the pathology of ad , since they are a key component of senile plaques ( peress et al . , 1993 ; rogers et al .
in the present formulation , we propose that virtually all forms of the ad syndrome ( ads ) result from targeting of the 4ec , whether it is the primary ( i.e. , initial event ) or a secondary insult resulting from either single causes of the sads ( i.e. , the very rare , so - called familial ad that shares some of the phenotypes of the sads , and quite often has clinical manifestations that are atypical for sads ) , or the proposed converging set of risk vs. protective factors that appear to be by far the most common precipitating events leading to ad ( fernndez et al .
this is relevant to a current , unresolved debate as to whether the primarily targeted element(s ) in ad are neurons ( wirths et al .
, 2004 ; bayer and wirths , 2010 ) , astrocytes ( selinfreund et al . , 1991 ; marshak et al . , 1992 ; sheng et al . , 1994 ;
, 2003 ; kashon et al . , 2004 ; pertusa et al . , 2007 ; simpson et al . ,
leung et al . , 2009 ; streit et al . , 2009 ; ager et al .
2010 ) , and the overarching concern as to whether the postulated and not mutually excluding roles of nucleic acid damage ( gleckman et al . , 1999 ;
myung et al . , 2008 ) , inflammation ( sastre et al . , 2006 ; rogers et al .
, 2007 ; rojo et al . , 2009 ; eikelenboom et al . , 2010 ; heneka et al . ,
, 2006a , b ; myung et al . , 2008 ) that are strongly felt to be central to the disease process target specific brain regions and circuits ( kulji , 2009b , 2010b ) , and/or cell types ( as stated above , loc.cit . ) ,
and/or components of cells such as synapses ( de kosky and scheff , 1990 ; terry et al . , 1991 ; bell and cuello , 2006 ; arendt , 2009 ) rather than any of the former elements of the brain , or , instead , specific molecules regardless of which cellular and subcellular components or compartments they are situated in , even outside the brain .
the recent finding that pancreatic lesions grossly resembling those in the brain occur in ad ( miklossy et al . , 2010 ) can in fact support the latter possibility , or , alternatively , when considering the similarities between neurons and pancreatic beta cells , the former cell - based explanations .
this unresolved debate is thus central to the elucidation of the key mechanisms underlying ad , as well as to additional , urgent public health issues such as the controversy about the potentially common mechanisms underlying the converging epidemics of diabetes and dementia worldwide ( kulji and alkovi - petrii , 2010 ) .
the present hypothesis is based on : ( 1 ) the evidence for glial participation in the pathophysiology of ad shown by alzheimer ( 1906 , 1907 ) , and confirmed repeatedly ever since ( perusini , 1910 , 1911 ; beach et al . , 1989 ; peress et al . , 1993 ; sheng et al . , 1994 ; simpson et al . , 2010 ) , since 4ec are considered by most as glial cells ( loc.cit ) , ( 2 ) a revision of the danger signals hypothesis of ad pathogenesis ( fernndez et al . , 2008 ) which , makes use of the massive convergent evidence for susceptibility and protective factors that appear to operate over decades to eventually produce ad , but that originally had no significant role for the increasingly apparent macroglial involvement in this condition ( summarized in the paragraph above , and discussed in more detail below ) , and ( 3 ) the experimentally unasserted involvement of the 4ec in ad , which is very likely because of their demonstrated hypersensitivity to dna damage ( reyners et al . , 1982 , 1986 ) which is know to occur in ad ( gleckman et al . , 1999 ;
myung et al . , 2008 ) but had not been linked yet to the involvement of 4ec hypothesized here .
this is consistent with the fact that dna damage has already been identified in closely related neurodegenerative disorders ( gleckman et al . , 1999 ) making our hypothesis plausibile
furthermore , addressing this possibility in ad but not considering the potential involvement of 4ec often confused with astrocytes
while the exact effect of the astrocytic dna damage on the functions and vitality of astrocytes is unclear , the presence of dna damage alone may not signify apoptosis but more likely identifies a subset of vulnerable astrocytes in which mechanisms of both cell death and repair have been activated ( the emphasis is ours ) .
this obviously opens the possibility that at least part of the astrocyte - like population susceptible to dna damage in ad are 4ec ( i.e. , those first recognized by reyners et al . , as described in the next subsection ) , rather than true astrocytes .
in a set of two studies that have received surprisingly little attention , reyners et al .
( 1982 , 1986 ) described their discovery of a large population of unconventional cells , that they felt erroneously is a previously unidentified astrocyte , that differs from the classically defined neuroglia in that : ( 1 ) they are antigenically different from astrocytes , oligodendrocytes and microglial cells , ( 2 ) express ng2 chondroitin sulfate proteoglycan - like immunoreactivity , ( 3 ) have no cytoplasmic intermediate filaments ( e.g. , gfap , vimentin , and s100 protein ) , and exhibit a variety of ultra structural features different from astrocytes although they superficially resemble astrocytes ( discussed below ) , ( 4 ) they can not be labeled with markers for microglia , although they do express mrna encoding proteolipid protein and myelin basic protein , ( 5 ) some of these cells appear to be oligodendrocyte progenitors and lose the ng2-like immunoreactivity as they mature , ( 6 ) they do not react with antibodies that recognize neurons , and ( 7 ) are more radiosensitive than conventional glial cells , which we presume may reflect their susceptibility to dna damage and/or free radical toxicity , or both , since there are far more mitotically active than all other types of glial cells .
these unusual properties were summarized by peters ( 2004 ) in an elegant study of their ultrastructurally unique characteristics .
4ec cells , which he dubbed the neuroglial cells , have pale , irregularly shaped nuclei with a thin rim of heterochromatin under the nucleolemma and a pale cytoplasm .
these cells are common in the brain of rodents and non - human primates , including the cerebral neocortex and hippocampus known to be targeted in ad ( kulji , 2009a , b , 2010b ) . in monkeys , there are at least two types of peters neuroglial cells ( a.k.a . ng2 cells ) , one with a darker nucleus than the more abundant type containing a lighter nucleus ( peters , 2004 ) , suggesting that there may be various types of cell within the broad category of 4ec . while it is clear that the 4ec are neither conventional microglia nor oligodendrocytes , it is very easy to confuse them with astrocytes due to their superficial resemblance to the latter ( reyners et al . , 1982,1986 ) . however , 4ec are said to differ from astrocytes not only in the absence of intermediate filaments characteristic of the latter , but , as mentioned , in that they have a more irregularly shaped nucleus , a more regularly shaped cell body than protoplasmic astrocytes , and their mitochondria are thinner that those in astrocytes .
furthermore , 4ec are separated from the basal lamina of cerebral microvessels by intervening processes of astrocytes ( peters , 2004 ) .
it is not known whether or how 4ec are involved in neurodegenerative disorders , whether ad or other conditions , such as multiple sclerosis which is intriguing due to their suspected role as precursors of oligodendrocytes among other cell lineages , including possibly even neurons ( nishiyama et al . , 2009 ) .
perhaps the most intriguing feature about 4ec is that they are the cells first identified by bergles et al .
( 2000 ) as a type of glia receiving glutamatergic input from hippocampal pyramidal cells ( known to be targeted in ad ) and by inhibitory interneurons ( also targeted in ad ) .
in fact , both in the gray and white matter , there is an ng2-expressing glial cell that express also ionotropic receptors for glutamate and gabaa and have direct synaptic junctions with axons that enable the transient activation of these receptors .
furthermore , these exhibit all the features of classical neuron - to - neuron synapses including rapid activation , quantized responses , depression and facilitation and presynaptic inhibition ( nishiyama et al . , 2009 ; bergles et al . , 2010
ng2-containing cells also express ampa receptors , exhibit permeability to calcium , and express also nmda receptors which raises the likelihood that they may be susceptible to excitotoxic injury .
it is therefore highly plausible that this novel mechanism for neuron - glia signaling is important not only for the normal brain , but also relevant for the diseased central nervous system ( bergles et al . ,
2010 ) although , to the best of our knowledge , they have never been implicated in ad .
this is undoubtedly due mainly to the lack of awareness of the existence of the 4ec .
however , among other possibilities , the targeting of 4ec we postulate may underlie the observed reduction in excitatory amino acid transporters that correlates with ad clinical manifestations , which has been attributed so far only to astrocyte damage , but not to 4ec degeneration ( simpson et al . , 2010 ) .
given the ongoing dramatic changes in the distinction between neurons and glial cells in the brain , the proposal that 4ec are pivotally involved in ad defies the former notion that the targeted element(s ) can be conceptualized simplistically as neurons vs. macroglia vs. microglia , vs. other cells that are not of a neural lineage .
in fact , considering the still unresolved nature of 4ec as precursors or variants of oligodendrocytes , their near - neuronal attributes ( e.g. , synapses and the participation on glutamatergic and gabaergic neurotransmission ) and their features in common with conventional astrocytes , their postulated primary , even pathogenic , involvement in ad blurs the distinction among all conventional cell types in ad .
even if the present proposal about the predominant targeting of 4ec in ad does not survive experimental testing , it casts doubt on the ability to postulate targeting mechanisms based exclusively on neuronal vs. glial phenotypes .
thus , it is also possible that it is not specific cell types , but key attributes shared between neuronal and non - neuronal cells that are targeted early on in the disease process , if this process attacks first or in any way selectively neural cells .
progress in conquering ad and related disorders is hampered by the so - called innovation gap ( ig ) , which is a collection of challenges that includes : ( a ) the fact that the number of approved treatments has not increased despite substantial increments in the investment toward the discovery and testing for this purpose over decades , ( b ) the frequent lack of predictive power for the efficacy in humans of the outcomes of testing in so - called models of the disorder , and ( c ) the failure of all trials published to date based on the overwhelmingly dominant amyloid hypothesis of ad pathogenesis ( kulji , 2009b , 2010a ) .
if the 4ec hypothesis is correct , it would open an essentially unexplored avenue for research and treatment development that could have a powerful effect in overcoming the ig .
a recent report provides powerful evidence that it may now possible to predict the outcome of patients at risk of developing ad , by measuring putative markers of the disorder that include -amyloid and both total and phosphorylated tau protein in the cerebrospinal fluid ( de meyer et al . , 2010 ) .
however , markers with proven diagnostic value do not presently include indicators of inflammation , macroglial alterations , and , much less , 4ec involvement , although there are numerous reports suggesting but not proving that the former may also be of diagnostic value ( crols et al .
, 1986 ; wallin et al . , 1996 ; fukuyama et al . , 2001 ; peskind et al . , 2001 ;
, 2008 ) , it is likely that an array of markers addressing different mechanisms involved in the condition may help to : ( 1 ) increase the sensitivity and specificity of the tests under development , ( 2 ) discern subtypes of patients in which different mechanisms mediate cognitive impairment , and ( 3 ) predict and monitor the response to various alternative mechanism - selective treatments . therefore , whether the hypothetical 4ec mechanism proposed here is universal , or unique to certain groups of patients , research aimed at testing this hypothesis may lead to the implementation of assays for 4ec involvement by cerebrospinal fluid analysis , or blood - borne indicators of 4ec involvement , or in vivo imaging assessment of such involvement ( with ligands for 4ec ) or a combination of these as well as a more refined histopathological assessment for patients with mild cognitive impairment and dementia postmortem .
the above considerations are relevant also to our recent assessment of the intersection of the epidemics of dementia and diabetes , since this is one additional situation in which the elucidation of the targeted vs. spared or disease - resistant elements is essential to move beyond conflicting interpretations of the same or closely related experimental observations ( kulji and alkovi - petrii , 2010 ) .
it has not escaped our attention that casting hypotheses on the primary targeting mechanisms causing ad in terms of one or another cell type or cellular component , such as synapses may be narrow - minded and ultimately inadequate .
in fact , given the : ( 1 ) seemingly unconventional nature of 4ec , which share cytological and molecular features with neurons , macroglia , and oligodendrocytes , ( 2 ) the evidence that markers previously felt to be unique to neurons such as doublecortin or macroglia such as s100b are expressed by a variety of other cell types ( steiner et al . , 2007 ; verwer et al . , 2007 ) , and ( 3 ) that the s100b / rage - mediated activation of microglia ( bianchi et al . , 2010 ) indicates there may be and inextricable interaction between 4ec , macro- and microglia in the genesis of brain inflammation that initially triggers neurodegeneration ( fernndez et al . , 2008 ;
2010 ) , it may be difficult to discern between a primary / initial macro- vs. microglial trigger of ad .
this challenge is in fact compounded by the 4ec hypothesis , since 4ec share properties with several neural cell types .
however , it would seem that casting the present hypothesis in terms of a putative primary involvement of 4ec is scientifically testable , and provides a heuristically useful counterbalance to the presently massive if appropriate
attention to the suspected role of microglia in the early mechanisms mediating ad ( fernndez et al .
, 2008 ; leung et al . , 2009 ; rojo et al . , 2009 ; streit et al . , 2009 ; ager et al . ,
this will require further characterization of 4ec in both the normal and diseased human and non - human brain , to approach the level of sophistication with which it is possible to study today the involvement of more conventional types of cell in the brain and other organs .
none of the above hypothetical scenarios are contradictory or incompatible with the eventual ( i.e. , secondary ) dysfunction , followed by the degeneration and death of neurons , as the final mediator of the clinical manifestations of ad and related disorders as proposed by most prior authors , including ourselves ( fernndez et al .
, 2008 ; kulji , 2009a , b , 2010b ; rojo et al . , 2009 ; kulji and alkovi - petrii , 2010 ) .
such secondary events include also inflammation and the ( intra- and extracellular ) accumulation of pathological forms of amyloid and tau proteins , regardless of whether these are cast as disease - compounding , or ultimately ineffective or counterproductive attempts at cellular and molecular defense .
the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | despite well over a century of research on all forms of the disorder known as alzheimer 's disease ( ad ) , it is still not known whether the condition targets initially neurons , glial cells , other cellular elements in the brain , or components of cells , such as synapses , or molecules independently of their cellular compartmentalization , or otherwise ( e.g. , specific neuronal circuits ) .
multiple lines of highly suggestive but as yet insufficient experimental evidence are discussed here to formulate the hypothesis that ad results from primary ( i.e. , direct and initial ) or secondary targeting of what we designate as the fourth element cell ( 4ec ) : a relatively recently identified type of brain cell that exhibits features in common with neurons ( e.g. , synapses , participation in glutamatergic , and gabaergic neurotransmission ) , astrocytes , oligodendrocytes , and their precursors , but is in other respects clearly distinct from all of them .
the 4ec is proposed to be the main target of both : ( 1 ) converging insults ( i.e. , not true
causes ) that over time cause sporadic forms of ad as postulated by the danger signal hypothesis which was not formulated with 4ec in mind as well as ( 2 ) the causes of inherited ( i.e. , familial ) forms of neurodegeneration that resemble certain aspects of the clinical manifestations of sporadic ad . |
quantitative genetic analyses using twin , family and adoption designs have demonstrated that familial resemblance in body mass index ( bmi ) is largely due to genetic similarity , with high heritability estimates reported from twin studies ( 4790% ) , and moderate - to - high estimates from family ( 2481% ) and adoption studies ( 2060% ) .
a recent meta - analysis of twin studies showed that heritability estimates were on average 0.07 higher in children than in adults .
genome - wide association studies ( gwas ) have made significant headway in identifying single - nucleotide polymorphisms ( snps ) that are related to the relative body weight , indexed using bmi . a large meta - analysis of 123 865 adults from 46 studies with follow - up in another 125,931 participants conducted by the genetic investigation of anthropometric traits ( giant ) consortium identified 32 snps robustly associated with adult bmi .
the majority of these snps ( 2328 ) demonstrated directionally consistent effects in age- and sex - adjusted bmi in children and adolescents . a subsequent meta - analysis of 14 studies with 5530 cases of obesity and 8318 controls identified another two snps associated with childhood and adolescent obesity that also showed directionally consistent effects in the previous meta - analysis of adult bmi .
however , even in combination , the 32 established snps explain < 2% of the variation in bmi in either adults or children ,
although there are some suggestions that the size of the association between combined genetic obesity risk and adiposity may vary over the lifespan , peaking during late childhood ( age 11 ) and early adulthood ( age 20 ) in line with heritability estimates .
the mismatch between the high heritability estimates from quantitative genetic analyses and the small proportion of variation explained through gwas findings across many complex traits have come to be known as the problem of missing heritability ' .
part of the missing heritability is likely to be due to rare genetic variants and some non - additive genetic effects .
these contribute to the estimated genetic effect in quantitative genetic studies , but are not detected in gwas analyses that only capture additive effects of common snps with minor allele frequencies of 5% .
a second possibility is that there are multiple additional common genetic variants that contribute to the genetic effect observed in quantitative genetic studies , but have such small effect sizes that they can not be detected even in the huge data sets used in contemporary gwas analyses .
however , until there is direct molecular genetic evidence for these additional sources of genetic influence , missing heritability is not clarified , and questions will remain about whether the heritability of obesity has been overestimated by quantitative genetic studies .
a novel approach called genome - wide complex trait analysis ( gcta ) takes advantage of the fact that the degree of genetic resemblance for common snps at the whole - genome level is normally distributed among unrelated individuals .
this can be used to quantify the proportion of the variation in a particular phenotype that is explained by the total common snp similarity , effectively a molecular genetic estimate of heritability .
the purpose of gcta is not to identify specific snps related to the target phenotype , but rather to estimate the total additive genetic effect of the common snps used on currently available dna arrays .
its value relative to gwas comes from the fact that the gcta estimate includes the effect of snps well below the current gwas threshold .
the first report found a genetic effect due to additive effects of common snps of 16.5% a remarkable order - of - magnitude increase compared with the effect of known genetic variants , and not far off the lower limit for additive genetic influence as estimated from family studies ( e.g. ) .
a second study produced very similar results , with gcta estimates of 14 and 10% in two independent adult samples .
in this study , we provide the first pediatric gcta estimate of additive genetic effects on adiposity in a sample of unrelated children .
we also include the twin - based estimate of heritability in the same sample for direct comparison with the gcta estimate by including data from the co - twin in the same families .
data were from the twins early development study ( teds ) , a population - based cohort of monozygotic and dizygotic twins ( > 11 000 pairs ) born between 1994 and 1996 in england and wales .
twins and parents provided informed consent for each part of the study prior to data collection .
genome - wide genotyping was completed in 2010 for one randomly selected child in each of 3665 families ; of these , 3152 ( 1446 male and 1706 female subjects ) survived quality control criteria for ancestry , heterozygosity , relatedness and hybridisation intensity outliers ( for details see ) . genotyping and
quality control was done using the affymetrix 6.0 genechip snp genotyping array ( affymetrix inc , santa clara , ca , usa ) using standard experimental protocols as part of the wtccc2 project .
snps were selected on their minor allele frequency ( > 0.01 ) , genotype call - rate ( > 0.80 ) , hardy weinberg equilibrium ( > 10 ) and plate effect p - value ( > 10 ) , which resulted in 700 000 quality - controlled genotyped snps . in addition , there were 2.5 million snps imputed from hapmap 2 and 3 , and wtccc controls , using the programme impute v.2 software .
imputed snps were screened using much more stringent quality control that resulted in reduction to 1 000 000 snps , giving a total of 1.7 million ( quality controled ) snps ( for details see ) . to control for ancestral stratification , we performed principal component analysis using eigenstrat from eigensoft package and identified significant axes using the tracy widom test .
this resulted in eight axes with p<0.05 that were used as covariates in gcta analyses .
height and weight data were obtained in 2005 when the children were 811 years old , as part of a study of the heritability of adiposity .
parents were sent detailed instructions and asked to record each child 's weight to the nearest pound or tenth of a kg , and height to the nearest cm , as well as the date of measurement .
parent- and researcher - measured heights and weights were correlated 0.90 and 0.83 in a subsample of 228 families .
scores ( bmi - sds ) that take into account the child 's age and sex , using 1990 uk growth reference data and computed with the programme imsgrowth .
reference values were used to exclude implausible heights ( < 1.05 or > 1.80 m ) , weights ( < 12 or > 80 kg ) and bmis ( < 11 or > 32 ) .
all analyses were conducted on bmi - sds that had been residualised for age and sex effects using a regression procedure .
we used the gcta package to quantify the proportion of variance in bmi - sds explained by 1.7 million snps for the unrelated children with genotype and bmi - sds data .
all possible pairs from a sample of 2269 individuals yields nearly 2.6 million pairwise comparisons ( 2 573 046 ) .
no pairs exceeded the gcta standard cutoff coefficient of 0.025 for genetic relatedness , confirming that no two children in the analysed sample appeared to be genetically related in the traditional sense .
we performed standard ace model - fitting analyses using openmx to estimate the heritability of bmi - sds for the same sample of children by including anthropometric data from their co - twin to provide a direct comparison for the estimate derived from gcta .
the fit of the model was not of primary interest in this study ; however , to assure a good fit ' , we used the full ace cholesky decomposition model ( including additive genetic ( a ) , shared environmental ( c ) and unique environmental ( e ) components ) , which is the full model and thus fits the data best and also provides estimates of the a , c and e parameters .
data were from the twins early development study ( teds ) , a population - based cohort of monozygotic and dizygotic twins ( > 11 000 pairs ) born between 1994 and 1996 in england and wales .
twins and parents provided informed consent for each part of the study prior to data collection .
genome - wide genotyping was completed in 2010 for one randomly selected child in each of 3665 families ; of these , 3152 ( 1446 male and 1706 female subjects ) survived quality control criteria for ancestry , heterozygosity , relatedness and hybridisation intensity outliers ( for details see ) . genotyping and
quality control was done using the affymetrix 6.0 genechip snp genotyping array ( affymetrix inc , santa clara , ca , usa ) using standard experimental protocols as part of the wtccc2 project .
snps were selected on their minor allele frequency ( > 0.01 ) , genotype call - rate ( > 0.80 ) , hardy weinberg equilibrium ( > 10 ) and plate effect p - value ( > 10 ) , which resulted in 700 000 quality - controlled genotyped snps .
in addition , there were 2.5 million snps imputed from hapmap 2 and 3 , and wtccc controls , using the programme impute v.2 software .
imputed snps were screened using much more stringent quality control that resulted in reduction to 1 000 000 snps , giving a total of 1.7 million ( quality controled ) snps ( for details see ) . to control for ancestral stratification , we performed principal component analysis using eigenstrat from eigensoft package and identified significant axes using the tracy widom test .
this resulted in eight axes with p<0.05 that were used as covariates in gcta analyses .
height and weight data were obtained in 2005 when the children were 811 years old , as part of a study of the heritability of adiposity .
parents were sent detailed instructions and asked to record each child 's weight to the nearest pound or tenth of a kg , and height to the nearest cm , as well as the date of measurement .
parent- and researcher - measured heights and weights were correlated 0.90 and 0.83 in a subsample of 228 families .
scores ( bmi - sds ) that take into account the child 's age and sex , using 1990 uk growth reference data and computed with the programme imsgrowth .
reference values were used to exclude implausible heights ( < 1.05 or > 1.80 m ) , weights ( < 12 or > 80 kg ) and bmis ( < 11 or > 32 ) .
all analyses were conducted on bmi - sds that had been residualised for age and sex effects using a regression procedure .
we used the gcta package to quantify the proportion of variance in bmi - sds explained by 1.7 million snps for the unrelated children with genotype and bmi - sds data .
all possible pairs from a sample of 2269 individuals yields nearly 2.6 million pairwise comparisons ( 2 573 046 ) .
no pairs exceeded the gcta standard cutoff coefficient of 0.025 for genetic relatedness , confirming that no two children in the analysed sample appeared to be genetically related in the traditional sense .
we performed standard ace model - fitting analyses using openmx to estimate the heritability of bmi - sds for the same sample of children by including anthropometric data from their co - twin to provide a direct comparison for the estimate derived from gcta .
the fit of the model was not of primary interest in this study ; however , to assure a good fit ' , we used the full ace cholesky decomposition model ( including additive genetic ( a ) , shared environmental ( c ) and unique environmental ( e ) components ) , which is the full model and thus fits the data best and also provides estimates of the a , c and e parameters .
of the 3152 children with genotyping data , 2402 families ( 76% ) had provided anthropometric data and recorded age when the children were measured . data from four children
were excluded because they were reported as being < 8 years old at the time they were measured ; and 80 data points were excluded for implausible anthropometric results .
data from 22 children whose zygosity was unknown were excluded from the analyses because they could not be included in the twin analyses , along with a small number of children ( n=27 ) with severe medical problems .
the sample characteristics for the children included in the gcta analysis are shown in table 1 .
the average age was 9.9 years , 53% were girls and 39% were from monozygotic ( identical ) twin pairs .
their average bmi - sds placed them close to the 1990 reference value , with comparatively low rates of overweight ( 8.7% ) and obesity ( 3.6% ) .
the twin estimate of heritability of bmi - sds in the sample was 82% ( 95% confidence interval : 0.740.88 ; p<0.001 ) .
the gcta estimate of genetic influence due to the additive effect of common snps was 30% ( 95% confidence interval 0.020.59 ; p=0.02 ) .
snp heritability was therefore equivalent to 37% of the twin - estimated heritability ( 30%/82% ) .
figure 1 plots the variance explained in bmi - sds from the twin analyses and the gcta .
this is the first pediatric study to use gcta to estimate the genetic influence on adiposity attributable to additive genetic effects from common snps across the entire genome .
consistent with findings in adults the gcta method gave an order - of - magnitude increase compared with the gwas estimates ( 1.5% ) , explaining 30% of the variance in bmi - sds .
this equated to 37% of the estimate of heritability derived from the twin design ( 82% ) in the same families , and is comparable to many estimates derived from family studies .
given that the 32 snps of the largest effect account for only 5% ( 1.5/30% ) of the total common additive genetic snp variance , these results suggest that 95% of the variation due to common snps have been undetected through gwas .
this gcta estimate is likely to be at the lower end of the true additive genomic influence because it is limited to snps with a minor allele frequency of 5% rarer variants are therefore excluded . in addition
, causal variants that were not genotyped or not highly correlated with the snps on the genotyping array will also have been missed . the gcta value ( 30% )
was larger than has been reported in studies in adults ( 1016.5% ) , suggesting that the additive genetic effect from common snps on bmi may be slightly higher for children .
this is consistent with the higher estimates of heritability from pediatric than adult twin studies .
this may reflect the fact that adults are more likely than children to be making deliberate attempts at weight control , thus , limiting the observed genetic effect .
the larger value may also be explained by the narrow age range of the sample , which reduces the effect of gene by age interaction .
although the method used in the gcta analysis can not be used to predict obesity risk for any one individual because the genetic variants involved are not identified , the results underline the importance of additive genetic effects in the development of adiposity in childhood .
this supports the current convention of using parental weight status as a proxy for childhood obesity risk .
targeting children of obese parents for early - life obesity - prevention interventions , given that these children are most at risk , might be a useful direction to take .
bmi tends to be lower in twins than singletons and consistent with this , the average bmi of the sample placed them close to the 1990 reference value , and therefore below contemporary levels of adiposity .
height and weight data were parent - reported , therefore may be less reliable than researcher - measured anthropometrics , although they were found to be reliable in a subsample of families .
lastly , the effects of pubertal status were not examined in this study ; differences in pubertal status may have resulted in a slightly lower gcta estimate of additive genetic effects on bmi - sds .
the study 's strengths included the opportunity to estimate heritability using the twin design in the same sample for which we carried out the gcta analysis .
these results find that gcta analysis explains a substantial proportion of the genetic effect identified as
they provide compelling evidence that additive genetic influence from multiple common snps is a powerful determinant of adiposity in childhood . | known single - nucleotide polymorphisms ( snps ) explain < 2% of the variation in body mass index ( bmi ) despite the evidence of > 50% heritability from twin and family studies , a phenomenon termed missing heritability ' .
using dna alone for unrelated individuals , a novel method ( in a software package called genome - wide complex trait analysis , gcta ) estimates the total additive genetic influence due to common snps on whole - genome arrays .
gcta has made major inroads into explaining the missing heritability ' of bmi in adults .
this study provides the first gcta estimate of genetic influence on adiposity in children .
participants were from the twins early development study ( teds ) , a british twin birth cohort .
bmi s.d .
scores ( bmi - sds ) were obtained from validated parent - reported anthropometric measures when children were about 10 years old ( mean=9.9 ; s.d.=0.84 ) .
selecting one child per family ( n=2269 ) , gcta results from 1.7 million dna markers were used to quantify the additive genetic influence of common snps . for direct comparison , a standard twin analysis in the same families estimated the additive genetic influence as 82% ( 95% ci : 0.740.88 , p<0.001 ) .
gcta explained 30% of the variance in bmi - sds ( 95% ci : 0.020.59 ; p=0.02 ) .
these results indicate that 37% of the twin - estimated heritability ( 30/82% ) can be explained by additive effects of multiple common snps , and provide compelling evidence for strong genetic influence on adiposity in childhood . |
subjects were recruited from the cacti study cohort described previously ( 38 ) for a hyperinsulinemic - euglycemic clamp substudy .
inclusion criteria for initial enrollment of type 1 diabetic subjects in the cacti study were age 19 to 56 , no history of cvd , insulin therapy within a year of diagnosis and current insulin therapy , diagnosis before age 30 and/or positive antibodies , and diabetes duration 10 years .
the nondiabetic controls were of similar age and free of cvd . within this cohort ,
inclusion criteria for the clamp substudy presented here included : ghb 9.5% , albumin excretion rate < 200 g / min , triglycerides < 400 mg / dl , blood pressure < 160/100 , and a cac measurement at the 6-year cacti follow - up .
recruitment of the subgroup was performed by a mailing to the full cacti cohort with the 6-year follow - up reminder .
the initial recruitment goal was 96 subjects , 24 each of type 1 diabetic men and women , and nondiabetic men and women with frequency matching for age ( by 5-year intervals ) , and bmi ( normal , overweight , and obese ) .
interested and eligible members of the cohort were accepted into the substudy as long as groups remained open and frequency - matching criteria were satisfied .
clamps were performed 10 to 967 ( median 207 ) days after the 6-year follow - up visit .
all participants provided informed consent , and the study was approved by the colorado combined institutional review board .
dual x - ray absorptiometry scans were performed for body composition and fat - free mass ( ffm ) just before the clamp study .
abdominal computed tomography scans for calculation of visceral and subcutaneous fat areas and liver to spleen density ratios were performed at the 6-year cacti follow - up and within 1 year of the clamp study .
anthropometric measures included waist and hip circumference , height , weight , and sagittal diameter .
type 1 diabetic subjects ( n = 44 ) underwent masked continuous glucose monitoring ( cgm ) ( medtronic minimed gold system ) for 3 days before the hyperinsulinemic - euglycemic clamp . reported cgm measures include 1 ) the overall mean glucose ( meant ) , the average of all glucose values ; 2 ) hypoglycemia , percentage of glucose values < 70 mg / dl ( 3.9 mmol / l ) ; 3 ) hyperglycemia , percentage of glucose values > 180 mg / dl ( 10 mmol / l ) ; and 4 ) glycemic variability within days , overall sd ( sdt ) , the sd of all glucose values .
subjects were asked to refrain from vigorous physical activity and provided a standardized diet ( 50% carbohydrate , 20% protein , 30% fat ) for 3 days before their study day .
caloric needs were based on ffm measured by dexa scan and a standard activity factor .
premenopausal women were scheduled for their study day within days 2 to 10 of their menstrual cycle .
subjects were admitted to the inpatient clinical research unit before dinner the evening before their study .
type 1 diabetic subjects were instructed to take their last long - acting insulin injections at least 12 h before admission .
dinner was provided on the unit and subjects then fasted overnight and through the clamp .
those on an insulin pump had the pump removed after dinner , and all type 1 diabetic subjects were maintained overnight on intravenous insulin with adjustments to achieve euglycemia by morning .
blood samples for determination of baseline hormone and substrate ( insulin , glucose , c - peptide , nefa , glycerol , and lactate ) concentrations were drawn before initiation of the clamp protocol .
a three stage hyperinsulinemic- euglycemic clamp was initiated and continued for the next 4.5 h using the method of defronzo et al .
briefly , a primed continuous infusion of insulin was administered at 4 , 8 , and then 40 mu / m / min for 1.5 h each . a variable infusion of 20% dextrose
arterialized blood was sampled every 5 min for bedside determination of glucose concentration ( analox , lunenberg , ma ) and the dextrose infusion adjusted as necessary . arterialized blood samples were taken twice during the last 10 min of each stage of the clamp for hormone and substrate measurements as above .
a hyperinsulinemic- euglycemic steady state was achieved during the last 30 min of the high insulin infusion stage and mean glucose infusion rate ( [ gir ] , mg / kg ffm / min ) during this time was used as the measure of insulin sensitivity .
all participants underwent two electron beam computed tomography scans without contrast within 5 min at baseline and two scans at follow - up visits at years 3 and 6 using the standard acquisition protocol .
calcium volume scores ( cvs ) were calculated using the volumetric method , which is based on isotropic interpolation , as previously described ( 40,41 ) .
variables were examined for normality , and non - normally distributed variables were log transformed for analysis .
differences in clinical and clamp parameters and unadjusted gir between type 1 diabetic and non - dm subjects were examined using unpaired student t tests .
multiply adjusted least squares means of gir and nefa and glycerol levels were calculated using generalized linear models .
correlation of insulin resistance measures to cac was examined using spearman rank test and partial adjustment was made for age , diabetes status , and sex .
subjects were recruited from the cacti study cohort described previously ( 38 ) for a hyperinsulinemic - euglycemic clamp substudy .
inclusion criteria for initial enrollment of type 1 diabetic subjects in the cacti study were age 19 to 56 , no history of cvd , insulin therapy within a year of diagnosis and current insulin therapy , diagnosis before age 30 and/or positive antibodies , and diabetes duration 10 years .
the nondiabetic controls were of similar age and free of cvd . within this cohort ,
inclusion criteria for the clamp substudy presented here included : ghb 9.5% , albumin excretion rate < 200 g / min , triglycerides < 400 mg / dl , blood pressure < 160/100 , and a cac measurement at the 6-year cacti follow - up .
recruitment of the subgroup was performed by a mailing to the full cacti cohort with the 6-year follow - up reminder .
the initial recruitment goal was 96 subjects , 24 each of type 1 diabetic men and women , and nondiabetic men and women with frequency matching for age ( by 5-year intervals ) , and bmi ( normal , overweight , and obese ) .
interested and eligible members of the cohort were accepted into the substudy as long as groups remained open and frequency - matching criteria were satisfied .
clamps were performed 10 to 967 ( median 207 ) days after the 6-year follow - up visit .
all participants provided informed consent , and the study was approved by the colorado combined institutional review board .
dual x - ray absorptiometry scans were performed for body composition and fat - free mass ( ffm ) just before the clamp study .
abdominal computed tomography scans for calculation of visceral and subcutaneous fat areas and liver to spleen density ratios were performed at the 6-year cacti follow - up and within 1 year of the clamp study .
anthropometric measures included waist and hip circumference , height , weight , and sagittal diameter .
type 1 diabetic subjects ( n = 44 ) underwent masked continuous glucose monitoring ( cgm ) ( medtronic minimed gold system ) for 3 days before the hyperinsulinemic - euglycemic clamp . reported cgm measures include 1 ) the overall mean glucose ( meant ) , the average of all glucose values ; 2 ) hypoglycemia , percentage of glucose values < 70 mg / dl ( 3.9 mmol / l ) ; 3 ) hyperglycemia , percentage of glucose values > 180 mg / dl ( 10 mmol / l ) ; and 4 ) glycemic variability within days , overall sd ( sdt ) , the sd of all glucose values .
subjects were asked to refrain from vigorous physical activity and provided a standardized diet ( 50% carbohydrate , 20% protein , 30% fat ) for 3 days before their study day .
caloric needs were based on ffm measured by dexa scan and a standard activity factor .
premenopausal women were scheduled for their study day within days 2 to 10 of their menstrual cycle .
subjects were admitted to the inpatient clinical research unit before dinner the evening before their study .
type 1 diabetic subjects were instructed to take their last long - acting insulin injections at least 12 h before admission .
dinner was provided on the unit and subjects then fasted overnight and through the clamp .
those on an insulin pump had the pump removed after dinner , and all type 1 diabetic subjects were maintained overnight on intravenous insulin with adjustments to achieve euglycemia by morning .
blood samples for determination of baseline hormone and substrate ( insulin , glucose , c - peptide , nefa , glycerol , and lactate ) concentrations were drawn before initiation of the clamp protocol .
a three stage hyperinsulinemic- euglycemic clamp was initiated and continued for the next 4.5 h using the method of defronzo et al .
briefly , a primed continuous infusion of insulin was administered at 4 , 8 , and then 40 mu / m / min for 1.5 h each . a variable infusion of 20% dextrose was infused to maintain blood glucose 90 mg / dl . arterialized blood was sampled every 5 min for bedside determination of glucose concentration ( analox , lunenberg , ma ) and the dextrose infusion adjusted as necessary .
arterialized blood samples were taken twice during the last 10 min of each stage of the clamp for hormone and substrate measurements as above .
a hyperinsulinemic- euglycemic steady state was achieved during the last 30 min of the high insulin infusion stage and mean glucose infusion rate ( [ gir ] , mg / kg ffm / min ) during this time was used as the measure of insulin sensitivity .
all participants underwent two electron beam computed tomography scans without contrast within 5 min at baseline and two scans at follow - up visits at years 3 and 6 using the standard acquisition protocol .
calcium volume scores ( cvs ) were calculated using the volumetric method , which is based on isotropic interpolation , as previously described ( 40,41 ) .
variables were examined for normality , and non - normally distributed variables were log transformed for analysis .
differences in clinical and clamp parameters and unadjusted gir between type 1 diabetic and non - dm subjects were examined using unpaired student t tests .
multiply adjusted least squares means of gir and nefa and glycerol levels were calculated using generalized linear models .
correlation of insulin resistance measures to cac was examined using spearman rank test and partial adjustment was made for age , diabetes status , and sex .
we present data from 87 subjects ( 40 with type 1 diabetes and 47 age- and sex - matched nondiabetic controls ) recruited for the clamp substudy between 2005 and 2008 .
four diabetic subject clamps were excluded from the final analysis due to errors in clamp and/or overnight insulin preparation detected by inappropriate insulin responses in the subjects and postclamp measurement of infusate insulin concentration .
subjects in the clamp study were representative of the full cacti cohort seen at the 6-year follow - up with no differences found for parameters known to impact insulin sensitivity , including age ( years : 44.8 7.9 vs. 45.0 9.1 , p = 0.87 ) , bmi ( kg / m : 26.2 4.2 vs. 26.8 4.9 , p = 0.19 ) , visceral fat ( log visceral fat area : 10.67 0.52 vs. 10.74 0.59 , p = 0.32 ) , and habitual daily physical activity ( log kcal : 7.28 1.00 vs. 7.15 1.45 , p = 0.24 ) . similarity between the clamp cohort and the full 6-year visit cohort was also found within sex and diabetes strata ( not shown ) .
in addition , type 1 diabetic subjects in the clamp study were similar to the full cacti cohort in diabetes duration ( 28.6 8.0 vs. 29.4 8.8 years , p = 0.57 ) . clinical characteristics of the study population by diabetes status are shown in table 1 .
in addition , the two groups were similar for other measures of body composition , including total percentage of body fat , waist circumference , waist to hip ratio , and habitual physical activity .
as expected , type 1 diabetic and nondiabetic groups differed significantly in diabetes - related parameters of ghb and fasting glucose .
total and ldl cholesterol and triglycerides also differed significantly by diabetes status , with the nondiabetic subjects exhibiting the higher cardiovascular risk phenotype of higher ldl and triglycerides .
statin use was significantly more frequent in type 1 diabetic subjects , but the difference in lipid profile remained after adjustment for statin use ( not shown ) .
type 1 diabetic subjects were more likely to be taking antihypertensive medication , but there was no difference in blood pressure between type 1 diabetic and nondiabetic subjects who were not taking medications ( not shown ) .
adiponectin was significantly higher in type 1 diabetic than nondiabetic subjects as has been reported previously ( 4245 ) .
baseline characteristics for clamp study cohort values are unadjusted mean sd except where otherwise indicated .
type 1 diabetic subjects in the clamp cohort exhibited stable glycemic control over the full duration of the study from baseline through the clamp visit ( mean ghb = 7.71 1.0 , 7.57 1.1 , 7.9 1.0 , and 7.51 0.87 at baseline , 3-year , 6-year , and clamp visits , respectively ) .
three - stage hyperinsulinemic - euglycemic clamps were performed in 87 subjects ( 46% with type 1 diabetes , 55% women ) .
final clamp glucose and insulin levels were not different between the groups ( table 2 ) .
whole - body insulin sensitivity represented by gir during the last 30 min of the clamp was significantly lower in type 1 diabetic than nondiabetic subjects ( 5.8 3.5 vs. 13.2 5.7 mg / kg ffm / min , p < 0.0001 ) .
this difference was only modestly attenuated after multivariate adjustment for age , fasting glucose , final clamp glucose and insulin , and bmi or visceral fat area ( table 2 ) .
insulin sensitivity by glucose infusion rate from hyperinsulinemic - euglycemic clamp insulin resistance in type 1 diabetes correlated with the expected parameters known to predict insulin resistance in nondiabetic and type 2 diabetic subjects , but relationships were left - shifted in type 1 diabetes .
for instance , triglyceride levels and bmi correlated with insulin resistance similarly in both groups ( fig .
, there was no interaction by diabetes for the relationship of gir to triglycerides or bmi ( p = 0.966 and 0.734 , respectively ) , but the y intercepts were significantly different for triglycerides and trended toward significance for bmi ( p = 0.002 and 0.156 , respectively ) .
similar relationships were found for waist circumference , visceral fat area , and total body fat ( not shown ) .
correlation of insulin sensitivity to triglycerides and bmi is retained , but left - shifted , in type 1 diabetes .
for the relationship of gir to triglycerides ( top panel ) the regression equation in type 1 diabetes is gir = 8.478 0.038 ( triglycerides ) , p = 0.022 ; for nondiabetic subjects gir = 17.140.037 ( triglycerides ) , p = 0.011 . in combined analysis ,
p = 0.966 for an interaction by diabetes and p = 0.002 for the difference in y intercept . for bmi ( bottom panel ) in type 1 diabetes , gir = 14.7320.33 ( bmi ) , p = 0.008 ; for nondiabetic subjects , gir = 23.870.41(bmi ) , p = 0.05 .
in combined analysis , p = 0.734 for an interaction by diabetes , p = 0.156 for the difference in the y intercept . insulin - mediated serum nefa suppression was also impaired in type 1 diabetic subjects ( fig .
the increase in serum nefa levels during the first clamp stage in diabetic subjects reflected an initial insulin infusion rate ( 4mu / m / min ) that was lower than their basal requirement .
the second stage insulin infusion rate ( 8mu / m / min ) was sufficient to lower glucose and nefa levels in all type 1 diabetic subjects . despite this
, nefa levels remained significantly higher in type 1 diabetic than nondiabetic subjects at the end of the second clamp stage ( least squares mean se : 370 27 vs. 185 25 mol / l , p < 0.0001 ) after adjustment for age , sex , bmi , fasting glucose , and time point insulin . the highest insulin infusion rate ( 40mu / m / min ) , was sufficient to similarly suppress nefa levels in type 1 and nondiabetic subjects .
nefa levels at all clamp stages were strongly inversely correlated with peripheral glucose uptake ( r = 0.56 , p < 0.0001 , r = 0.63 , p < 0.0001 , r = 0.40 , p = 0.0002 for stage 1 , 2 , and 3 nefa levels , respectively ) .
data are least squares mean se ( adjusted for age , sex , bmi , fasting glucose , and time point insulin ) .
, type 1 diabetic subjects ; , nondiabetic controls ; * p < 0.0001 , p < 0.05 .
insulin - mediated suppression of glycerol followed a similar pattern to nefa levels ( fig .
1 , bottom panel ) . the defect in glycerol suppression in type 1 diabetic subjects remained significant in the unadjusted data during the third clamp stage , though this was attenuated at the final time point by adjustment for age , sex , bmi , fasting insulin , and time point insulin level .
a relationship between insulin resistance and cac is suggested by a plot of the raw data of peripheral glucose utilization ( gir ) versus cac volume at the 6-year visit ( fig .
3 ) . in fact , after adjustment for age , gir correlated inversely with cac volume at the 6-year cacti follow - up visit and with an increase in cac volume from baseline to the 6-year visit and from 3-year to 6-year follow - up visits ( , p = 0.42 , < 0.0001 ; 0.41 ,
< 0.0001 ; 0.24 , < 0.028 , respectively ) ( table 3 ) . in a logistic regression analysis adjusted for age , sex , bmi , and diabetes status , lower gir
was independently associated with the presence of cac : odds ratio ( or ) 0.45 , 95% ci ( 0.220.93 ) for a one standard deviation ( sd ) increase in gir .
thus , for every 6.1 mg / kg ffm / min increase in gir ( signifying greater insulin sensitivity ) , the odds of having cac decreased by 55% .
plot of raw data for cac volume at 6-year follow - up visit as a function of gir ( n = 87 ) .
open ( white ) squares = nondiabetic subjects , black triangles = type 1 diabetic subjects .
correlation of insulin resistance to cac volume ; or of any cac at 6-year visit associated with insulin resistance by gir and by failure of nefa suppression in clamp stage 2 the nefa levels during stage 2 of the clamp were similarly but positively correlated with cac volume at the 6-year visit , and cac increase from baseline to 6-year and from 3-year to 6-year follow - up visit ( table 3 ) .
partial adjustment for sex and diabetes status attenuated all correlations somewhat ( table 3 ) , but there was no significant interaction with diabetes status . in a logistic regression analysis adjusted for age , sex , bmi , and diabetes status , higher stage 2 nefa levels ( insulin resistance ) , were independently associated with the presence of cac , or 2.40 , 95% ci ( 1.085.32 ) for one sd difference in nefa level .
thus , for every 186 mol / l increase in stage 2 nefa ( signifying insulin resistance ) , the odds of having cac increased by 140% . these one sd differences in measures of insulin action are comparable to the differences seen between type 1 diabetic and nondiabetic subjects in this study . in other logistic regression models ldl and hdl levels did not have a significant effect on the odds ratio for the presence of cac in the whole cohort , nor did insulin dose in logistic regression analysis of the diabetic group ( not shown ) .
however , statistical significance was lost for the 3-year to 6-year visit change and otherwise attenuated to near statistical significance in the diabetic group only ( table 3 , models 3 and 4 ) .
similarly , logistic regression analysis of the odds ratio for the presence of cac at the 6-year follow - up visit associated with a change in gir was analyzed separately for diabetic and nondiabetic subjects .
odds ratios for the two groups were very similar , but statistical significance was lost for the diabetic group ( e.g. , for gir : or = 0.38 [ 95% ci : 0.111.37 ] and 0.40 [ 95% ci : 0.180.90 ] , respectively ) .
neither peripheral glucose uptake nor insulin - mediated nefa suppression correlated with measures of current or recent glycemic control in this study ( table 4 ) .
pearson coefficients revealed no significant correlation between gir or stage 2 nefa levels and ghb or cgm measures of mean glucose , percentage of values > 180 mg / dl , percentage of values < 70 mg / dl , and overall sd ( glycemic variability ) .
pearson correlation coefficients are shown for correlation of gir and stage 2 nefa levels to ghb and cgm measures of glycemic control for all type 1 diabetic subjects in addition , gir and stage 2 nefa did not differ by ghb quartile ( anova , p = 0.89 ; fig . 4 and data not shown ) over the range of ghb values represented by this cohort ( < 9.5% ) .
bmi also did not differ by ghb quartiles ( 26.4 , 28.1 , 27.4 , and 26.0 kg / m , anova , p = 0.27 ) , excluding the possibility that glycemia - related changes in insulin sensitivity were countered by glycemia - related changes in bmi .
insulin sensitivity is expressed as glucose infusion rate per fat - free mass ( gir , mg / kg ffm / min ) and shown by quartile of ghb measured 3 days before the clamp study day .
pairwise comparisons are all nonsignificant with p values ranging from 0.49 ( 2nd and 4th quartiles ) to 0.96 ( 1st and 4th quartiles ) .
three - stage hyperinsulinemic - euglycemic clamps were performed in 87 subjects ( 46% with type 1 diabetes , 55% women ) .
final clamp glucose and insulin levels were not different between the groups ( table 2 ) .
whole - body insulin sensitivity represented by gir during the last 30 min of the clamp was significantly lower in type 1 diabetic than nondiabetic subjects ( 5.8 3.5 vs. 13.2 5.7 mg / kg ffm / min , p < 0.0001 ) .
this difference was only modestly attenuated after multivariate adjustment for age , fasting glucose , final clamp glucose and insulin , and bmi or visceral fat area ( table 2 ) .
insulin resistance in type 1 diabetes correlated with the expected parameters known to predict insulin resistance in nondiabetic and type 2 diabetic subjects , but relationships were left - shifted in type 1 diabetes . for instance , triglyceride levels and bmi correlated with insulin resistance similarly in both groups ( fig .
, there was no interaction by diabetes for the relationship of gir to triglycerides or bmi ( p = 0.966 and 0.734 , respectively ) , but the y intercepts were significantly different for triglycerides and trended toward significance for bmi ( p = 0.002 and 0.156 , respectively ) .
similar relationships were found for waist circumference , visceral fat area , and total body fat ( not shown ) .
correlation of insulin sensitivity to triglycerides and bmi is retained , but left - shifted , in type 1 diabetes .
for the relationship of gir to triglycerides ( top panel ) the regression equation in type 1 diabetes is gir = 8.478 0.038 ( triglycerides ) , p = 0.022 ; for nondiabetic subjects gir = 17.140.037 ( triglycerides ) , p = 0.011 . in combined analysis ,
p = 0.966 for an interaction by diabetes and p = 0.002 for the difference in y intercept . for bmi ( bottom panel ) in type 1 diabetes , gir = 14.7320.33 ( bmi ) , p = 0.008 ; for nondiabetic subjects , gir = 23.870.41(bmi ) , p = 0.05 . in combined analysis ,
p = 0.734 for an interaction by diabetes , p = 0.156 for the difference in the y intercept .
insulin - mediated serum nefa suppression was also impaired in type 1 diabetic subjects ( fig .
the increase in serum nefa levels during the first clamp stage in diabetic subjects reflected an initial insulin infusion rate ( 4mu / m / min ) that was lower than their basal requirement .
the second stage insulin infusion rate ( 8mu / m / min ) was sufficient to lower glucose and nefa levels in all type 1 diabetic subjects . despite this
, nefa levels remained significantly higher in type 1 diabetic than nondiabetic subjects at the end of the second clamp stage ( least squares mean se : 370 27 vs. 185 25 mol / l , p < 0.0001 ) after adjustment for age , sex , bmi , fasting glucose , and time point insulin .
the highest insulin infusion rate ( 40mu / m / min ) , was sufficient to similarly suppress nefa levels in type 1 and nondiabetic subjects .
nefa levels at all clamp stages were strongly inversely correlated with peripheral glucose uptake ( r = 0.56 , p < 0.0001 , r = 0.63 , p < 0.0001 , r = 0.40 , p = 0.0002 for stage 1 , 2 , and 3 nefa levels , respectively ) .
data are least squares mean se ( adjusted for age , sex , bmi , fasting glucose , and time point insulin ) . ,
type 1 diabetic subjects ; , nondiabetic controls ; * p < 0.0001 , p < 0.05 .
insulin - mediated suppression of glycerol followed a similar pattern to nefa levels ( fig .
1 , bottom panel ) . the defect in glycerol suppression in type 1 diabetic subjects remained significant in the unadjusted data during the third clamp stage , though this was attenuated at the final time point by adjustment for age , sex , bmi , fasting insulin , and time point insulin level .
a relationship between insulin resistance and cac is suggested by a plot of the raw data of peripheral glucose utilization ( gir ) versus cac volume at the 6-year visit ( fig .
3 ) . in fact , after adjustment for age , gir correlated inversely with cac volume at the 6-year cacti follow - up visit and with an increase in cac volume from baseline to the 6-year visit and from 3-year to 6-year follow - up visits ( , p = 0.42 , < 0.0001 ; 0.41 , < 0.0001 ; 0.24 , < 0.028 , respectively ) ( table 3 ) .
in a logistic regression analysis adjusted for age , sex , bmi , and diabetes status , lower gir was independently associated with the presence of cac : odds ratio ( or ) 0.45 , 95% ci ( 0.220.93 ) for a one standard deviation ( sd ) increase in gir .
thus , for every 6.1 mg / kg ffm / min increase in gir ( signifying greater insulin sensitivity ) , the odds of having cac decreased by 55% .
plot of raw data for cac volume at 6-year follow - up visit as a function of gir ( n = 87 ) .
open ( white ) squares = nondiabetic subjects , black triangles = type 1 diabetic subjects .
correlation of insulin resistance to cac volume ; or of any cac at 6-year visit associated with insulin resistance by gir and by failure of nefa suppression in clamp stage 2 the nefa levels during stage 2 of the clamp were similarly but positively correlated with cac volume at the 6-year visit , and cac increase from baseline to 6-year and from 3-year to 6-year follow - up visit ( table 3 ) .
partial adjustment for sex and diabetes status attenuated all correlations somewhat ( table 3 ) , but there was no significant interaction with diabetes status . in a logistic regression analysis adjusted for age , sex , bmi , and diabetes status , higher stage 2 nefa levels ( insulin resistance ) ,
were independently associated with the presence of cac , or 2.40 , 95% ci ( 1.085.32 ) for one sd difference in nefa level .
thus , for every 186 mol / l increase in stage 2 nefa ( signifying insulin resistance ) , the odds of having cac increased by 140% . these one sd differences in measures of insulin action are comparable to the differences seen between type 1 diabetic and nondiabetic subjects in this study . in other logistic regression models ldl and hdl levels did not have a significant effect on the odds ratio for the presence of cac in the whole cohort , nor did insulin dose in logistic regression analysis of the diabetic group ( not shown ) .
however , statistical significance was lost for the 3-year to 6-year visit change and otherwise attenuated to near statistical significance in the diabetic group only ( table 3 , models 3 and 4 ) .
similarly , logistic regression analysis of the odds ratio for the presence of cac at the 6-year follow - up visit associated with a change in gir was analyzed separately for diabetic and nondiabetic subjects .
odds ratios for the two groups were very similar , but statistical significance was lost for the diabetic group ( e.g. , for gir : or = 0.38 [ 95% ci : 0.111.37 ] and 0.40 [ 95% ci : 0.180.90 ] , respectively ) .
neither peripheral glucose uptake nor insulin - mediated nefa suppression correlated with measures of current or recent glycemic control in this study ( table 4 ) .
pearson coefficients revealed no significant correlation between gir or stage 2 nefa levels and ghb or cgm measures of mean glucose , percentage of values > 180 mg / dl , percentage of values < 70 mg / dl , and overall sd ( glycemic variability ) .
pearson correlation coefficients are shown for correlation of gir and stage 2 nefa levels to ghb and cgm measures of glycemic control for all type 1 diabetic subjects in addition , gir and stage 2 nefa did not differ by ghb quartile ( anova , p = 0.89 ; fig . 4 and data not shown ) over the range of ghb values represented by this cohort ( < 9.5% ) .
bmi also did not differ by ghb quartiles ( 26.4 , 28.1 , 27.4 , and 26.0 kg / m , anova , p = 0.27 ) , excluding the possibility that glycemia - related changes in insulin sensitivity were countered by glycemia - related changes in bmi .
insulin sensitivity is expressed as glucose infusion rate per fat - free mass ( gir , mg / kg ffm / min ) and shown by quartile of ghb measured 3 days before the clamp study day .
pairwise comparisons are all nonsignificant with p values ranging from 0.49 ( 2nd and 4th quartiles ) to 0.96 ( 1st and 4th quartiles ) .
our study demonstrates significant insulin resistance in subjects with type 1 diabetes relative to nondiabetic controls of similar age , bmi , and habitual physical activity levels .
importantly , type 1 diabetic patients were profoundly insulin resistant despite similar overall adiposity , body fat composition , and hdl cholesterol levels , and , paradoxically , lower fasting triglycerides and higher adiponectin levels .
these findings , consistent with previous smaller studies ( 28,29,31 ) , suggest a novel component in the etiology of insulin resistance in type 1 diabetes .
our type 1 diabetic cohort exhibits the expected relationship between insulin resistance and bmi , waist circumference , and triglycerides .
however , at any given value , the type 1 diabetic subjects are likely to be more insulin resistant than nondiabetic subjects , thus effectively left - shifting the relationship between insulin sensitivity and insulin resistance - associated clinical parameters in type 1 diabetes . beyond hyperglycemia and the chronic exogenous peripheral hyperinsulinemia of type 1 diabetes , plausible explanations for this
we have found that in these relatively well controlled type 1 diabetic patients with mean ghb 7.5% 0.9% , the degree of insulin resistance was not explained by recent glycemic control defined by ghb and cgm measures .
since ghb was measured at four points during the study and remained consistent , it also does not seem likely that periods of loss of control are responsible for the insulin resistance measured in this study .
these data do challenge the general belief that poor glycemic control is the entire etiology of insulin resistance in type 1 diabetes .
however , our data do not explicitly contradict existing data . in the pittsburgh epidemiology of diabetes
study , where ghb was found to be a significant predictor of insulin resistance , ghb < 9.510 defined the low risk group , and a ghb difference representing the full range of our diabetic subjects would result in only a 1 mg / kg / min change in gir ( 27 ) .
our entire diabetic clamp cohort was controlled to the level of the improved ghb in these previous reports .
finally , in the important 1986 study of yki - jarvinen , ( 46,47 ) the authors found improved insulin sensitivity with initiation of treatment and improved glycemic control in newly diagnosed type 1 diabetes .
however , the scatter plots in this report indicate that improved mean gir was largely driven by subjects who no longer needed insulin at the 3-month and 1-year follow - ups . including only the insulin - dependent subjects at these visits appears to yield a remarkably consistent 40% reduction in gir across ghb values from 8.8 to 13% and durations of diabetes from 2 weeks to 20 years
. these results are more consistent with some aspect of nonphysiologic insulin delivery rather than glycemic control as the etiology of insulin resistance .
our data do not , however , rule out the possibility that very poor glycemic control may contribute to insulin resistance .
we also make the novel observation that insulin resistance in type 1 diabetic subjects also included impaired insulin - mediated suppression of serum nefa and glycerol levels .
the parallel lack of suppression of nefa and glycerol levels suggests that lipolysis was the source of most of the nefa measured .
however , the patterns were not identical , suggesting that regulation of re - esterification may also be altered between groups .
insulin resistance has been strongly implicated as a cardiovascular risk factor in the general population ( 1725 ) .
our demonstration of insulin resistance in type1 diabetic subjects in terms of both glucose and nefa metabolism suggests that insulin resistance may also contribute to increased cvd risk in type 1 diabetes .
the pittsburgh epidemiology of diabetes study demonstrated a correlation between estimated insulin resistance and coronary artery disease ( 10 ) .
in support of this finding , we report significant associations between directly measured insulin resistance and both presence and progression of cac in our full cohort , as well as in our nondiabetic subgroup .
this is a novel finding , representing the only existing demonstration of a correlation between a direct measure of insulin sensitivity by modern validated methodology and an accepted measure of cvd .
the finding of statistical significance with this small study group is surprising and supports the hypothesis that insulin resistance is an important factor in cac development .
though we failed to achieve statistical significance to the level of p < 0.05 in the diabetic group , the near significance ( p = 0.060.09 ) for the correlation of gir and nefa suppression to cac volume at 6 years and to cac progression from 0 to 6 years suggests that the relationship between insulin resistance and cac is also present in type 1 diabetes .
furthermore , the striking similarity in correlation coefficients between the diabetic and nondiabetic populations in the separate analyses suggests that the slope of the relationship between insulin resistance and cac is similar in subjects with and without type 1 diabetes .
the failure to achieve statistical significance in the diabetic group is a limitation of this study . because of
the near significance and the similarity in correlation coefficients and odds ratios to the statistically significant relationship found in the nondiabetic group , we believe this is most likely a power issue related to the smaller range in insulin resistance values and the smaller subject number .
however , we can not rule out the possibility that the association between insulin resistance and cac is weaker in individuals with type 1 diabetes than those without diabetes .
because of the left - shift in the relationship between gir and metabolic syndrome criteria described above , type 1 diabetic subjects in our study had a phenotype less consistent with
for instance , type 1 diabetic subjects had a healthier lipid profile than nondiabetic controls , even after adjustment for statin use . similarly , hypertension treatment was more common among type 1 diabetic subjects , but overall blood pressure was not higher , even after adjustment for medication use .
thus , standard characteristics that predict insulin resistance in the general population were not present in our insulin - resistant type 1 diabetic patients .
this is consistent with a previous report that standard prediction models of cvd risk ( highly dependent on hypertension and lipids ) do not accurately predict cvd risk in the type 1 diabetic population ( 16 ) .
we also find that about half the cvd risk in our type 1 diabetic cohort is not predicted by standard cvd risk prediction models ( j.k.s .- b .
we have reported differences in lipoprotein cholesterol subfraction distribution in this clamp group that may explain some of this excess risk ( 48 ) .
unfortunately , this suggests that traditional approaches to control these risk factors may be inadequate to slow premature formation of coronary atherosclerosis in type 1 diabetes .
the main limitations of this study are the single time point measurement of insulin action , the late study time of this measurement , and the lack of power to reach significance in the type 1 diabetic cohort .
we assumed that the single point measure of insulin resistance reflected a parameter that has been fairly constant over the time of the cacti follow - up . supporting this assumption ,
cacti follow - up has demonstrated essentially stable weight , glycemic control , and lifestyle over the 6-year study span , but a prospective design with repeat clamp studies would be optimal .
we are developing an insulin sensitivity prediction model based on this study , and will apply this model to baseline and follow - up data to allow a prospective analysis of the correlation of estimated insulin resistance with cac and to increase the power of the analysis in the diabetic cohort . in summary
, we found that profound insulin resistance in type 1 diabetes extends beyond glucose control to regulation of fat metabolism , may be associated with increased coronary atherosclerosis , and can not be easily identified using standard clinical predictors , including poor glycemic control .
these findings suggest that insulin resistance , possibly through effects on overall nefa exposure and lipotoxicity , may play a role in the residual risk of cvd in type 1 diabetes , as well as in the absence of diabetes , and thus may represent an important therapeutic target that is not currently considered in type 1 diabetes .
in summary , we found that profound insulin resistance in type 1 diabetes extends beyond glucose control to regulation of fat metabolism , may be associated with increased coronary atherosclerosis , and can not be easily identified using standard clinical predictors , including poor glycemic control .
these findings suggest that insulin resistance , possibly through effects on overall nefa exposure and lipotoxicity , may play a role in the residual risk of cvd in type 1 diabetes , as well as in the absence of diabetes , and thus may represent an important therapeutic target that is not currently considered in type 1 diabetes . | objectiveto assess insulin action on peripheral glucose utilization and nonesterified fatty acid ( nefa ) suppression as a predictor of coronary artery calcification ( cac ) in patients with type 1 diabetes and nondiabetic controls.research design and methodsinsulin action was measured by a three - stage hyperinsulinemic - euglycemic clamp ( 4 , 8 , and 40 mu / m2/min ) in 87 subjects from the coronary artery calcification in type 1 diabetes cohort ( 40 diabetic , 47 nondiabetic ; mean age 45 8 years ; 55% female).resultsperipheral glucose utilization was lower in subjects with type 1 diabetes compared with nondiabetic controls : glucose infusion rate ( mg / kg ffm / min ) = 6.19 0.72 vs. 12.71 0.66 , mean se , p < 0.0001 , after adjustment for age , sex , bmi , fasting glucose , and final clamp glucose and insulin .
insulin - induced nefa suppression was also lower in type 1 diabetic compared with nondiabetic subjects : nefa levels ( m ) during 8 mu / m2/min insulin infusion = 370 27 vs. 185 25 , p < 0.0001 , after adjustment for age , sex , bmi , fasting glucose , and time point insulin .
lower glucose utilization and higher nefa levels , correlated with cac volume ( r = 0.42 , p < 0.0001 and r = 0.41 , p < 0.0001 , respectively ) and predicted the presence of cac ( odds ratio [ or ] = 0.45 , 95% ci = 0.220.93 ,
p = 0.03 ; or = 2.4 , 95% ci = 1.085.32 , p = 0.032 , respectively ) .
insulin resistance did not correlate with ghb or continuous glucose monitoring parameters.conclusionstype 1 diabetic patients are insulin resistant compared with nondiabetic subjects , and the degree of resistance is not related to current glycemic control .
insulin resistance predicts the extent of coronary artery calcification and may contribute to the increased risk of cardiovascular disease in patients with type 1 diabetes as well as subjects without diabetes . |
the pathophysiology of thrombogenesis is not fully understood yet and the risk of venous thrombotic complications varies according to age , histologic type of renal disease , and level of serum albumin .
deep venous thrombosis , renal vein thrombosis , and pulmonary embolism are common in nephrotic syndrome , whereas portal vein and superior mesenteric vein ( smv ) thrombosis in nephrotic syndrome are rare .
there were several cases of portal vein thrombosis reported as a complication of nephrotic syndrome treated with anticoagulation therapy alone . here
, we describe a case of portal , splenic , and smv thrombosis which occurred in a patient with a relapsed steroid dependent minimal change disease ( mcd ) and who was treated with anticoagulation and thrombolytic therapy .
a 31-year - old man presented to our hospital because of severe diffuse abdominal pain and vomiting for the past 2 days .
he was diagnosed with mcd , which was confirmed by renal biopsy 4 years ago .
he had been treated with corticosteroid and cyclosporine 4 years ago and then stopped the corticosteroid treatment when complete remission was achieved .
however , relapse of nephrotic syndrome occurred soon , and his medication was changed to tacrolimus and corticosteroid . after achieving complete remission again , he had been maintained with tacrolimus .
he was an ex - smoker ( 5 pack - year ) and he quit smoking 4 years ago . on physical examination ,
his body temperature was 36.8c , blood pressure 140/70 mmhg , respiratory rate 26 breaths / min , and heart rate 102 beats / min .
there was diffuse and rebound tenderness on the abdomen and facial edema and moderate pitting edema on lower limbs .
total protein was 4.4 g / dl and serum albumin was 1.6 g / dl .
his total cholesterol was 489 mg / dl , and triglyceride was 270 mg / dl .
the urinalysis showed an isolated proteinuria and urine protein creatinine ratio of 4,353 mg / g .
other laboratory results were as follows : hemoglobin of 19.0 g / dl , hematocrit of 54.0% , white blood cells of 10,280/mm and platelets of 157,000/mm .
the blood urea nitrogen was 19.3 mg / dl and creatinine was 0.96 mg / dl .
enhanced abdomen computed tomography ( ct ) scan was performed and showed diffuse portal , splenic , and smv thrombosis accompanied with bowel edema probably due to bowel ischemia ( fig .
we evaluated the coagulation status with prothrombin time , activated partial thromboplastin time , d - dimer , fibrinogen , protein c , protein s , antithrombin iii , antiphospholipid antibody , and the factor v leiden mutation .
his prothrombin time was 11.6 seconds , inr 1.03 ( international normalized ratio , normal range : 0.851.30 ) , the activated partial thromboplastin time was 36.4 seconds and the antithrombin iii was 48.6% ( normal range : 80120% ) .
protein c showed 163.2% ( normal range : 72150% ) and protein s 79.0% ( normal range : 72150% ) .
urokinase was injected via selective catheterization of the superior mesenteric artery ( sma ) as thrombolytic therapy at the same time .
an angiography showed no visualization of the portal vein , smv , and splenic vein .
the catheter was placed on the tip in the sma and a continuous thrombolytic therapy was started with urokinase 50,000 iu / h . during the prolonged infusion of urokinase , the follow - up smv venography via the infusion catheter was performed every 24 hours for 5 days .
there was no visualization of the main portal vein and smv , but collateral vessel formation was observed at the follow - up smv venography and symptoms were gradually improved .
thrombolytic therapy was stopped and anticoagulation therapy with heparin was switched to warfarin . while anticoagulation with thrombolytic therapy was on - going , we did not control the underlying nephrotic syndrome because of infection risk and other complications related to ischemic colitis .
we started mycophenolate mofetil ( 1,000 mg / d ) and 1 mg / kg of prednisolone to control nephrotic syndrome when the patient could eat food and was symptom free with regards to his abdomen .
six months later , complete remission ( reduction of proteinuria to < 300 mg / g ) of the nephrotic syndrome was maintained and the follow - up ct scan showed disappearance of all portal vein , smv , and splenic vein thrombi ( fig .
it has been known why thromboembolic complications increase in patients with nephrotic syndrome ; the alterations in coagulation factors , fibrinolysis , and platelet function lead to a hypercoagulability in nephrotic syndrome .
in addition , low levels of plasma albumin , dyslipidemia , and the therapeutic use of diuretics and corticosteroids are thought to contribute to the formation of thrombi , .
smv thrombosis is an uncommon and potentially lethal disease because its presenting symptoms overlap with those of many other diseases , leading to significant delays in diagnosis and therapy .
anticoagulation therapy alone could be sufficient in a mild form of portal and smv thrombosis .
the main treatments of portal and smv thrombosis are anticoagulation and thrombolytic therapy and these should be considered and started as soon as possible after diagnosis .
thrombolytic therapy is a safe and effective method for patients with symptomatic acute portal vein and smv thrombosis , and urokinase is currently the most established thrombolytic agent for venous thrombosis , .
thrombolytic therapy can be administered directly via percutaneous - transhepatic or transjugular - transhepatic routes or indirectly via sma infusion of thrombolytic agents .
direct thrombolytic therapy is more efficient and decreases the dose of the thrombolytic agent , lowering the risk of related complications .
however , an indirect method is technically easy compared to a direct method and there are potential benefits in infusing thrombolytic agents into small mesenteric venous branches .
advanced age , membranous nephropathy , severe proteinuria , hypovolemia , hypoalbuminemia , and dyslipidemia are all now recognized to be correlated with an increased risk of thromboembolism development .
therefore , primary prophylactic anticoagulation needs to be carefully considered for such high - risk patients and an acute mesenteric venous thrombosis should be suspected when severe abdominal pain , vomiting , and ascites develop in patients with nephrotic syndrome . in this case , although the patient had recurrent episodes of relapse , he was young and had states of normoalbuminemia , euvolemia , and mild proteinuria as an outpatient .
an immediate surgical intervention is needed if the patient is suspected of having intestinal infarction and necrosis .
the indirect thrombolytic therapy via sma was successfully administered and the patient s symptoms improved , albeit the thrombi remained . in sma angiography ,
regression of the thrombi was not observed , however , thrombolytic therapy prevents the progression of a thrombus formation and helps to develop a collateral vessel formation that prevents the progression of bowel ischemia .
six months later , we discontinued the anticoagulation , because a complete remission of the nephrotic syndrome was maintained and the thrombi had disappeared . in summary ,
the possibility of an acute portal vein or smv thrombosis should be considered when severe acute abdominal pain and nausea occur in patients with nephrotic syndrome , especially in high risk patients .
anticoagulant therapy with thrombolytic therapy should be considered and provided when bowel ischemia or infarction is suspected .
interventional treatment , including direct or indirect portal vein and smv thrombolysis , is a safe and effective strategy for patients with symptomatic acute portal vein and smv thrombosis prior to the development of bowel infarction .
the authors report no conflict of interest . the authors alone are responsible for the content and writing of the paper . | thromboembolism is a major complication of nephrotic syndrome .
renal vein thrombosis and deep vein thrombosis are relatively common , especially in membranous nephropathy .
however , the incidence of portal vein and superior mesenteric vein ( smv ) thrombosis in patients with nephrotic syndrome is very rare . to date
, several cases of portal vein thrombosis treated by anticoagulation therapy , not by thrombolytic therapy , have been reported as a complication of nephrotic syndrome . here
, we report a case of portal , splenic , and smv thrombosis in a patient with a relapsed steroid dependent minimal change disease who was treated successfully with anticoagulation and thrombolytic therapy using urokinase .
radiologic findings and his clinical conditions gradually improved .
six months later , a complete remission of the nephrotic syndrome was observed and the follow - up computed tomography scan showed the disappearance of all portal vein , splenic vein , and smv thrombi . |
there is an urgent
need to better understand the molecular factors
that govern influenza a virus ( iav ) virulence .
seasonal ( human - adapted )
iav is a major cause of morbidity and mortality each year , but the
pandemic potential of iav strains that originate in animal hosts poses
an even more serious threat to public health .
there have been four
influenza pandemics in the past century , in 1918 , 1957 , 1968 , and
2009 .
the precise origin of the 1918 pandemic strain , which killed
roughly 50 million people , is unclear .
however , the three subsequent
pandemic strains resulted from the process of genetic reassortment ,
in which gene segments from different viruses mix during host coinfection
to produce novel viral strains . there is much concern that a new pandemic strain might result from
reassortment involving the highly pathogenic h5n1 and h7n9 avian influenza
viruses currently circulating in asia .
the majority of these viruses
undergo a deletion in the stalk of the neuraminidase surface protein ,
a characteristic that is associated with virulence when causing outbreaks
in domestic poultry .
it is thus vitally
important to understand the effect of stalk deletion on viral function .
influenza a strains are named according to the alleles coding for
their two surface antigens , the hemagglutinin ( ha ) and neuraminidase
( na ) glycoproteins .
hemagglutinin
( ha , 18 antigenically distinct alleles identified to date ) facilitates
viral adhesion to human host cells by binding to cellular sialylated - oligosaccharide
receptors , and neuraminidase ( na , 11 antigenically distinct alleles )
facilitates viral release by cleaving sialic acid linkages .
for example , hyperglycosylation
and active - site mutation tend to reduce ha / sialic acid binding . na
activity is generally proportional to the length of the highly variable
na stalk , and recent neuraminidase stalk deletions often produce highly pathogenic
avian influenza viruses such as h5n1 .
influenza virulence is determined in large part by the balance
between na and ha activities at the virion surface .
recent viral strains
with stalk - deletion na glycoproteins ( nadel ) are more virulent
because the reduced nadel activity alters this balance .
for example , the virulence of both a pandemic 2009 h1n1 virus and
a more recent 2013 h7n9 strain was enhanced when deletion mutations
containing 20 and 5 amino acids , respectively , were introduced into
the corresponding na stalks . in these strains ,
reduced na activity due to a stalk deletion may
enhance viral infectivity by reducing oligosaccharide - receptor cleavage
and thus increasing the number of receptors available for ha binding .
this strategy is particularly advantageous when ha activity is itself
compromised ( e.g. , due to hyperglycosylation , active - site mutations ,
etc . ) , leading many to express concerns that a current h5n1 strain
might obtain a long - stalk na through reassortment with a human - adapted
virus .
optimal viral replication is achieved only when the activities
of na and ha are ideally complementary .
the mechanism by which reduced
na stalk height impacts na activity
remains uncertain .
one prevailing theory suggests that nadel glycoproteins have reduced sialidase activity relative to wild type
( nawt ) because their diminished height hinders access to
cellular sialylated - oligosaccharide receptors ( i.e. , the
the
implication is that a towering canopy of ha effectively buries the
short - stalk nadel such that cell - bound oligosaccharides
simply can not reach the na enzymatic pockets .
while there is merit
to the limited - access theory , especially in cases where the stalk
length is profoundly reduced , experiment and computational modeling
suggest it may not fully explain the reduced nadel activity .
we hypothesize that , in addition to any limited - access effects ,
na stalk length also reduces na enzymatic activity by altering the
binding affinity of sialic acid , the native substrate , to the enzymatic
pocket .
given that the stalk is roughly 40 from the active
site , we propose an indirect mechanism by which stalk length impacts
binding - pocket dynamics at a distance .
other studies have demonstrated
that the na enzymatic pocket is highly flexible and so samples many
conformations .
for example , x - ray crystallography and molecular dynamics
simulations of the isolated neuraminidase catalytic domain suggest
that key loops surrounding the active site ( i.e. , the 150- and 430-loops )
are highly mobile , permitting large cavities to form immediately adjacent
to the main pocket .
given that the enzyme is known to adopt a variety of conformational
states , it is reasonable to hypothesize that stalk length may have
a quasi - allosteric impact on ligand binding affinity by shifting the
binding - pocket conformational population toward a subtly different
distribution . to properly study the impact of stalk length
, we built a full
model
of 2009 h1n1 neuraminidase as well as a model that included a 20 amino
acid stalk - deletion mutation similar to one known to enhance virulence
in a recent strains .
, who
replaced the long - stalk nawt of a 2009 h1n1 pandemic virus
with a short - stalk h5n1 nadel .
they attributed the resulting
decrease in transmissibility at least in part to a reduction in the
ability of the short - stalk nadel to cleave tethered substrates
and penetrate the host mucus barrier . rather than exactly replicating
the work of blumenkrantz et al .
, who inserted a foreign na ( from h5n1 )
into a h1n1 virus , we made our deletion directly in the h1n1 nawt stalk itself .
our models were constructed using data from
both x - ray crystallography ( used to model the enzymatic head ) and
also protein protein docking and modeling ( used to model the
stalk , transmembrane , and intravirion domains , which have never been
crystallized ) .
all na models were embedded in complex - mixture lipid
bilayers ( figure 1 ) .
equilibrated
neuraminidase models used in the current study : ( a )
wild - type ( long - stalk ) nawt and ( b ) stalk - deletion nadel .
subsequent atomistic molecular
dynamics simulations of these systems
support the theory that the na stalk length impacts the dynamics of
the na binding pocket and therefore the affinity for the endogenous
substrate .
furthermore , these simulations reveal previously uncharacterized
druggable hotspots that may open up additional avenues
for structure - based drug design .
previous simulations have included
only the na head ; our models , which include additional na domains
as well as the associated lipid bilayer , allow us to take the next
steps toward more realistic atomic simulations of viral components
to generate a
complete model of long - stalk ( wild - type ) apo nawt , a monomer
of the tetrameric na head was built by homology using schrdinger s
prime and the na sequence of the 2009 h1n1 pandemic reference strain
a / california/04/2009 ( genbank accession fj966084 ) , which codes for
a na protein identical to that of the a / england/195/09 virus used
by blumenkrantz et al .
default prime
parameters were used . the monomeric homology model , based on the 3nss
crystal structure , was missing a single
lysine residue on the c terminus , which was added manually . the appropriate
tetramer was formed by aligning copies of the homology - model monomer
to chains a d of the 2hu4 crystal structure using multiseq , as implemented
in vmd .
the resulting tetrameric head
was then processed with schrdinger maestro s protein
preparation wizard and subjected to repeated minimization in the maestro
environment .
both sspro
4.5 , a neural - network - based algorithm
for predicting secondary structure from primary sequence , and abtmpro , a svm algorithm for determining whether a given
transmembrane segment is helical or barrel , suggested
that the stalk has an -helical structure .
we therefore used
the amber xleap module to generate a single helix with the
appropriate long - stalk sequence .
rosettadock 3.4 was used to assemble four identical copies of this helix into a
four - helix bundle , one helix at a time . at each step ,
the top 200 best - scoring structures were
clustered by root - mean - square distance ( rmsd ) using cutoffs ranging
from 0.75 to 3.0 , and representative dockings were examined
visually .
the top representative docked pose that positioned the newly
introduced helix in the expected parallel conformation was retained .
when no such pose was identified by clustering , all docked poses were
examined sequentially , starting with the pose that had the highest
score , until an acceptable pose was identified .
the resulting four - helix
bundle was then processed with schrdinger maestro s
protein preparation wizard and subjected to repeated minimization
in the maestro environment .
we assumed that the cysteine residues located in the extra - virion
region of the stalk likely participate in interchain disulfide bonds , but that the cysteine residues located in the
transmembrane region likely do not .
the tetrameric - head and stalk / transmembrane - region models
were next positioned near each other using vmd .
the combined models
were subsequently loaded into maestro and again subjected to multiple
minimizations .
finally , maestro was used to build and position
the residues of
the short intravirion topological domain .
based on the assumption
that this domain lies at the interface of the inner membrane leaf
and bulk intravirion water , all hydrophobic side chains were manually
pointed upward toward the bilayer , and all polar side chains were
pointed downward . to generate a model of apo na with a virulence - enhancing
stalk
deletion (
hereafter called nadel ) , the appropriate mutant
amino acid sequence was first identified .
these residues were manually removed from the nawt model , and the flanking na segments were juxtaposed and fused using
vmd and schrdinger s maestro , followed by multiple
minimizations as needed .
our efforts
to construct a
complex - mixture lipid - membrane model were guided by a recent paper
describing the composition of the influenza viral envelope .
we identified and classified all listed membrane
components with mol % > 1.5 , taking care to keep the composition
of
the inner and outer leaflets separate .
as our ultimate goal was to
perform molecular dynamics simulations of this system , we were limited
to the lipids of the charmm 36 force field .
consequently , we had to substitute some experimentally identified
lipids with available lipid models .
3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylethanolamine ( pope ) was used for any phosphatidylethanolamine
( pe ) variant , 2,3-distearoyl - d - glycero-1-phosphatidylserine
( dsps ) for phosphatidylserine ( ps ) , 3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylglycerol ( popg ) for the forssman glycolipid
hapten , and 3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylcholine
( popc ) for sphingomyelin ( sm ) . cholesterol and phosphatidylcholine ,
also major components of the envelope membrane , are included in the
charmm 36 force field .
the na models were then positioned within this generated bilayer ,
and clashing lipid residues were deleted with pymolecule . in preparation for molecular dynamics
simulations , we resolved multiple steric clashes in both the long - stalk
and stalk - deletion systems using serial iterations of minimization
and geometry optimization in schrdinger s maestro ,
coupled with manual atomic and molecular manipulation in vmd .
partial
charges were
assigned to all atoms according to the specifications of the charmm22 all - hydrogen force field for proteins and the charmm36 all - hydrogen force field for lipids .
the vmd plug - in cionize(47 ) was used to position sodium and chloride ions as required to bring
the systems to electrical neutrality and to simulate a 20 nm solution .
limited manual adjustments to the positions of some ions that had
been placed far from any protein or lipid molecule were made .
for both the nawt and nadel systems , amber s
xleap module was used to generate a water
box extending 15 beyond any atom .
water molecules were subsequently
removed if they were not positioned directly above the bilayer , so
that the 15 margin was ultimately retained only along the z
axis , perpendicular to the bilayer itself .
the nawt and
nadel systems had 366 293 and 351 699 atoms ,
respectively .
psfgen
was used to fully
parametrize each system according to the charmm22 , charmm36 , and tip3p force fields for proteins , lipids , and water molecules ,
respectively .
namd 2.9 running on the
stampede supercomputer at the texas advanced computing center was
used for all md simulations .
periodic boundary conditions were employed
with the particle mesh ewald method to account for electrostatic effects
( smoothing cutoff : 14 ) . because of observed instabilities when
our standard minimization protocols were used , we subjected the initial
nawt system to only 1000 steps of unrestrained conjugate
gradient minimization , with the first and max initial steps for the
line minimizer reduced to 1.0 10 and 1.0
10 , respectively .
the structure was
minimized in four distinct steps : hydrogen atoms were first relaxed
for 5 000 steps ; hydrogen atoms , water molecules , and ions
were next relaxed for 5 000 steps ; hydrogen atoms , water molecules ,
ions , protein side chains , and lipid tails were then relaxed for 10 000
steps ; and , finally , all atoms were relaxed for 25 000 steps . following minimization ,
each system was subjected to a constrained
equilibration using an npt ensemble at 310 k. the
equilibration was realized in four steps , using stepwise harmonic - constraint
force constants of 4 , 3 , 2 , and 1 kcal / mol / on the
protein backbone . for each force
constant ( 1 fs time step ) , 250 000
steps of md simulation were performed .
langevin dynamics were applied
to maintain the temperature , and a modified langevin piston nos
the nawt model underwent an additional 5 million
2 fs steps of unconstrained simulation , followed by another 21
million 1 fs steps .
the nadel model underwent an additional
5.5 million 2 fs steps of unconstrained simulation , followed
by another 36.5 million 1 fs steps .
satisfied that both systems
were sufficiently equilibrated , five
distinct 100 ns productive runs were performed for each ( 100
million 1 fs steps ) using distinct random seeds in order to sample
diverse protein configurations .
( 5 simulations ) ( 100 ns per simulation )
( 2 systems ) ( 4 monomers per system ) = 4 s of
monomeric simulation total .
5000 and 7000 frames were extracted from
the last 50% and 70% of the tetrameric nawt and nadel simulations , respectively , for subsequent analysis . for comparison purposes
, we also analyzed an archived simulation
of the isolated apo na head ( hereafter called nahead ) ,
described previously . in brief , the na
head ( pdb i d : 3nss(26 ) ) was simulated for 100 ns using the
amber99sb force field , yielding 400 ns of monomeric trajectory .
for
the current study , we extracted 5000 frames from this simulation for
subsequent analysis .
we used root - mean - square - distance ( rmsd ) calculations
to judge
the geometric similarity between the primary na enzymatic pockets
of crystallographic holo and simulated apo models .
we first identified
active - site residues as those that came within 5.0 of a crystallographic
oseltamivir ligand ( pdb i d : 2hu4:a , h5n1 ) .
these residues are arg118 , glu119 , asp151 ,
arg152 , arg156 , trp178 , ser179 , ile222 , arg224 , glu227 , ser246 , glu276 ,
glu277 , arg292 , asn294 , tyr347 , gly348 , arg371 , and tyr406 . for the
purposes of our rmsd analysis , we discarded tyr347 because that tyrosine
is an asparagine in h1n1 na .
hydrogen atoms were also removed prior
to performing all rmsd calculations ; rmsd alignment was performed
only on the remaining heavy atoms .
we performed principal
component analysis ( pca ) on these same active - site heavy atoms .
after
rmsd aligning the nawt trajectories by these atoms using
vmd , we used the python modules scikit - learn and numpy to calculate their first two principal components
and to project each trajectory frame onto those components .
the frames
of the nadel trajectory were similarly projected onto the
same two nawt principal components to facilitate comparison .
the povme algorithm was used to measure the volume of the primary
sialic - acid - binding pocket over the course of the nawt ,
nadel , and nahead simulations . to calculate
the volume of the binding pocket for a given trajectory frame , we
monitored the space within a pocket - centered sphere of radius 16.0
. in all cases ,
the pocket volume was calculated from the portion
of the sphere that was unoccupied by protein atoms . only volumetric
regions contiguous with the pocket
grid spacing and
padding parameters were set to 1.0 and 1.09 , respectively .
all - atom , residue root - mean - square
fluctuations ( rmsf ) were calculated for
each monomer of each simulation . for the nawt and nadel simulations ,
for the nahead simulation ,
there were 4 associated monomeric simulations ( 1 run 4 monomers ) .
in all cases , the monomeric trajectories were first aligned by the
carbons of residues ser90 to asp469 ( i.e. , the residues of
the na head )
. the residue center - of - mass rmsf values were calculated
for each monomeric simulation separately . to identify druggable
hotspots , we first identified representative protein conformations
from the md simulations . the heads of the aligned monomeric trajectories
were subjected to rmsd clustering , as
implemented in the gromacs computer package .
we adjusted the rmsd cutoff until the trajectory frames were separated
into five clusters .
the required cutoffs were 1.72 , 1.74 , and 1.74
for the nawt , nadel , and nahead simulations , respectively .
the centroid members of each of these
clusters were considered representative of the corresponding simulated
model .
the 15 representative na structures ( 3 models
5 cluster centroids ) were then submitted to the ftmap server to identify druggable hotspots . to simplify the analysis , all the
docked molecular probes associated with each simulation ( nawt , nadel , and nahead ) were considered collectively .
to identify druggable hotspots that were specific to a given model
,
the associated docked probes were superimposed , and any probe atom
associated with one model that came within 5 of any probe atom
associated with the other was deleted .
the remaining probe atoms congregated
at hotspots that were unique to their respective models .
as described
in the introduction , the limited - access theory
holds that stalk - deletion neuraminidase glyocproteins ( nadel ) have reduced sialidase activity relative to nawt because
their diminished height hinders access to cellular sialylated - oligosaccharide
receptors . the fact that na enzymatic activity varies depending on
ligand bulkiness is perhaps the most compelling evidence in favor
of this theory .
studies have shown that short - stalk nadel glycoproteins have significantly reduced activity when a bulky substrate
( e.g. , fetuin ) is used , presumably because
the large substrate can not physically reach the na enzymatic sites .
in contrast , na activity is largely stalk - independent when cleaving
the small molecule munana ( essentially a single sialic acid with a
4-methylcoumarin fluorophore attached to the 2-carbon ) , apparently because this smaller substrate is not subject to the
same large - scale steric hindrance .
however , several lines of
evidence suggest that the limited - access theory may not fully explain
the reduced nadel activity .
binding to
these receptors , which are extended , large , and cell bound , is likely
diffusion limited ; in contrast , munana is a relatively small molecule
with an affinity that is likely little dependent on kon .
while munana may be a fine substrate for measuring
relative na enzymatic activity in the context of a small - molecule
screen , it is arguably an inadequate surrogate for the actual receptor .
second , fetuin ( -2-hs - glycoprotein ) is also very different
than the endogenous receptor , and the theory that its bulk alone prevents
it from accessing the nadel enzymatic sites is hardly certain .
we note that all available monoclonal antibodies known to target na
antigenic sites bind equally well to virions with nadel and nawt glycoproteins , despite
the fact that antibodies are much larger than fetuin ( 160
and 1000 kda for igg and igm , respectively , vs 40
kda for fetuin ) .
furthermore ,
recent cryo - electron microscopy studies of whole - virion particles
demonstrate that the na glycoproteins are not evenly interspersed
among the more prevalent ha s as the limited - access theory
suggests ; rather , the na particles tend to cluster together on the
viral surface .
even when stalk mutations
substantially reduce the na height relative to ha , this clustering
effect alone should in theory preserve much access to the na enzymatic
head .
computational evidence likewise suggests that simple steric
considerations
are not solely responsible for reduced nadel activity .
our models of 2009 h1n1 glycoproteins demonstrate that each stalk
amino acid contributes only 1.2 to the total na height
( data not shown ) .
the 2013 h7n9 five - amino - acid stalk deletion with
enhanced virulence mentioned in the introduction therefore reduced the na height by only 6 ( roughly
4% ) .
more substantial stalk deletions
do not necessarily force the na enzymatic head to retreat entirely
behind a towering canopy of ha glycoproteins either . in our models
of h1n1
ha and na , we found the height of nawt was nearly
equal to that of ha ( 149 vs 154 , respectively ) .
when a virulence - enhancing
20 amino acid na stalk deletion was modeled
( nadel ) , the height of the na fell to only 125 ,
still 81% of the ha height .
given that the na head is itself a boxlike
structure 80 square , the resulting 24 height reduction
seems unlikely to substantially interfere with access .
we suggest
that , beyond limited - access effects , actual differences
in the affinities of nawt and nadel for the
endogenous sialylated - oligosaccharide receptors contribute to the
differences in enzymatic activity . as support for our reduced - affinity
hypothesis
, we analyze atomistic molecular dynamics simulations of
three na models : five 100 ns simulations of 2009 h1n1 nawt ; five 100 ns simulations of 2009 h1n1 na with a 20-amino - acid deletion
in the stalk ( nadel ) ; and
one 100 ns simulation of the isolated h1n1 na head , nahead ( figure 1 ) .
these
simulations indicate that stalk length does in fact impact the dynamics
of the enzymatic sialic acid binding pocket , suggesting a potential
mechanism for the proposed stalk - length - dependent differences in affinity .
to
verify that the simulations of the three na systems ( i.e. , nawt , nadel , and nahead ) had properly equilibrated ,
we rmsd aligned each tetrameric trajectory by its c atoms using vmd .
the rmsd was then
calculated between each trajectory frame and a corresponding reference
structure ( figure s1 ) . despite extensive
pre - equilibration ,
the rmsd values of the whole - glycoprotein nawt and nadel simulations continued to drift slightly
throughout the productive runs .
to further investigate the source
of this drift , we calculated separate c rmsd values
for the residues of the na head ( figure 2 ) , the lower ( membrane - embedded ) stalk , and
the upper stalk .
the dynamics of the head and lower stalk were in
fact stable in both the nawt and nadel simulations .
it was the upper stalk that was dynamically unstable . given that the
upper stalk serves as a linker connecting two fairly ridged bodies
( the lower stalk and the head ) , it is reasonable to suppose that this
region is legitimately hypermobile .
rmsds of all trajectory monomers to their
respective reference
structures . in all cases ,
only the c of the head
domain ( i.e. , the c of all residues homologous to
the 388 residues present in the 3nss crystal structure ) were considered .
( a ) nawt ( 5 tetrameric trajectories 4 monomers per
tetramer = 20 runs ) ; ( b ) nadel ( 5 tetrameric trajectories
4 monomers per tetramer = 20 runs ) ; and ( c ) nahead ( 1 tetrameric trajectory 4 monomers per tetramer = 4 runs ) .
subsequent analyses ( described
below ) focused primarily on the
residues of the na binding pocket , located on the enzymatic head .
given the instability of the upper stalk , however , we chose to discard
the initial 50% and 30% of the nawt and nadel trajectories , respectively , to ensure that subsequent analysis focused
on the most equilibrated portion of the simulations .
in contrast ,
nahead , based on the 3nss crystal structure , equilibrated far faster .
we therefore discarded
only the first 20% of the nahead simulation ( figures 2 and s1 ) .
we next
sought to characterize the effects of the stalk on binding - pocket
dynamics by comparing the geometries of our simulated apo binding
sites to that of holo ( ligand - bound ) na .
we calculated the rmsd values
between the simulated binding - pocket conformations and that of a reference
crystallographic structure , 2hu4 ( h5n1 ) ,
which shares in common with h1n1 a total of 18 out of 19 binding - pocket
residues . in calculating these rmsd values , the single differing residue
was ignored .
a histogram of the rmsd values suggests that the
long - stalk nawt pocket exists in two conformational states
( figure 3a ) , with rmsds
to the apo ( reference ) conformation of roughly 1.8 and 2.7 ,
respectively .
in contrast , the pocket conformations sampled during
the nadel and nahead simulations were not binary ,
suggesting the pocket is less flexible in these systems .
( a ) simulated apo binding - pocket
conformations are compared to the crystallographic ( 2hu4 ) holo conformation .
geometric similarity was judged by calculating the rmsds between simulated
and crystallographic pocket residues .
.
geometric similarity was judged by calculating the rmsds between simulated
and crystallographic triad conformations .
( c ) representative
nawt arginine - triad conformations sampled from the 2.4
state ( in yellow ) and the 3.4 state ( in red ) .
the crystallographic
position of the triad residues is shown in thick , white licorice .
an oseltamivir molecule has been positioned within the binding pocket
for reference ( in orange ) , though the simulations did not include
any ligand .
( d ) the simulated conformations of nawt and
nadel active - site heavy atoms , in black and red , respectively ,
projected onto the first two principal components of the nawt simulation .
the nawt site assumes a greater variety of
conformations , in harmony with the rmsd analysis .
an arginine triad formed by arg118 , arg292 , and arg371 is
a key
feature of the enzymatic pocket .
this triad forms a positively charged
arginine nest that surrounds the negatively charged carboxylate groups
of the endogenous substrate as well as all fda - approved na inhibitors .
as with the whole pocket ,
the average rmsd between the atom positions
of the simulated apo triad configurations and the holo 2hu4 conformation were 2.5 and 2.4 for the
nawt and nadel simulations , respectively .
a t - test led us to reject the null hypothesis that these two
averages are statistically equal ( p = 0.0 ) .
a histogram of the arginine - triad rmsd values suggests that , unlike
the nadel triad , the nawt triad again assumes
two distinct conformations ( figure 3b ) .
to further characterize these two nawt states , we visually inspected representative conformations from
each ( figure 3c ) . in
the state with rmsd = 2.4 , arg371 is near its crystallographic
conformation , and arg292 is displaced only slightly in the direction
opposite of arg371 .
in contrast , in the state with rmsd = 3.4
, arg371 is generally displaced in the direction of arg118 ,
and arg292 is even further from arg371 .
arg118 possesses roughly the
same conformation in both states . to further verify that the
nawt pocket assumes a greater
variety of conformational states than the nadel pocket
,
we next performed a principal component analysis ( pca ) of the nawt active - site heavy atoms ( figure 3d ) .
pocket conformations from both the nawt and nadel simulations were projected onto the
first two principal components . the nawt pocket ( in black )
sampled the same region of pca space as the nawt simulation
( in red ) , but the nawt pocket additionally sampled other
conformations not seen in the nadel simulation , confirming
that the nawt pockets adopted a greater number of conformational
states .
having studied the impact of stalk length on binding - pocket
dynamics generally , we next considered the dynamics of specific binding - pocket
residues .
we calculated the associated all - atom , residue root - mean - square
fluctuations for each of the 20 monomeric
trajectories associated with the nawt and nadel simulations . for each residue
, we compared the ensembles of nawt and nadel rmsf values using a t - test . a number of residues exhibited statistically significant average
rmsf differences when a liberal significance level was used ( t - test , = 0.10 ) .
most interestingly , the rmsf values
of two of the three arginine residues of the key active - site triad
( arg371 and arg118 ) differed significantly ( table 1 ) .
this analysis again implicates the arginine
triad and 371 loop as contributing to differential , stalk - length - dependent
binding - pocket dynamics .
data from the
corresponding nahead ( stalk - absent ) simulations are also
included . the average
rmsf value is shown plus or minus the standard deviation .
the residue - specific
populations of all nawt and nadel rmsf values
were compared using t - tests .
to better visualize differences in active - site dynamics ,
we colored
the residues of a nawt conformation by rmsf = rmsfwt rmsfdel ( figure 4 ) .
the 371 , 406 , and 430 loops were generally
more flexible in the nawt simulation ( shown in red ) . however ,
a portion of the 150 loop was more flexible in the nadel simulation ( shown in blue ) .
na active - site residues colored by rmsf
= rmsfwt rmsfdel , from 0.2
to 0.2 .
residues in
red and blue were more flexible in the nawt and nadel simulations , respectively .
a crystallographic oseltamivir molecule
is shown for reference , though no ligand was included in the simulations .
finally , we also used our
molecular dynamics simulations to study
na druggability . to get an initial impression of the full extent of
pocket dynamics , we first performed a volumetric analysis of the primary
sialic - acid - binding pocket using the povme algorithm .
the trajectory frames with both the largest and smallest
volumes from the nawt , nadel , and nahead simulations were compared separately ( figure 5 ) . in all cases , the pocket fluctuated between
a closed conformation ( figure 5a ) and an open conformation ( figure 5b ) , suggesting a highly dynamic pharmacophore .
interestingly , the nawt pocket tended to be larger than
the nadel pocket ( figure 5c , p = 0.0 per a t - test ) , implying possible differences in the pharmacophore distributions
of these two na variants that may have important implications for
drug discovery . to further study variant - dependent differences in
na - pocket druggability .
ftmap seeks to replicate in silico the experimental
multiple solvent crystal structures ( mscs ) method developed by mattos
et al . in brief , the ftmap algorithm uses computer
docking to flood the surface of a protein with small organic molecular
probes .
these probes are then clustered to identify regions of the
protein surface that are most likely to bind small molecules .
( a ) the smallest and ( b ) largest binding - pocket
conformations sampled by the nawt simulation .
the smallest
and largest conformations from the nadel and nahead simulations were similar ( data not shown ) .
( in licorice ) have been placed in each pocket for reference , though
the simulations included no ligand .
( ) histograms of the volumes sampled
over the course of the nawt and nadel simulations ,
in black and gray , respectively .
we used ftmap to identify druggable hotspots on the surfaces
of
five representative na structures extracted from each of our molecular
dynamics simulations .
for the most part , the druggable regions of
the enzymatic site did not differ substantially between the nawt and nadel simulations .
however , ftmap did position
some molecular probes near a 430-loop - adjacent region of the nawt pocket that was not deemed druggable when
the nadel conformations were considered , suggesting subtle
differences that may be pharmacologically relevant ( figure 6a ) .
unique druggable hotspots
( outlined ) . the 371 , 430 , and 150 loops
are shown in mauve , pink , and green , respectively .
( a ) a druggable
hotspot was identified near the 430 loop of the nawt pocket .
( b ) a second druggable hotspot was identified beneath the 371 loop
of both the nawt and nadel pockets .
( c ) a large
druggable hotspot was identified near the base of the nawt and nadel heads .
the nawt and nadel hotspots were then
merged
and similarly compared to the nahead hotspots . surprisingly ,
in both stalk - inclusive na simulations ( nawt and nadel ) , the 371 loop occasionally flipped upward , opening a novel
druggable hotspot that was not apparent when the representative structures
of the nahead simulation were considered ( figure 6b ) .
this pocket has been described
previously , but the nawt and nadel simulations confirm its importance . to date , no known na
inhibitor exploits this pocket , and it has not been the focus of previous
cadd efforts .
finally , another large druggable hotspot located at
the base of the na head was unique to the stalk - inclusive ( i.e. , nawt and nadel ) simulations ( figure 6c ) .
factors
that alter the sialic acid binding / association mechanism
may profoundly alter na affinity and activity in additional ways not
explained by the theory of limited access .
the length
of the stalk appears to influence the dynamics of the 150 , 430 , 371 ,
and 406 binding - pocket loops .
the 371 loop and the critical arginine
catalytic triad are particularly affected . as a consequence
,
the apo nawt pocket may adopt a greater
variety of conformational states than does that of nadel .
specifically , the nadel pocket tends to sample a single
conformational state that is generally similar to that of the holo
conformation .
in contrast , nawt additionally samples more
distant pocket geometries ( figure 3 ) .
while the impact of stalk length on binding - pocket
dynamics may
well be partially responsible for the reduced nadel affinity ,
we note also that stalk length likely has a profound influence on
the long - range electrostatics of the whole - virion surface , which could
also impact kon .
future efforts will focus
on elucidating stalk - length - dependent differences in this effect as
well . finally , the predicted druggable hotspots evident in the
stalk - inclusive
simulations also suggest novel and urgently needed opportunities for
drug discovery . prior to the 2009 h1n1 pandemic , seasonal h1n1 in
the united states was almost entirely oseltamivir resistant . fortunately , that pandemic strain itself remained
largely susceptible to oseltamivir , but
given the viral proclivity for rapid mutation and adaptation , multidrug
combinatorial therapies may be necessary in the future .
these novel hotspots also provide means
for experimentally testing
some of the hypotheses generated in the current study .
virtual screening
into the na pocket conformations sampled over the course of the nawt and nadel simulations could identify compounds
that are uniquely suited to the corresponding pocket geometries .
if
subsequent experimental validation confirms binding , x - ray crystallography
could be used to determine if the novel ligand(s ) stabilize one of
the predicted na conformations . | deletions in the
stalk of the influenza neuraminidase ( na ) surface
protein are associated with increased virulence , but the mechanisms
responsible for this enhanced virulence are unclear . here
we use microsecond
molecular dynamics simulations to explore the effect of stalk deletion
on enzymatic activity , contrasting na proteins from the a / swine / shandong / n1/2009
strain both with and without a stalk deletion . by modeling and simulating
neuraminidase apo glycoproteins embedded in complex - mixture lipid
bilayers ,
we show that the geometry and dynamics of the neuraminidase
enzymatic pocket may differ depending on stalk length , with possible
repercussions on the binding of the endogenous sialylated - oligosaccharide
receptors .
we also use these simulations to predict previously unrecognized
druggable hotspots on the neuraminidase surface that
may prove useful for future efforts aimed at structure - based drug
design . |
one of the most important ways to manage the behavior of children is pain control .
since a deep anesthesia is necessary in all dental treatments , a comfortable injection with minimal pain and no injury has a considerable importance .
in fact , a local anesthesia induced by a single injection not only provides the patient 's comfort and decreases pain but also increases patient 's trust to dentist . the most common method of inducing anesthesia for maxilla is infiltration or supra - periosteal injection technique . in this method , needle is penetrated in deep vestibular mucosa above the apex of the tooth .
inferior alveolar ( i.a ) nerve block is the common technique of anesthetizing the mandible .
the only difference is that the injection must be a little lower , and more posteriorly for children because the mandibular foramen is lower to occlusal surface of deciduous teeth .
one of the most common complaints following i.a nerve block is soft - tissue injury due to biting lips , tongue , and buccal mucosa .
today i.a nerve block is commonly used to treat the pulp of mandibular primary molar teeth .
block injection for hemophilic patients who have not received recombinant factor may lead to cervical hematoma or even death . in these patients , infiltration may be used . infiltration technique because of direct vision of practitioner on it , less depth penetration of needle , less technical errors , less amounts of anesthetic solution , easier application , limited anesthesia of soft - tissues outside the operation field and shorter duration of being anesthetized might be used as an alternative to block .
on the other hand because the anesthetized area is limited , bilateral anesthesia is possible .
sharaf selected pediatric patients who needed bilateral multiple dental treatments ( amalgam filling , pulpotomy and tooth extraction ) . the researcher compared the effectiveness of infiltration to block techniques .
results showed that infiltration is an appropriate technique to anesthetize primary mandibular first molar for pulpotomy .
donohue et al . showed that anesthetizing primary mandibular molar region for tooth filling , pulpotomy and extraction with infiltration technique is possible .
jones et al . in a study on 308 patients concluded that block injection is significantly more painful than infiltration .
ram and peretz evaluated the patients responses to pain during block and infiltration injections ; they showed that children responded more severely in the form of crying to mandibular block injection . in a study by oulis et al .
it was shown that sufficiency of infiltration technique for mandibular tooth extraction and pulpotomy was less than a block .
aminabadi et al . by evaluating the relation between the site of injection and pain in 5 - 6 year old children showed that the most severe response was related to nasopalatine nerve block and the least was to posterior superior and i.a nerves block , and also maxillary injections , in general , was more painful than mandibular injections .
they assessed tooth sensitivity by pulp tester at the end of 5 , 8 , 11 , 15 , 20 , 25 , and 30 min following injecting articaine 4% .
the results of their study showed the faster appearance of anesthesia with infiltration injection compared to block while the efficacy was identical . to assess the behavior and comprehensive response of a child to the dental office environment
, it must be considered that what response is appropriate for his / her age .
therefore , suitable tests and certain methods have to be applied . in the present study ,
the degree of pain during injection and on pulpal exposure of the two mandibular anesthetizing methods were assessed and compared . to assess the pain , sound - eye - motor ( sem ) scale was used which is valid and easy to apply .
the protocol of this clinical trial was registered ( irct201009084714n1 ) at the clinical trial administration office research vice chancellor of ministry of health of iran .
the study was approved by the school of dentistry ethics committee at the isfahan university of medical sciences , iran . among the patients referred to isfahan specialized clinics for dental pediatric procedures , 40 pediatric patients candidate for bilateral pulpotomy of mandibular primary first molars
inclusion criteria were general health , lack of painful illness , able to be placed in the grades + or + + of frankl behavioral scale . according to this rating scale , grade +
is referred to the children who negotiate first but accept dental treatment ; grade ( + + ) is referred to those who have a good relation with the dentist and are interested in dentistry with laughing and enjoying .
in this crossover study ,
the patients were placed randomly into the groups a and b. on the first session , the children in the group a received infiltration for mandibular right first molar and in group b i.a nerve block for the left side . on the second session ,
all the patients of group a received i.a nerve block on the left side and all patients of group b received infiltration technique on the right side .
all patients received lidocaine hydrochloride anesthetic ( darupakhsh , tehran , iran ) using 27 gage needles ( supa , tehran , iran ) .
infiltration injection with the volume of 0.2 ml of lidocaine 2% was done at the depth of buccal vestibule between the roots of teeth .
i.a nerve block injection was performed with the volume of 1 ml of lidocaine 2% in a standard manner at mandibular jaw .
the amount of pain was measured through patient 's expressions , behaviors , or biologic markers .
one of the valid indices to assess the pain is sem scale , which is accepted to assess the comfort or existence of pain in children . by reading the table of this four score scale for the recorded eye and body movements and also voice ,
for example , a child whose voice was the lack of the sign of pain earned grade 1 , or would earn grade 4 if he / she was crying .
since all the variables of sem scale are qualitative and also are not distributed normally , the non - parametric tests were used to analyze and compare the grade of variables .
the amount of pain during injection and on pulpotomy was assessed using this scale and recorded by a calibrated and trained dentist who was blind .
mann - whitny test was used to compare the grades of sem scale for different techniques in two groups independently , and wilcoxon signed rank tests for each group and the total of patients .
the range and mean value of patients age were 5 - 8 and 6.25 years respectively .
eighteen out of 40 patients ( 45% ) earned + and 22 ( 55% ) + + for cooperation .
the scores of sem during injection and on pulpal exposure for infiltration technique and also i.a nerve block technique are summarized in tables 1 and 2 respectively .
efficacy of infiltration technique , sem scores during injection and pulpal exposure efficacy of i.a nerve block technique , sem scores during injection and on pulpal exposure no significant differences were found between the scores of injections and also pulpal exposure of two groups when infiltration technique was applied ( p = 0.766 ) comparing the results only showed a significant difference between two groups regarding eye movements and also the sum of sem scores during injection .
thus in group b , which received block injection first , score of eye movements during block injection was higher .
however , comparing the efficacy of block induced anesthesia between two groups did not show significant differences .
the sem score of infiltration injection was significantly lower than a block in both groups regardless the order of injections but was not significant on pulpal exposure .
comparison between the sem scores of two different techniques in groups a and b the scores of sem in infiltration technique were significantly lower than block .
no significant difference was seen between block and infiltration induced anesthesia on pulpal exposure ( p = 0.917 )
in this study , the efficacy of infiltration and i.a nerve block induced anesthesia to perform mandibular primary first molars pulpotomy were compared .
since injection is known as a stressful phase in pediatric dentistry , well controlled pain during injection is an important effective factor in getting the cooperation of the child .
thus , the dentist who can perform an injection with minimum pain , stress , and anxiety is more successful .
considering that block injection induces relatively sustained anesthesia and may also cause soft - tissue traumatic injuries , the necessity of an alternative technique is felt . according to this study infiltration technique
this finding is consistent with the results of sharaf , jones et al . , and ram and peretz . in the study of sharaf with the use of sem scale
, it was revealed that block injection , especially in 3 - 7 year old children , was more painful than infiltration technique and caused negative behaviors .
although studied on a wider range of age ( 3 - 16 ) and used vas scale to evaluate the severity of pain , but showed that block injection was more painful than infiltration technique .
the present study showed no significant difference between the efficacy of block and infiltration induced anesthesia during injection and on pulpotomy .
in the studies of sharaf and donohue et al . who used lidocaine , although the 3 - 9 year old children were evaluated the same results were demonstrated .
used articaine 4% and tested the sensitivity of teeth by a pulp tester and reached the same results as the present study .
they revealed that the appearance of anesthesia in infiltration technique is earlier with the same efficacy .
the results of oulis et al . were different , although the efficacy of two techniques for restorative treatments of mandibular primary teeth was similar , but block technique was more efficient for pulpotomy .
the difference between the results of theirs and present studies might be related to the difference in type of teeth .
their study was on mandibular first and second primary molar teeth while this study was on mandibular first primary molar .
since block injection induces sustained anesthesia that is potentially traumatic to soft - tissues , it seems that infiltration anesthesia could be used as a suitable alternative technique for mandibular primary first molar pulpotomy . | background : the most common technique to anesthetize mandibular primary teeth is inferior alveolar ( i.a ) nerve block injection which induces a relatively sustained anesthesia and in turn may potentially traumatize soft - tissues .
therefore , the need of having an alternative technique of anesthesia with a shorter term but the same efficacy is reasonable .
the aim of this study was a comparison of the efficacy of two anesthetic techniques of mandibular primary first molar.materials and methods : in this randomized crossover clinical trial , 40 children with ages ranged from 5 years to 8 years whose mandibular primary first molars were eligible for pulpotomy , were selected and divided randomly into two groups .
the right and left mandibular first molars of group a were anesthetized with infiltration and i. a nerve block techniques in the first and second sessions respectively .
the left and right mandibular first molars of group b were anesthetized with i.a nerve block and infiltration techniques in the first and second sessions respectively .
the severity of pain were measured and recorded according to sound - eye - motor scale by a certain person .
data was analyzed using wilcoxon signed rank and mann - whitney u tests ( p > 0.05).results : the severity of pain was lower in infiltration technique versus i.a nerve block .
there were no significant differences between the severities of pain on pulpal exposure of two techniques.conclusion:it seems that infiltration technique is more favorable to anesthetize the mandibular primary first molar compared to i.a nerve block . |
lung cancer is one of the absolute priorities in oncology because of its mortality , incidence , social and economic impact .
the lack of clinical signs at the early stages and therapeutic inefficiency for advanced stages , lead to the evidence that early diagnosis should reduce lung cancer related mortality , as in early stages 70% of cases can be treated successfully with surgery .
this justifies the need to study the impact on survival of a lung cancer screening program with stronger motivation than those used for assessing and spreading the standard protocols for breast , prostate and colonic cancer screening .
opportunities for screening for lung cancer have been debated for years , but several studies have shown that conventional chest x - ray and sputum examination did not satisfy the primary criteria of a screening test [ 4 , 5 ] , until the value of low - dose spiral ct in detecting small parenchymal lesions was demonstrated by some reports [ 6 , 7 ] . the diagnostic imaging technique to be applied was stated in the first elcap report , which describes the ct tumour detection rate as four times higher than chest x - ray , and by studies demonstrating similar accuracy of low - dose spiral ct and conventional ct in detecting pulmonary nodules . while awaiting the new promising techniques of molecular biology ,
multidetector computed tomography [ 911 ] , with the use of low dose protocols , is nowadays widely recognized as the best technique for lung tumour screening .
the high accuracy , together with the low biological and economical costs , justify the success of the protocols for the early detection of lung cancer by low - dose spiral ct .
this is confirmed by the growing number of ongoing studies all over the world .
although the various screening campaigns sometimes apply different inclusion criteria , the epidemiologic data identify the high risk population in asymptomatic subjects of both genders , over 50 years of age , smokers or ex - smokers ( within the previous 1015 years ) of at least 20 cigarettes a day for at least 20 years . in order to reduce the cost / benefit ratio of the test
, lung cancer screening is based on the application of low - dose protocols to avoid exposure to excessive amounts of radiation .
the low - dose technique ensures good spatial resolution and high contrast resolution , and guarantees high sensitivity for the detection of lung nodules up to 2 mm in diameter [ 12 , 13 ] .
ct protocols differ according to different scanners : we report an example applied to lightspeed ct 16-rows ( general electric , milwaukee , wl , usa ) : thickness of acquisition 2.5 mm ; standard reconstruction filter ; 40 ma ; 140 kv ; rotation time 0.8 s ; speed 35 mm / rotation ; collimation 10 mm .
the introduction of multidetector ct , with the use of thin sections , resulted in an increase in the number of lung nodules detected [ 10 , 11 ] , thus making the definition of diagnostic algorithms very relevant .
the most important parameter to define the diagnostic work - up is the nodule diameter and its changes over time .
although there is no clinical evidence , it is widely accepted that lung nodules less than 5 mm in size can be safely checked at 1 year intervals : this follow - up schedule allows identification of early stage malignancies , while avoiding unnecessary anxiety and useless radiation in the majority of those being screened [ 14 , 16 ] .
the evaluation of the nodule characteristics ( solid , partially solid , non - solid ) , depending on the ability to obscure the lung parenchyma , and its morphologic appearance [ 1820 ] are important to determine the grade of suspicion .
apart from follow - up to evaluate volume doubling time , diagnosis can be reached by pet or pet / ct , ct enhancement with contrast media via i.v .
several authors have suggested different protocols for the management of lung nodules [ 2527 ] : we suggest the algorithm shown in
fig .
1 . the preliminary results of observational studies show high sensitivity of the technique in detecting lung nodules .
in the elcap report , 23% of patients had one or more non - calcified nodules ; the european institute of oncology study detected 284 nodules in 199/1035 subjects ( 19% ) .
a large number of these nodules were benign and the prevalence of lung cancer was 2.7% and 1.1% in the two series . at the second year
ct , the incidence of lung cancer was 0.59% and 1.1% [ 16 , 28 , 29 ] ( table 1 ) .
the results from the observation of the stage at diagnosis are encouraging : 81% of patients had a stage i tumour , with a good prognosis ( survival rate at 5 years of 63% for stage ia and 46% for stage ib ) .
epidemiologic data in the us show that the diagnosis at stage i is made in only 20% of cases not resulting from screening projects .
the percentage of invasive procedures performed for benign lesions ranges from 21% to 55% .
the high prevalence of benign nodules detected by ct and reported as false positive results is the major criticism against programs for early detection of lung cancer .
the use of a dimensional cut - off should reduce the number of false positives and the recall rate with no major impact on the cancer detection rate .
some authors state that the screening results are affected by an overdiagnosis bias , as ct reveals a significant percentage of non - aggressive tumours that would never cause the death of the patient , because of slow growth and other risk factors related to age and smoking habits .
this could explain the increase of survival in cancer patients enrolled in screening campaigns , without a corresponding reduction of mortality .
early diagnosis does not necessarily result in a reduction of mortality ; there are no definitive data on the outcome of the subjects enrolled in screening programs . at the end of their screening project at the mayo clinic , swenson and
colleagues did not demonstrate any significant difference in mortality when comparing their results with those from a study performed in the 1970s by conventional chest x - ray . our data on 1035 volunteers , enrolled in a screening project in 20002001 , with only 30 subjects lost to follow - up at 5 years , show 42 lung cancers detected , 9 deaths from lung cancer , 11 for other cancers , 7 for cardiovascular diseases and 6 for other causes , resulting in a mortality rate of 3.2% in 5 years .
the costs are extremely variable from one study to another , and data reported range from 2500 to 2,300,000$per year of saved life [ 3436 ] . to assess the real efficacy of ct in reducing lung cancer related mortality and to introduce this test into clinical practice , we are all waiting for the results of the randomized clinical studies recently started all over the world .
prevalence , incidence and percentage of stage i tumours detected by low - dose ct in published lung cancer survey studies | the lethality of lung cancer is related to the advanced stage at diagnosis .
initial studies have demonstrated that screening computed tomography ( ct ) is effective in diagnosing lung cancer at an earlier stage when compared with current clinical practice , however the best clinical approach for screening detected nodules has to be defined .
the population to be identified as high risk should be over 50 years of age and should have smoked at least one pack / day for 20 years .
ct protocols should use multidetector ct , low dose and a 2.5 reconstruction interval .
diagnostic work - up on detected nodules should be designed according to size and consider ct at 3 or 12 months to evaluate doubling time , ct enhancement , pet / ct and/or fnab or vats . the prevalence of lung cancer in the screened population is 1.1%2.7% , and the incidence is 0.2%1.1% .
eighty - one percent of cancers are diagnosed in stage i. the percentage of surgery performed for benign lesions ranges from 21% to 55% . in our series ,
the overall mortality rate was 3.2% in 5 years .
the results of randomized clinical studies , when available , will assess the real efficacy of ct in reducing lung cancer related mortality . |
the role of aggregate formation in the pathophysiology of huntington s disease ( hd ) remains uncertain .
however , the temporal appearance of aggregates tends to correlate with the onset of symptoms and the numbers of neuropil aggregates correlate with the progression of clinical disease .
using highly sensitive immunohistochemical methods we have detected the appearance of diffuse aggregates during embryonic development in the r6/2 and yac128 mouse models of hd .
these are initially seen in developing axonal tracts and appear to spread throughout the cerebrum in the early neonate . | |
the diagnosis in verrucous lesions of the oral cavity can range from verrucous hyperplasia to verrucous proliferative leukoplakia ( vpl ) , and verrucous carcinoma .
vpl is a distinct entity among these , and it has a high potential for malignant transformation . the present study evaluates the clinico - pathological features of verrucous lesions of the oral cavity and analyzes the treatment outcomes .
this is a retrospective study of 15 consecutive patients who presented with verrucous lesions of the oral cavity , during the 5-year period from january 2006 to december 2010 .
an institutional review board approval was obtained for this study . a prospectively maintained tumor board database : electronic medical records of outpatient clinic visits as well as inpatient records , operative notes , histopathology reports , and clinical photographs
fifteen patients with verrucous lesions of the oral cavity [ figure 1 ] presented during the study period .
eleven ( 73% ) of them were males and four ( 27% ) were females .
six patients had history of smoking , 11 chewed tobacco with or without areca nut , two had history of alcohol abuse , and four patients had multiple habits ( tobacco in different forms as well as alcohol ) .
nine cases presented with lesions in the buccal mucosa and five cases with lesions on the lateral border of the tongue .
mean size of the lesion was 3.1 cm in the greatest dimension ( range 15 cm ) .
two patients had clinically palpable ipsilateral level i cervical lymph nodes . at presentation , the preoperative biopsy was reported as verrucous carcinoma in seven patients and verrucous hyperplasia in eight patients .
verrucous lesion on lateral border tongue demographics and tumor characteristics all these patients were offered surgery as definitive treatment and adjuvant radiotherapy if necessary , depending on the nodal and margin status in the histopathological evaluation .
wide local excision of the primary lesion , with adequate mucosal and soft - tissue margins in all three dimensions , was carried out .
eleven cases could be resected via a per oral approach , while four cases were approached using a midline lip - split after raising the cheek flap , for better access to the posterior margin of the tumour in the posterior tongue and retromolar trigone regions .
a marginal mandibulectomy was done in four patients to get an adequate margin as the lesions were abutting the lower alveolus .
the indications for neck dissection in this series were clinically palpable nodes , invasive carcinoma at presentation , or tumor size more than 4 cm .
fasciocutaneous free - flaps , either anterolateral thigh or radial artery forearm flap , were used to cover the defect after ablative surgery in eight patients .
one case of oral tongue proliferative verrucous leukoplakia , where the margins were close , had to be taken for re - excision .
five out of eight verrucous hyperplasia , on final histopathology , were found to have an invasive component .
all patients remain loco - regionally controlled at the end of follow - up period .
all these patients were offered surgery as definitive treatment and adjuvant radiotherapy if necessary , depending on the nodal and margin status in the histopathological evaluation .
wide local excision of the primary lesion , with adequate mucosal and soft - tissue margins in all three dimensions , was carried out .
eleven cases could be resected via a per oral approach , while four cases were approached using a midline lip - split after raising the cheek flap , for better access to the posterior margin of the tumour in the posterior tongue and retromolar trigone regions .
a marginal mandibulectomy was done in four patients to get an adequate margin as the lesions were abutting the lower alveolus .
the indications for neck dissection in this series were clinically palpable nodes , invasive carcinoma at presentation , or tumor size more than 4 cm .
fasciocutaneous free - flaps , either anterolateral thigh or radial artery forearm flap , were used to cover the defect after ablative surgery in eight patients .
one case of oral tongue proliferative verrucous leukoplakia , where the margins were close , had to be taken for re - excision .
five out of eight verrucous hyperplasia , on final histopathology , were found to have an invasive component .
all patients remain loco - regionally controlled at the end of follow - up period .
verrucous hyperplasia [ figure 2 ] is a forerunner of verrucous carcinoma , and the transition is so consistent that the hyperplasia , once diagnosed , should be treated like verrucous carcinoma .
there is a slight female predilection with the sixth to eighth decades , the years of peak frequency .
these are followed , in order , by the buccal mucosa , tongue , floor of mouth , lip , and palate .
, in a study of 60 patients of oral verrucous hyperplasia concluded that the lesions occur more commonly on the buccal mucosa and are highly associated with the areca quid chewing and cigarette smoking habits .
occasionally , lesions with the general morphology of verrucous carcinoma may contain foci of invasive squamous cell carcinoma of varying grade .
verrucous hyperplasia : h and e 100 , section shows verrucous proliferation of epithelium with minimal atypia proliferative verrucous leukoplakia ( pvl ) [ figure 3 ] , defined by hansen and colleagues in 1985 , consists generally of proliferative , irregular white patches or plaques that progress slowly and multifocally on the oral mucous membrane .
pvl is a form of field cancerization in which the tissues that appear clinically normal progress through advanced stages to dysplasia , to culminate in some form of epithelial cancer .
pvl is more common in women than in men , with a peak incidence at 60 to 70 years of age .
no apparent link between human papilloma virus ( hpv ) and use of tobacco products has been firmly established with regard to pvl there is high degree of malignant transformation .
silverman and gorsky reported that 70.3 % of 54 patients developed a squamous cell carcinoma .
its aggressiveness relates not only to a high recurrence rate , but also to a relentless progression from a localized simple keratosis to extensive oral disease and squamous carcinomas of verrucous or conventional squamous cell type
, in a study of 19 patients have demonstrated the high frequency of osccs on many occasions manifesting several cancers at different oral locations , thus demonstrating the field cancerization of this entity .
oral proliferative verrucous leukoplakia : h and e 100 , section shows mucosal hyperkeratosis , acanthosis , and papillomatosis verrucous carcinoma [ figure 4 ] is a distinct low - grade variant of squamous cell carcinoma of the oral cavity . it was first identified as a clinical and histologic entity by ackermann in 1948 in a study of 31 patients .
it is a rare tumor representing only 34% of all oral carcinomas , with an annual incidence of one to three cases for every one million persons .
the tumor typically appears in the sixth decade of life and accounts for 28% of all squamous cell carcinoma .
it is characterized by a slow - growing , painless , broad - based verrucous or wart - like papillary lesion .
in contrast to oral squamous cell carcinoma , verrucous carcinoma does not metastasize regionally or distally .
rekha et al . , studied 133 cases of verrucous carcinoma and concluded that they accounted for 16.08% among oral squamous cell carcinoma .
walvekar et al . , have studied 101 cases of oral verrucous carcinoma and have found excellent prognosis with surgical management .
they have emphasized the surgical resection with adequate margins and need for close follow - up .
verrucous carcinoma : h and e 100 , section shows bulbous proliferation of edges , acanthosis , and hyperkeratosis .
the cells show intercellular bridges , vesicular bland nucleus , and prominent nucleoli , with abundant eosinophilic cytoplasm treatment modalities for verrucous lesions have included surgery , radiation therapy , chemotherapy , cryotherapy , laser therapy , photodynamic therapy , and treatment with recombinant alpha interferon . surgical excision remains the preferred treatment for the primary lesion .
femiano et al . , reported an open trial of surgery in 25 patients with oral hpv - positive pvl , compared with combined therapy using surgery and methisoprinol , a synthetic agent with immunomodulatory properties and some antiviral activity against hpv , in another group of 25 patients with oral pvl .
after months of follow - up , there were 18 recurrences in the group treated by surgery alone , compared with four in those also receiving methisoprinol .
lin et al . , has demonstrated the effectiveness of topical photodynamic therapy in oral verrucous hyperplasia .
, in a study of 39 patients have found that surgical treatment is effective in oral verrucous carcinoma .
walvekar et al . , have studied 101 cases of oral verrucous carcinoma and have found excellent prognosis with surgical management .
they have emphasized the surgical resection with adequate margins and need for close follow up .
five out of eight cases of verrucous hyperplasia in our series showed invasive carcinoma later .
free vascularized flaps were used for reconstruction in eight of our cases , as the defects after ablative surgery were large and deep .
a surgical excision with wide margins and an appropriate reconstruction is necessary to optimize the disease and functional outcome . | objective : verrucous lesions of the oral cavity can be of varied histopathology .
the present study evaluates the clinico - pathological features of verrucous lesions of the oral cavity and analyzes the treatment outcomes.materials and methods : this is a retrospective study of 15 consecutive patients who presented with verrucous lesions of the oral cavity , during the 5-year period from january 2006 to december 2010 .
demographic , clinico - pathological features , treatment details , and outcomes were analyzed.results:fifteen patients with verrucous lesions of the oral cavity were treated with surgery as the primary modality .
the mean age was 62.8 years ( range 3585 years ) .
wide excision of the primary lesion with adequate mucosal and soft - tissue margins was carried out .
free - flap reconstruction was done in eight patients .
all patients remain loco - regionally controlled with good functional speech and swallowing outcome.conclusions:verrucous lesions of the oral cavity are a distinct clinical entity with varied histopathology .
a surgical excision with wide margins and appropriate reconstruction is necessary to optimize the disease and functional outcome . |
aspergillus ochraceoroseus ( atcc 38873 ) and aspergillus rambellii ( atcc 42001 ) are two fungal organisms that were first characterized in 1978 ( bartoli and maggi 1978 ) .
both were named a. ochraceoroseus until a more thorough comparison , decades later , revealed differences which resulted in renaming one of the isolates a. rambellii ( frisvad et al . 2005 ) .
these two isolates are the sole representatives of their respective species ever sampled , because constant civil unrest in the ivory coast makes further sampling a dangerous task , and also because no isolates of either species have been found anywhere else ( klich et al .
these two species have not been found in agricultural or clinical environments , but they both secrete two important secondary metabolites ( sms ) : aflatoxin b1 ( afb1 ) and sterigmatocystin ( st ) .
afb1 is considered the most serious of the mycotoxins produced by any organism ( samuel et al .
( 2005 ) reported that a. rambellii secreted a greater concentration of afb1 than any other aflatoxigenic species .
large quantities of st are also secreted by both species . in many of the aspergillus species from subgenus circumdati , this less potent sm is most often an intermediate compound in the aflatoxin ( af ) pathway , serving as a precursor in the synthesis of b1 and g1 afs ( yu et al .
st has its own pathway of clustered genes in aspergilli , mostly from subgenus nidulantes , many of which are homologous to af cluster genes but organized differently ( ehrlich et al .
2005 ) . whether or not these organisms are the ancestors to modern - day af- and st - producers , or the result of hybridization or horizontal gene transfer ( hgt ) events between af and st species , has yet to be determined .
the genomes described here will allow for further studies that result in greater understanding of the evolution of aflatoxigenic aspergillus species .
the draft genome assembly for a. ochraceoroseus was 23.9 mb , composed of 2,644 contigs with an n50 of 330 and n50 length of 21,980 bp , and 48.48% g+c content .
the a. rambellii genome was larger ( 26.1 mb ) , composed of 3,102 contigs with an n50 of 206 and n50 length of 38,345 bp , and 46.16% g+c content .
ochraceoroseusgeneral assembly size ( bp)23,940,954 cegma % completeness92.74 average depth36 g+c ( % ) 48.9 protein - coding genes7,837 protein - coding genes > 100 amino acids7,631predicted protein - coding sequences > 100 amino acids gene density ( 1 gene every n bp)3,054 median gene length ( bp)1,799.9 mean gene length ( bp)1,502 average number of exons per gene3.34a .
rambelliigeneral assembly size ( bp)26,069,208 cegma % completeness95.97 average depth79 g+c ( % ) 46.2 protein - coding genes7,807 protein - coding genes > 100 amino acids7,566predicted protein - coding sequences > 100 amino acids gene density ( 1 gene every n bp)3,339 median gene length ( bp)1,826.6 mean gene length ( bp)1,517.5 average number of exons per gene3.37 genome characteristics for the aspergillus ochraceoroseus and aspergillus rambellii type strains the genomes of a. ochraceoroseus and a. rambellii were approximately 10 mb smaller , and appeared to encompass 3,0005,000 fewer predicted genes , than those for many other sequenced aspergillus species ( table 2 ) .
one species with the closest genome size and predicted gene counts to a. ochraceoroseus and a. rambellii was aspergillus fumigatus ( 29 mb and 9,296 , respectively ) .
one reason for a. ochraceoroseus and a. rambellii exhibiting smaller genome sizes likely related to the number of sm clusters found within these fungi .
for example , a. fumigatus contained 18 putative sm clusters and aspergillus nidulans contained 35 putative sm clusters , while species such as aspergillus flavus ( l - type ) and aspergillus nomius contained 45 and 53 sm clusters , respectively ( moore et al .
the a. ochraceoroseus genome contained 18 putative sm clusters and the a. rambellii genome contained 20 putative sm clusters .
it has been postulated that high genetic drift , coupled with deletions , in eukaryotes refines their genomes by removing regions that are not integral to the organism s survival ( kelkar and ochman 2012 ) . from an evolutionary standpoint
this could indicate that a. ochraceoroseus and a. rambellii represent a more streamlined genomic condition for aflatoxigenic aspergilli .
unfortunately , no other isolates have been sampled for either species to aid with population - scale inferences , so their geographic isolation coupled with their lack of ubiquity adds to their value .
rambellii267,807 genomic comparison of six aspergillus species the availability of whole - genome data could help us to understand the origins , or evolution , of af- and st - producing ( af+st ) fungi .
phylogenomic comparison of a. ochraceoroseus and a. rambellii , with other aspergillus species and the outgroup taxa penicillium chrysogenum , indicated that these two species share a most recent common ancestor with a. nidulans ( fig .
1 ) . even farther back in evolutionary time was their divergence from the af - producing species representing section flavi , preceded by divergence from a shared common ancestor with a. fumigatus ( a nonproducer of af or st ) .
sequencing the genomes of more aflatoxigenic species will improve our understanding of how the af or st chemotype has evolved .
this tree was inferred using multiple af and/or st producing aspergillus species ( a. flavus l , a. nidulans , a. nomius , a. ochraceoroseus , and a. rambellii ) and one af and st nonproducing species ( a. fumigatus ) , with penicillium chrysogenum as the outgroup taxa .
this tree was inferred using multiple af and/or st producing aspergillus species ( a. flavus l , a. nidulans , a. nomius , a. ochraceoroseus , and a. rambellii ) and one af and st nonproducing species ( a. fumigatus ) , with penicillium chrysogenum as the outgroup taxa .
potential recombination or hgt among fungi may cloud a species true lineage ; therefore , whole - genome comparisons offer a more holistic approach to lineage - based inferences .
previous single - locus phylogenies have shown the cladal relatedness of these st - producing fungi ( klich et al .
2005 ) , but the coproduction of afb1 by a. ochraceoroseus and a. rambellii could indicate that the distant common ancestor shared with the strictly aflatoxigenic species may have been able to secrete both sms , and that this ability was lost in a. nidulans at the time of divergence .
coalescence analysis of species containing the st pathway may help to determine whether or not the ancestor of modern - day st producers could also produce afb1 .
an alternative scenario could be hgt of one or more genes , that allow for further synthesis of the st intermediate to afb1 , from an aflatoxigenic species into these af+st producing species .
further research is required to confirm or refute the hgt scenario . with regard to the af+st biosynthesis pathway , clustered genes in a. ochraceoroseus and a. rambellii share 99.4% identity and the same orientation .
they found it to contain the same genes as the st cluster of a. nidulans , but with a portion of the cluster ( afln to aflw ) having reversed orientation ( cary et al .
the basis for afb1 synthesis in a. ochraceoroseus was unknown because two of the genes required for conversion of st to afb1 ( aflp and aflq ) were not found within its af+st gene cluster .
recently , however , an ortholog of the aflq gene was found to exist within the a. ochraceoroseus genome ( cary et al .
blast query of the a. ochraceoroseus aflq ortholog to the a. rambellii genome sequence revealed a putative aflq ortholog sharing 99.95% identity .
phylogenetic analysis involving the aflq gene from multiple af and st producing fungi revealed the ortholog in a. ochraceoroseus to be most similar to the most recent common ancestor than those of af- or st - producing species ( cary et al .
2012 ) , which supports the existence of an ancestor that was able to produce and secrete both toxic sms .
previous research reported a possible heterothallic existence for a. ochraceoroseus and a. rambellii , with each species containing a single mating - type ( mat ) idiomorph ( cary et al . 2012 ) .
our findings from sequencing the genomes of these closely related species confirmed that a. ochraceoroseus contains a single ( mat1 - 1 ) gene and a. rambellii contains a single ( mat1 - 2 ) gene .
the other heterothallic aspergillus species such as a. flavus and a. parasiticus have a mat gene flanked by two conserved genes in close proximity : one for dna lyase ( apn ) and one for cytoskeleton assembly control ( sla ) .
the relative distances of these genes to the mat1 - 1 gene in a. flavus and a. parasiticus are approximately 2,500 bp and 2,000 bp , respectively ( ramirez - prado et al .
for the a. ochraceoroseus mat1 - 1 gene , there are over 51,000 bp of nucleotide sequence between it and the apn gene , and 2,300 bp of nucleotide sequence separating it from the sla gene .
the distances between each of the apn and sla genes and the mat1 - 2 gene in a. rambellii measured over 33,000 bp and 3,600 bp , respectively .
the chromosomal location of the mat locus in a. flavus and a. parasiticus is reported to be chromosome vi ( ramirez - prado et al .
2008 ) , but this has not yet been determined for a. ochraceoroseus and a. rambellii .
if the pathway cluster genes within these two af+st producing species are reflections of an ancestral organization that predates the divergence of af and st producers , then perhaps their mat loci are also examples of a more relaxed [ ancestral ] organization and could impact their ability to outcross .
there are conflicting theories about which form of sex ( heterothallism vs. homothallism ) is ancestral in fungi ( ni et al .
2011 ) . if a. ochraceoroseus and a. rambellii are indeed reflective of an ancestor that produced afb1 and st , and that contained a more disorganized heterothallic mating type locus , then perhaps these species support the argument that heterothallism is ancestral to homothallism
we sequenced the genomes of the a. ochraceoroseus and a. rambellii type strains species using a personal genome machine ( pgm ) from life technologies ( grand island , new york ) .
template preparation and sequencing was conducted according to previously reported protocols ( moore et al .
totals of 2.92 m and 7.43 m reads were obtained for a. ochraceoroseus and a. rambellii , respectively .
maker was then used to integrate ab initio gene predictions with protein homology evidence from the uniref50 protein database ( ftp://ftp.ncbi.nlm.nih.gov/refseq/release/fungi/ , last accessed july 1 , 2014 ) for a. rambellii , and the ncbi fungal refseq database ( ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref50/uniref50.fasta.gz , last accessed july 1 , 2014 ) for a. ochraceoroseus .
the annotations were converted to ncbi submission format using genome annotation generator [ https://github.com/genomeannotation/gag , last accessed october 14 , 2015 ] and deposited at ncbi under biosample project numbers prjna275128 ( a. ochraceoroseus ) and prjna275129 ( a. rambellii ) .
the antibiotics - secondary metabolite analysis shell ( anti - smash ) program was used to predict sm clusters in a. ochraceoroseus and a. rambellii ( medema et al .
this program is modeled to look for many types of backbone genes , but our estimates accounted for the presence of only three types : polyketide synthase ( pks ) , nonribosomal peptide synthetase ( nrps ) , and pks - nrps hybrid .
phylogenomic analysis involved protein orthologs , from multiple aspergillus species that were identified by proteinortho , and was performed using similar techniques as discussed in moore et al .
penicillium chrysogenum was used as the outgroup taxa and we inferred the evolutionary relationships of a. ochraceoroseus and a. rambellii with two aflatoxigenic species from section flavi ( a. flavus l and a. nomius ) , an st producer from section nidulantes ( a. nidulans ) and a. fumigatus .
the protein sequences for a. nidulans , a. flavus , and a. fumigatus were retrieved from fungidb ( accessed august 28 , 2014 ) .
the a. nomius protein sequences were obtained from ncbi ( last accessed august 28 , 2014 ) .
the mat locus comparisons were performed by blast query of a. flavus mat , apn , and sla genes to the a. ochraceoroseus and a. rambellii genomes .
distance mapping between the mat genes and each flanking gene were performed using sequencher software ( gene codes corporation , ann arbor , mi ) .
we sequenced the genomes of the a. ochraceoroseus and a. rambellii type strains species using a personal genome machine ( pgm ) from life technologies ( grand island , new york ) .
template preparation and sequencing was conducted according to previously reported protocols ( moore et al .
totals of 2.92 m and 7.43 m reads were obtained for a. ochraceoroseus and a. rambellii , respectively .
maker was then used to integrate ab initio gene predictions with protein homology evidence from the uniref50 protein database ( ftp://ftp.ncbi.nlm.nih.gov/refseq/release/fungi/ , last accessed july 1 , 2014 ) for a. rambellii , and the ncbi fungal refseq database ( ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref50/uniref50.fasta.gz , last accessed july 1 , 2014 ) for a. ochraceoroseus .
the annotations were converted to ncbi submission format using genome annotation generator [ https://github.com/genomeannotation/gag , last accessed october 14 , 2015 ] and deposited at ncbi under biosample project numbers prjna275128 ( a. ochraceoroseus ) and prjna275129 ( a. rambellii ) .
the antibiotics - secondary metabolite analysis shell ( anti - smash ) program was used to predict sm clusters in a. ochraceoroseus and a. rambellii ( medema et al . 2011 ) .
this program is modeled to look for many types of backbone genes , but our estimates accounted for the presence of only three types : polyketide synthase ( pks ) , nonribosomal peptide synthetase ( nrps ) , and pks - nrps hybrid .
phylogenomic analysis involved protein orthologs , from multiple aspergillus species that were identified by proteinortho , and was performed using similar techniques as discussed in moore et al .
penicillium chrysogenum was used as the outgroup taxa and we inferred the evolutionary relationships of a. ochraceoroseus and a. rambellii with two aflatoxigenic species from section flavi ( a. flavus l and a. nomius ) , an st producer from section nidulantes ( a. nidulans ) and a. fumigatus .
the protein sequences for a. nidulans , a. flavus , and a. fumigatus were retrieved from fungidb ( accessed august 28 , 2014 ) .
the a. nomius protein sequences were obtained from ncbi ( last accessed august 28 , 2014 ) .
the mat locus comparisons were performed by blast query of a. flavus mat , apn , and sla genes to the a. ochraceoroseus and a. rambellii genomes .
distance mapping between the mat genes and each flanking gene were performed using sequencher software ( gene codes corporation , ann arbor , mi ) . | aspergillus ochraceoroseus and aspergillus rambellii were isolated from soil detritus in ta national park , ivory coast , africa .
the type strain for each species happens to be the only representative ever sampled .
both species secrete copious amounts of aflatoxin b1 and sterigmatocystin , because each of their genomes contains clustered genes for biosynthesis of these mycotoxins .
we sequenced their genomes using a personal genome machine and found them to be smaller in size ( a. ochraceoroseus = 23.9 mb and a. rambellii = 26.1 mb ) , as well as in numbers of predicted genes ( 7,837 and 7,807 , respectively ) , compared to other sequenced aspergilli .
our findings also showed that the a. ochraceoroseus type strain contains a single mat1 - 1 gene , while the type strain of a. rambellii contains a single mat1 - 2 gene , indicating that these species are heterothallic ( self - infertile ) .
these draft genomes will be useful for understanding the genes and pathways necessary for the cosynthesis of these two toxic secondary metabolites as well as the evolution of these pathways in aflatoxigenic fungi . |
rosacea is a persistent and recurrent inflammatory condition of the middle face as a rule .
classical morphology rosacea frequent subtypes are the erythematotelangiectatic ( et ) rosacea and the papulopustular ( pp ) rosacea , while the other anatomoclinical forms are the granulomatous ( or lupoid ) rosacea , the phymatous rosacea or rhinophyma , and rosacea fulminans or pyoderma faciale . in general , a persistent stasis erythema may precede by years the development and establishment of either an et form or a pp form .
rosacea may appear in any immunocompetent patient and has to be differentiated from demodicosis of either immunocompetent or immunodeficient patients .
semeiology , symptomatology , and pathologic features of rosacea suggest that its pathogenesis is mediated by sensory nerves via antidromically released neuromediators , therefore constituting a model of neurogenic inflammation .
after emotional stimulation or ingestion of alcohol , catecholamine is released from gastric mucosa or adrenal medulla and kallikrein is secreted by salivary glands , increasing bradykinin levels in blood .
substance p ( sp ) is a neuropeptide , secreted by sensory neurons of human epidermis and dermal papillae .
sp biologic functions consist in local axon reflex vasodilatation , acceleration of epidermal and endothelial cell proliferation , mast cell degranulation , antidromic vasodilatation , and liberation of inflammation mediators from macrophages and t lymphocytes .
biopsies on papular lesion and healthy skin of volunteer rosacea patients have shown that , in genetically predisposed persons , sp vasodilatory activity may be involved in the induction and/or maintenance of pp rosacea .
vessel dilatation in rosacea is not due to yielding to damaged connective tissue but to fusion of vessels by a gradual breakdown of adjacent vascular walls .
vascular endothelial growth factor ( vegf ) staining on skin sections has detected no differences between pp rosacea and et rosacea .
laser doppler imager has detected elevated blood flow in affected skin compared with nonaffected skin of the same patient in the pp form but not in the et form of rosacea .
normal skin demodex folliculorum ( a head sebaceous follicle lumen mite ) , usually located at the upper part of the infundibulum of the follicle , has been indirectly involved in rosacea , although a possible causal correlation has not been established up to this day .
the majority of rosacea medications do not affect demodex but do improve rosacea , while treatment with lindane , which is effective on the mite , does not improve the condition .
density of demodex was demonstrated to be significantly higher in the pp form of rosacea , although no correlation was ascertained between demodex intrafollicular inflammation and perifollicular inflammation .
possibly increased blood flow in rosacea skin dilated vessels constitutes an ideal condition for dermis demodex multiplication .
demodex folliculorum antigens reacting with rosacea patients ' sera have been considered to be responsible for stimulating mononuclear cell proliferation .
the number of mast cells ( which promote vasodilatation , angiogenesis , and fibrosis ) was significantly higher in lesional than in nonlesional skin of patients with rosacea .
all things considered , it is evident that neuropeptides such as sp and/or bradykinin may play a decisive role in increasing blood flow and/or vascular density in rosacea .
consequently , it looked reasonable to explore the involvement of single nucleotide polymorphisms ( snps ) within neuromediators and their receptors in rosacea .
tachykinins ( tacs ) are characterized by pharmacological similarity to their endogenous inflammatory mediator , bradykinin , yet tacs are not of similar structure to that of bradykinin .
heparin - initiated bradykinin formation plays an important role in mast cell - mediated diseases , such as rosacea .
tacs are a family of closely related peptides such as sp , neurokinin a , and neurokinin b , which act as neuropeptides in nonneuronal cells and noninnervated tissues . biological actions of tac1 and tac3 are mediated by three types of g protein - coupled receptors ( tacr1 , tacr2 , and tacr3 ) .
tacr3 or nk - r3 or neurokinin b receptor belongs to a family of genes which function as receptors for tacs , characterized by interaction with g proteins , variations in the 5-end of the sequence , and 7-hydrophobic transmembrane regions interactions .
tac1 gene is localized on chromosome 7q21-q22 , tac3 is localized on 12q13-q21 , tacr1 is localized on 2p12 , tacr2 is localized on 10q11-q21 , and tacr3 is localized on 4q25 .
circulating neurokinin b is elevated in women with preeclampsia during the third trimester of pregnancy .
the increased placental expression of tac3 gene belongs to the mechanism responsible for the elevated circulating neurokinin b levels in preeclampsia . in preeclampsia
complicated pregnancies , elevated neurokinin b plasma levels have been associated with elevated nitric oxide metabolite levels possibly in a compensatory mechanism to improve blood flow to the uteroplacental unit . in familial hypogonadotropic hypogonadism ,
mutations of neurokinin b gene and its respective receptor tacr3 suggested an eventual key role for neurokinin b in the central control of reproduction .
neurokinin b ( endogenous ligand of tacr3 ) is involved in pathogenesis of parkinson 's disease by interacting with brain dopaminergic transmission .
kisspeptin / neurokinin b / dynorphin - expressing neurons of hypothalamus , promoting skin vasodilatation , participate in the estrogen modulation of the body temperature and contribute to the induction of ensuing hot flushes in menopausal women ; such flushes are clinically similar to those experienced in rosacea .
the role of mast cells in tachykinin neurogenic skin inflammation induction has been shown and thus their intense presence in pp rosacea [ 21 , 22 ] may be explained .
we chose to study the tacr3 gene polymorphism rs3733631 , because tacr3 endogenous ligand , neurokinin b , by increasing blood flow ( anatomoclinical feature of rosacea ) , is involved in the pathophysiology of ( a ) parkinsonism ( frequently coexists with rosacea ) and ( b ) hot flushes in menopausal women ( clinically similar to rosacea vasomotor crises ) .
possible involvement of the tacr3 gene in rosacea would most probably be associated with either upregulation or downregulation of this gene .
snps such as rs3733631 , localized within the promoter area , upstream from the coding region , make prime candidates to reflect any association between gene and disease .
certain additional tacr3 polymorphisms , such as a29v , g59e , s455 g , a449s , w275x , and rs4580655 , have been associated with idiopathic central pubertal disorders [ 23 , 24 ] , predicting and improving learning and memory in aged organism , alcohol and cocaine dependence , and pediatric slow transit constipation .
one hundred and twenty - eight patients , examined at the outpatient clinic of the university department of dermatology at the university hospital of alexandroupolis , 53 males ( 41.4% ) and 75 females ( 58.6% ) , were included in this study .
patients ' age ranged from 14 to 86 years with a median age of 56.25 16.35 years .
all patients were clinically diagnosed by the same physician and suffered from either the et form ( n = 67 ) or the pp form ( n = 61 ) of central face rosacea .
rosacea patients with accompanying disease were excluded from the study to avoid possible interactions with potentially coexisting genetically determined disorders .
one hundred and twenty - one unrelated subjects , free from rosacea , not suffering from any potentially genetically determined disorder , individually matched to patients by both gender and age , were recruited as controls [ 54 ( 44.6% ) males and 67 ( 55.4% ) females ] . there were no significant differences in gender ( p = 0.608 ) and age ( p = 0.783 ) between patients and controls ( table 1 ) .
we genotyped the tacr3 gene polymorphism rs3733631 c / g by pcr - rflp as follows : primers f : 5-cttgcagcgaatgaatgaaa-3 and r : 5-gggtaatcgagtcacatcagg-3 generated a 216p long amplicon after 40 cycles of pcr at 95 30 , 55 30 , and 72 30. the pcr product was subsequently digested with restriction enzyme haeiii which generates three bands 107 , 86 , and 21 bp for allele g and two bands 128 and 86 bp for allele c. restriction digests were analyzed by electrophoresis in 2.5% agarose gel .
statistical analysis of the data was performed using the statistical package for the social sciences ( spss ) ( inc , chicago , il , usa ) .
the chi - square test was used to assess differences in genotype and allele frequencies between patients suffering from rosacea and matched controls .
it was also used to compare the observed frequency of each genotype with that expected for a population in the hardy - weinberg equilibrium .
multivariate unconditional logistic regression analysis was used to estimate age - adjusted odd rations ( or ) and 95% confidence intervals ( ci ) as the measure of association of the studied polymorphism with the appearance of rosacea .
all tests were two - tailed and statistical significance was considered for values less than 0.05 .
the distribution of genotypes and alleles in patients and healthy controls is shown in table 2 .
c / c , c / g , and g / g genotypes were found in 84.4% , 13.3% , and 2.3% of patients and in 87.6% , 11.6% , and 0.8% of healthy controls , respectively .
the genotype distribution was in hardy - weinberg equilibrium in controls ( = 0.481 , df = 1 , and p = 0.488 ) but not in patients ( = 4.512 , df = 1 , and p = 0.034 ) .
no statistically significant differences were detected in either genotype or allele frequencies between the two groups ( p = 0.433 and p = 0.295 , resp . ) .
however , a trend of higher frequency of g carriers ( g / g or c / g genotype ) and g allele was substantiated within males ( 22.7% versus 13.0% , p = 0.139 for g / g , g / c genotypes , and 14.2% versus 6.5% , p = 0.071 for the g allele ) ( table 2 ) . more specifically , in male patients , an increased ( but not statistically significant ) risk of rosacea incidence was associated with g allele ( or , 2.38 ; 95% ci , 0.936.11 ) .
further stratification for the clinical form of rosacea in each patient suggested a statistically significant predominance of g carriers suffering from pp form of rosacea versus controls ( 70.5% c / c , 26.2% c / g , and 3.3% g / g in patients versus 87.7% c / c , 11.6% c / g , and 0.8% g / g in controls ) as well as a higher frequency of the allele g ( 83.6% c and 16.4% g in patients versus 93.4% c and 6.6% g in controls ) ( tables 3(a ) and 3(b ) ) .
therefore , the frequency of c / g or g / g genotype on one hand is statistically significantly higher in pp rosacea patients ( p = 0.006 ) and g allele is also more frequent in pp patients ( p = 0.004 , table 3(b ) ) .
consequently , a statistically significant increased risk of pp rosacea incidence was associated with g allele ( or , 2.77 ; 95% ci , 1.385.57 ) . in patients with et rosacea ,
the predominance of g allele was not significant ( p = 0.087 ) but the higher frequency of c / g or g / g genotype in patients was closer to being statistically significant ( p = 0.052 ) ( table 3(a ) ) .
regarding the distribution of genotypes in pp rosacea males , the higher frequency of c / g or g / g genotype in patients was statistically significant ( p = 0.021 ) and the frequency of g allele was significantly higher ( p = 0.008 , tables 3(a ) and 3(b ) ) .
genetic polymorphisms have not been previously studied extensively in rosacea . specifically , in one study of bsm1 polymorphism of the vitamin d receptor ( vdr ) , the frequency of allele 1 was increased in patients with rosacea fulminans , but that increase was not statistically significant .
another study showed a statistically significant relationship between both null combination of glutathione - s - transferase m1 ( gctm1 ) and gstt1 genotype polymorphisms and rosacea , in the setting of a comparative genetic study , in a group of rosacea patients and a group of matched healthy controls .
a recently published genome - wide association study reported correlation of rosacea with hla - drb1 , hla - dqb1 , and hla - dqa1 as well as rs763035 , providing evidence regarding contribution of genetic component to pathogenesis of rosacea . in our study
we observed a trend ( p = 0.071 ) within male patients to carry g allele of rs3733631 with higher frequency than controls regardless of the type of rosacea .
there have been no previous studies in the literature reporting association or not of rs3733631 tacr3 gene polymorphism with rosacea ; however , it has been studied in alcohol and cocaine dependence .
the toll - like receptor 2 gene ( tlr2 ) is located at 4q32 , adjacent to tacr3 at 4q25 . in pp rosacea , serine protease kallikrein-5 ( klk5 ) and two abnormal cathelicidin peptides ( ll-37 and fa-29 ) are upregulated in the skin and induce erythema and vascular dilatation when injected in mouse skin .
ll-37 is characterized by lower antimicrobial activity than normal smaller cathelicidin in healthy skin and it promotes angiogenesis and consequently secondary inflammation .
it is possible that the polymorphism at the adjacent tacr3 may be somehow involved in increasing expression of tlr2 in rosacea and therefore in upregulation of klk5 in a calcium - dependent manner ; possibly pp rosacea patients overreact , resulting in the histogenesis of rosacea papules and pustules , even though bacterial diversity and quantities are similar in rosacea lesions and in normal skin .
tlr2 stimulation occurs only as a response to specific triggers , such as emotional stimulation or ingestion of alcohol and kallikrein , secreted by salivary glands , increasing vasodilatory bradykinin blood levels [ 5 , 33 ] .
the vitamin d - dependent amplification mechanism might induce increase of tlr2 susceptibility , as 1,25 ( oh)2 vitamin d gene polymorphism has been correlated with the intensity of inflammation .
this gene participates in the n - terminal caspase recruitment ( nact ) protein domain family , being involved in the response of toll - like receptors transduced inflammatory stimuli .
our study suggested a statistically significant predominance of c / g or g / g genotype ( and g allele ) in patients suffering from pp rosacea versus matched healthy controls .
the number of patients included in our study constitutes a relatively small size sample ; additional studies are needed to confirm our reported results .
pp rosacea is characterized by elevated blood flow and increased sp vasodilatory activity in affected skin .
genotypes carrying g allele may be accompanied by alterations of kisspeptin / neurokinin b / dynorphin ( kndy ) neurons of the hypothalamus and these altered neurons ( via projections to rostral hypothalamic structures such as the medial preoptic area and medial preoptic nucleus which control thermoregulatory effectors ) could maximize the vasodilatation of the skin as well as the inflammation ( induced by vasodilatation ) , resulting in development of pp rosacea [ 6 , 20 , 33 ] .
genotypes carrying g allele may provide a genetic predisposition to pp rosacea and not to the et form .
therefore , pp rosacea may be characterized by a genetic uniqueness , posing the possibility of its nosological distraction from the et form . | background .
rosacea is a chronic skin disease , possibly following the neurogenic skin inflammation model .
neurokinin b , involved in the pathogenesis of parkinson 's disease , frequently coexisting with subsequent onset of rosacea , is an endogenous ligand of the tachykinin receptor 3 ( tacr3 )
. methods .
128 rosacea patients and 121 matched controls were genotyped for rs3733631 by pcr - rflp and analyzed by chi - square test .
results .
we observed statistically significant predominance of the c / g or g / g genotype ( p = 0.006 ) and of the g allele ( p = 0.004 ) in the papulopustular ( pp ) form of rosacea and statistically marginal significance of the c / g or g / g genotype in erythematotelangiectatic ( et ) rosacea ( p = 0.052 ) .
significantly higher frequency of the c / g or g / g genotype and g allele in pp rosacea ( p = 0.021 and p = 0.008 , resp . ) was ascertained within male patients .
conclusion .
tacr3 rs3733631 g allele possibly predisposes the evolution of the initial phase of rosacea to the pp and not the et form in male patients . |
in normal human epidermis , each melanocyte associates with approximately 35 keratinocytes to form an
the keratinocytes in each unit exert considerable control over the behavior of the associated melanocyte , via interactions of cell - cell adhesion molecules and release of paracrine factors . ultraviolet radiation ( uvr )
uvr exposure enhances keratinocyte production of a wide variety of paracrine factors , including interleukins , growth factors , interferons , and chemokines , that may profoundly affect melanocyte and melanoma cell proliferation , migration , and gene expression [ 14 ] .
within the cell , s100 proteins , in the form of heterodimers or homodimers , control the localization and activity of a variety of target proteins ; however , the s100 proteins s100a8 and a9 are also secreted from cells as calprotectin , a heterodimeric proinflammatory cytokine .
calprotectin exerts its effects by binding to a variety of receptors on the surface of target cells , including toll - like receptor 4 ( tlr4 ) , the receptor for advanced glycation end - products ( rage ) , and extracellular matrix metalloproteinase inducer ( emmprin ) [ 57 ] .
s100a8 and a9 are expressed at extremely low levels in unperturbed keratinocytes , but their expression is readily stimulated by a variety of insults , including uvr exposure .
calprotectin is chemotactic for leukocytes and keratinocytes ; thus , it stimulates keratinocyte motility and recruits inflammatory cells to the skin .
melanocytes and melanoma cells themselves do not appear to express significant levels of s100a8 and a9 , although they do express the calprotectin receptors rage , tlr4 , and emmprin [ 7 , 11 , 12 ] .
based on these observations , we investigated a possible role for calprotectin in the paracrine activation of melanocytes and melanoma cells .
normal human melanocytes ( nhm ) and normal human keratinocytes of neonatal origin were obtained from the american type culture collection ( manassas , va ) and were maintained in dermal basal medium supplemented with a melanocyte or keratinocyte growth kit ( atcc ) as appropriate . the wc62 melanoma cell line ( coriell institute , camden , nj ) , derived from the primary melanoma of a patient with metastatic disease , was grown in eagle 's modified essential medium ( atcc ) supplemented with 10% normal calf serum ( thermo scientific , hudson , nj ) .
cells grown on coverslips were fixed briefly in paraformaldehyde and nonspecific binding was blocked with 5% bsa in tris - buffered saline containing 0.05% tween .
coverslips were incubated with a rabbit polyclonal anti - rage primary antibody ( abcam , cambridge , ma ) diluted in blocking buffer for 2 hours at room temperature and then with alexa 488-labeled goat anti - rabbit fab secondary antibody ( life technologies , grand island , ny ) for 1 hour in the dark at room temperature .
control slides were incubated with normal goat igg ( r&d systems , minneapolis , mn ) at a protein concentration equivalent to the primary antibody before addition of secondary antibody .
cells were examined by confocal microscopy . to validate expression of rage , mrna was isolated from normal human keratinocytes , nhm , and wc62 cells using the qiagen rneasy kit followed by qiashredder treatment ( qiagen , valencia , ca ) .
after reverse transcription and dnase treatment , rage and gapdh mrna levels were determined using taqman gene expression assays ( hs00542584_g1 and hs02758991_g1 , life technologies , grand island , ny ) as directed together with taqman universal master mix ( 4304437 , applied biosystems ) .
amplifications were performed on a 7900ht real - time pcr analyzer ( applied biosystems ) .
relative gene expression was calculated using the comparative c(t ) method , using gapdh expression to calculate delta ct and determining fold change compared to expression in normal human keratinocytes . to assay the effect of calprotectin on melanocyte and melanoma cell proliferation
, we treated the cells with calprotectin , using recombinant s100a8 and s100a9 ( abnova , taiwan ) allowed to dimerize in vitro .
cells were seeded at 15 10 cells per well in 96-well dishes in standard medium or medium containing calprotectin at a final concentration of 100 pg / ml s100a8 and 1 ng / ml s100a9 . on the succeeding 5 days , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide ( mtt ) ( sigma , st .
louis , mo ) dissolved in freshly prepared 0.1 n hcl in anhydrous isopropanol at a concentration of 5 mg / ml was added and cells were incubated for 3 hours .
absorbance was measured at 570 nm . to evaluate the effect of calprotectin on migration
, cells were plated into uncoated or matrigel - coated transwell chambers ( becton , dickinson and company biosystems , bedford , ma ) .
lower chambers were filled with medium alone or with medium containing recombinant s100a8 and s100a9 .
after 24 hours , upper chambers were cleaned of remaining cells and membranes were fixed and stained to visualize migrating cells ( diffquick kit , sigma , st .
dye was extracted from transwell membranes using methanol and absorbance measured with a spectrophotometer at 550 nm . all migration assays were conducted in the presence of 10 g / ml mitomycin c ( sigma , st .
as shown in figure 1(a ) , both cell types expressed detectable levels of surface rage , the canonical calprotectin receptor , in agreement with previous reports .
expression of rage mrna was confirmed by qrt - pcr ( figure 1(b ) ) .
compared to normal human keratinocytes , nhm expressed more than twice the level and wc62 cells expressed a comparable level of rage mrna .
both immunofluorescence and qrt - pcr results suggest higher expression of rage in nhm than in wc62 cells . as shown in figure 2(a ) , a significant difference in cell number between control and calprotectin - treated cells appeared at 4 days of treatment in nhm and at 2 days of treatment in wc62 cells .
calprotectin stimulated a maximum increase in cell number of 1.8-fold in nhm and 2.5-fold in wc62 cells compared to control cells .
movement through untreated membranes ( migration ) and through matrigel - coated membranes ( invasion ) was significantly enhanced by 2- to 3-fold in both melanocytes and melanoma cells treated with calprotectin compared to control cells ( figure 2(b ) ) .
it is important to note that these studies do not indicate that rage is the calprotectin receptor responsible for the effect of calprotectin on nhm and wc62 melanoma cell proliferation and migration , only that these cells express at least one calprotectin receptor .
another study has shown that overexpression of rage in a human melanoma cell line is associated with not only increased migration but also reduced proliferation in contrast with our study , .
other calprotectin receptors , including tlr4 and emmprin , may play important roles in mediating calprotectin effects on cells of melanocytic origin .
indeed , a recent study demonstrates that s100a9 , probably also calprotectin , is a ligand for emmprin and that emmprin overexpression enhances and emmprin blockade suppresses the migration of melanoma cell lines in response to s100a9 treatment .
however , it does not appear that emmprin is expressed at appreciable levels on normal human melanocytes .
it is clear from these studies that exogenous calprotectin can activate melanocytes and melanoma cells to proliferate and to migrate .
thus , calprotectin appears to be one of the numerous paracrine factors released by uvr - exposed keratinocytes that may promote melanomagenesis .
blocking the induction , release , or activity of calprotectin may thus represent a potential preventative or therapeutic strategy for melanoma .
tlrs and rages , receptors for calprotectin and for a variety of other ligands , are being considered as therapeutic targets for a wide array of diseases , including sepsis , asthma , and diabetes [ 19 , 20 ] . a number of approaches to blocking signaling through these receptors are under investigation and may prove valuable in preventing or treating melanoma
. however , further studies are clearly needed to determine the importance of calprotectin in melanoma development and progression and the therapeutic benefit of blocking its activity .
we have shown that melanocytes and melanoma cells express rage , the canonical calprotectin receptor , and that calprotectin stimulates these cells to proliferate and to migrate .
because calprotectin activates melanocytes and melanoma cells , it is a potential target for intervention in melanomagenesis . | calprotectin , a heterodimer of s100a8 and s100a9 , is a proinflammatory cytokine released from ultraviolet radiation - exposed keratinocytes .
calprotectin binds to toll - like receptor 4 , the receptor for advanced glycation end - products , and extracellular matrix metalloproteinase inducer on target cells to stimulate migration .
melanocytes and melanoma cells produce little if any calprotectin , but they do express receptors for the cytokine .
thus , keratinocyte - derived calprotectin has the potential to activate melanocytes and melanoma cells within the epidermis in a paracrine manner .
we examined the ability of calprotectin to stimulate proliferation and migration in normal human melanocytes and melanoma cells in vitro .
we first showed , by immunofluorescence and quantitative rt - pcr , that the melanocytic cells employed expressed a calprotectin receptor , the receptor for advanced end - products .
we then demonstrated that calprotectin significantly enhanced proliferation , migration , and matrigel invasion in both normal human melanocytes and melanoma cells .
thus , calprotectin is one of the numerous paracrine factors released by ultraviolet radiation - exposed keratinocytes that may promote melanomagenesis and is a potential target for melanoma prevention or therapy . |
idiopathic adulthood ductopenia ( iad ) was first proposed as a condition characterized by chronic cholestasis by ludwig et al . in 1988 .
currently , the diagnosis of iad is considered with adult or adolescent onset , biochemical evidence of cholestatic liver disease , evidence of ductopenia by liver biopsy with normal cholangiography , and an unknown etiology .
a diagnosis of iad requires exclusion of other conditions with chronic cholestasis that are associated with ductopenia [ 1 - 3 ] . to date , less than 100 cases have been reported in the medical literature , with only 1 prior case in korea .
we report a case of idiopathic adulthood ductopenia with a brief review of the medical literature .
a 19-year - old man who had abnormal liver function tests ( lfts ) was referred to the outpatient clinic .
four years and 1 month earlier , abnormal lfts were detected repeatedly , but the patient remained asymptomatic .
he had no history of neonatal or infantile liver disease , previous drug treatments , jaundice , itching , or abdominal pain .
laboratory tests revealed the following findings : hemoglobin 14.5 g / dl , white blood count 7090/mm , platelets 376,000/mm , alanine aminotransferase ( alt ) 147 iu / l , aspartate aminotransferase ( ast ) 123 iu / l , alkaline phosphatase ( alp ) 1345 iu / l , -glutamyl transpeptidase ( ggt ) 576 iu / l , total bilirubin 0.9 mg / dl and albumin 4.0 g / dl .
serum iron , transferrin saturation , ferritin , alpha 1-antitrypsin , and ceruloplasmin were normal .
serological tests for anti - smooth muscle , anti - neutrophil - cytoplasm , anti - nuclear , anti - mitochondrial , anti - liver - kidney microsome antibodies , hepatitis b surface antigen , and hepatitis c virus serology were negative .
an endoscopic retrograde cholangiopancreatography ( ercp ) demonstrated completely normal extrahepatic bile ducts and minimal irregularities of the intrahepatic bile ducts in the left lobe ( fig .
mild chronic inflammatory infiltrates consisting primarily of lymphocytes and liver parenchymal spotty necrosis were also found ( fig .
treatment was started with ursodeoxycholic acid at doses of 300 mg twice daily and changed to 200 mg twice daily with vitamins .
iu / l ; alt , 64 iu / l ; ggt , 112 iu / l ; alp , 208 iu / l ; and total bilirubin 0.9 mg / dl ( fig .
primary biliary cirrhosis ( pbc ) and primary sclerosing cholangitis ( psc ) are the most common causes of chronic progressive cholestasis in adults . however , a small number of patients who present with chronic cholestasis , lack anti - mitochondrial antibodies for pbc and have cholangiographic abnormalities consistent with psc .
these groups of patients often are thought to have ama - negative pbc , small - duct psc , or idiopathic adulthood ductopenia .
ludwig reported the causes of small duct biliary diseases in 2,082 cases diagnosed between 1988 and 1994 at the mayo clinic .
while pbc and psc accounted for more than 90% of the cases with small duct biliary diseases , iad was present in only 1% of cases .
in korea , only one case in 2004 , a 28-year - old woman who presented with pruritus and jaundice , has been reported .
several possible causes have been suggested , including late onset nonsyndromic paucity of the intrahepatic bile ducts , small - duct psc without large duct involvement and without evidence of inflammatory bowel disease , nonsuppurative viral cholangitis , hepatitis c virus , and autoimmune cholangitis or cholangitis in autoimmune hepatitis , in the absence of typical autoantibodies .
typically , iad is more common in males ( m : f , 1.8:1 ) , and it is usually diagnosed in young or middle - aged adults ( 15 - 77 years , median age : 34 years , 30 years for male , 36 years for female )
late onset nonsyndromic paucity of intrahepatic bile ducts usually is diagnosed in the teenage years and nonsuppurative viral cholangitis in adults more than 40 years of age .
this case appears to belong to the late - onset nonsyndromic paucity of intrahepatic bile ducts , as abnormal lfts have been identified in teenagers .
ductopenia is defined as the loss of interlobular and septal bile ducts of more than 50% among 20 or more portal tracts .
approximately one - third of patients present with episodic jaundice and pruritus , which gradually worsens . in other patients , cholestasis progresses incessantly , although the rate of deterioration varies .
iad related to autoimmune hepatitis or viral hepatitis requires specific treatments . in advanced cases ,
liver transplantation is the treatment of choice . in cases that are not severe , ursodeoxycholic acid ( udca )
can be used for treatment . a beneficial effect with the treatment of udca has been suggested , but its impact on the progression of the disease is unknown . the clinical outcome may be dependent on the cause of the disease and on the degree of ductopenia .
most patients with a late - onset nonsyndromic paucity of the intrahepatic bile ducts and small - duct primary sclerosing cholangitis may have progressive disease , but this may be self - limited in patients with virus - related cholangitis and autoimmune cholangitis . typically , iad eventually progresses to end - stage liver disease .
however , some patients with mild to moderate loss of intrahepatic bile ducts , less than 50% based on a liver biopsy , have a benign course .
mild iad , also termed mild idiopathic biliary ductopenia , does not meet the current diagnostic criteria for iad as no cases have had ductopenia involving 50% of the portal tracts .
our patient had only abnormal lfts without any symptoms , but had a ductopenia of more than 50% .
iad is a progressive disease and our patient may have been diagnosed at an early stage . in conclusion , little is known about iad .
iad is not a specific single entity but a syndrome associated with variable etiologies and variable clinical outcomes . | idiopathic adulthood ductopenia ( iad ) is a chronic cholestatic liver disease of unknown etiology characterized by adult onset , an absence of autoantibodies , inflammatory bowel disease , and a loss of interlobular bile ducts . in the present report
, a case fulfilling the iad criteria is described .
a 19-year - old man was admitted to the hospital for persistent elevation of transaminases and alkaline phosphatase without clinical symptoms .
viral hepatitis markers and autoantibodies were absent .
the patient had a normal extrahepatic biliary tree and had no evidence of inflammatory bowel disease .
a liver biopsy specimen showed absence of interlobular bile ducts from 58% of the portal tracts .
he was diagnosed with iad and was treated with ursodeoxycholic acid . |
the present study aimed to collate the findings from observational studies reporting psychological distress among amputees in india . the search strategy included electronic search engine databases : pubmed , psychinfo , and google scholar .
the keywords for the search were amputation , india , and psychological distress or depression or anxiety or posttraumatic stress disorder .
unpublished material and nonpeer - reviewed material were not included as a part of the review , and hand searches of libraries were not conducted as a part of the present review .
the present review included observational studies published in peer - reviewed english language journals pertaining to psychological distress .
nonlimb amputation was also not included in the present review . those studies which did not report the prevalence of psychological distress in the study sample
full texts of these abstracts were searched . upon careful reading of the studies , 4 of 16 were not included in the review as they did not report the prevalence of psychological distress in the study sample . they were analysis and discuss the epidemiology , services and rehabilitation of amputees in india , prevalence , etiology , levels of amputation , rehabilitation after amputation , and effect of anxiety , and depression on unilateral transtibial amputees .
the review included 12 published articles based on ten study samples as mentioned in table 1 .
the causes of amputation had been primarily traumatic ones ( accidents - motor vehicle accident and railway track accidents , machinery injury , blasts , etc . ) although other causes were also represented in the sample of amputees .
in addition , lower limb amputation was noted in majority of cases than upper limb amputation and among them , most amputation performed with one limb .
assessment methods included clinical impression and diagnosis , as well as used of structured scales such as beck depression inventory , beck hopelessness scale , carroll rating scale for depression , general health questionnaire , hamilton anxiety rating scale , hamilton depression rating scale , hospital anxiety depression scale , mini - international neuropsychiatric interview state - trait anxiety inventory , structured clinical interview for dsm disorder , and trauma symptom inventory .
follow - up studies show that rates of anxiety and depression decrease over a period of time .
phantom limb phenomenon had been observed in a considerable proportion of patients with amputations ( 14%92% ) associated with pain , telescopy , and movement .
the search strategy included electronic search engine databases : pubmed , psychinfo , and google scholar .
the keywords for the search were amputation , india , and psychological distress or depression or anxiety or posttraumatic stress disorder .
, further studies were identified from the cross references . unpublished material and nonpeer - reviewed material
were not included as a part of the review , and hand searches of libraries were not conducted as a part of the present review .
the present review included observational studies published in peer - reviewed english language journals pertaining to psychological distress .
nonlimb amputation was also not included in the present review . those studies which did not report the prevalence of psychological distress in the study sample
full texts of these abstracts were searched . upon careful reading of the studies , 4 of 16 were not included in the review as they did not report the prevalence of psychological distress in the study sample . they were analysis and discuss the epidemiology , services and rehabilitation of amputees in india , prevalence , etiology , levels of amputation , rehabilitation after amputation , and effect of anxiety , and depression on unilateral transtibial amputees .
the review included 12 published articles based on ten study samples as mentioned in table 1 .
the causes of amputation had been primarily traumatic ones ( accidents - motor vehicle accident and railway track accidents , machinery injury , blasts , etc . ) although other causes were also represented in the sample of amputees . in addition , lower limb amputation was noted in majority of cases than upper limb amputation and among them , most amputation performed with one limb .
assessment methods included clinical impression and diagnosis , as well as used of structured scales such as beck depression inventory , beck hopelessness scale , carroll rating scale for depression , general health questionnaire , hamilton anxiety rating scale , hamilton depression rating scale , hospital anxiety depression scale , mini - international neuropsychiatric interview state - trait anxiety inventory , structured clinical interview for dsm disorder , and trauma symptom inventory .
the rates of depression reported in the studies varied from 10.4 to 63% . rates of ptsd have ranged from 3.3% to 56.3% .
follow - up studies show that rates of anxiety and depression decrease over a period of time .
phantom limb phenomenon had been observed in a considerable proportion of patients with amputations ( 14%92% ) associated with pain , telescopy , and movement .
the present review suggests that indian literature pertaining to psychological effects of amputation has gradually accrued over time .
the studies have been heterogeneous and have attempted to understand the psychological impairment and psychiatric disorders among the amputees from different perspectives .
the studies though have been consistent in finding that a substantial proportion of those individuals who undergo amputation do suffer from psychological distress and psychiatric disorders .
the prevalence of psychiatric disorders among amputees has been found to be in the range of 32%84% .
these rates are generally high as compared to population - based psychiatric prevalence studies conducted in india .
however , the case controls studies in the present study provided an inconclusive picture .
the study by bhojak and nathawat found a lower level of hopelessness among surgical controls , while the study by mozumdar and roy found a comparable level of depression among non - amputated healthy males . the rates of depression and ptsd also have been alarming in some of the studies .
the findings of this review call for adept management of the psychological distress among the amputees . depression and anxiety are common problems among patients who have suffered from amputation .
both the antecedent circumstances preceding the amputation and the irreversible procedure of amputation itself may contribute to the occurrence of symptoms of psychological distress .
the surgical treatment providers need to liaise with psychiatrists and psychologists so that a comprehensive evaluation occurs when required and treatment of psychiatric disorders if identified can be initiated .
previous literature suggests that symptoms of anxiety and depression become better over the course of time although patient may need support from various sources while coming to terms with the impairment .
the numbers of studies which have followed up patients over a period of time are quite limited .
since indian social support system and health - care delivery services are quite different from the western world , the changes with time in the psychological distress and psychiatric morbidity can give a glimpse of remedial and accentuating factors .
moreover , studies need to consistently need to use standardized methods of making psychiatric diagnoses and eliciting symptoms using scales .
consistent subgroup analysis of traumatic and nontraumatic amputations can help discern the acuity of the psychological distress in these two groups .
moreover , intervention studies need to be conducted to assess the effectiveness of interventions among the amputees in the local context .
| amputation is a major health burden on the families , society , and on medical services as well .
traumatic limb amputation is a catastrophic injury and an irreversible act which is sudden and emotionally devastating for the victims .
in addition , it causes inability to support self and the family and driving many patients toward various psychiatric disorders .
extensive information regarding the effects of amputation has not been ascertained and therefore it was decided to do a systematic review . the goal of this review was to provide comprehensive information of peer - reviewed papers examining the psychological distress among amputees in india .
a search of the literature resulted in a total of 12 articles with varied sample size from 16 to 190 .
the sample has been largely comprised males with lower limb amputation caused by primarily traumatic ones , i.e. , motor vehicle accident , railway track accidents , machinery injury , blasts , etc .
, the prevalence of psychiatric disorders among amputees has been found to be in the range of 32% to 84% including depression rates 10.4%63% , posttraumatic stress disorder 3.3%56.3% , and phantom limb phenomenon 14%92% . although the studies reported that symptoms of anxiety and depression become better over the course of time , however surgical treatment providers need to liaise with psychiatrists and psychologists to support and deal with the psychological disturbances . |
this process includes dna and chromatin modifications by methylation , acetylation , phosphorylation , ubiquitylation , and sumolyation .
epigenetic processes are natural and essential to many organism functions , but if they occur improperly , they may result in major adverse health effects .
the best known epigenetic process is dna methylation , which involves the addition of a methyl group ( ch3 ) , which primarily occurs at the 5 position of the pyrimidine ring of the cytosine .
the presence of 5-methylcytosine in the promoter of specific genes alters the binding of transcriptional factors and other proteins to dna and recruits methyl - dna - binding proteins and histone deacetylases that compact the chromatin around the gene - transcription start site . both mechanisms block transcription and cause gene silencing .
chromatin is the complex of histone proteins and dna that is tightly bundled to fit into the nucleus .
the complex can be modified by histone acetylation , which alters chromatin structure to influence gene expression . in general
, tightly folded chromatin tends to be shut down , or not expressed , while more open chromatin is functional , or expressed . among all the epigenetic research conducted so far , the most extensively studied disease family is cancer , and the evidence linking epigenetic processes with cancer continues to grow [ 1 , 4 , 5 ] .
post - transcriptional histone modification plays an important role in regulating gene transcription , and is mediated by a variety of enzymes , including histone acetyltransferases ( hats ) and histone deacetylation ( hdacs ) .
these enzymes mediate acetylation and deacetylation of specific lysine amino acid residues on histone tails . in general ,
histone h3 and h4 acetylation is associated with open chromatin status favoring gene transcription , whereas histone deacetylation is associated with closed chromatin status favoring transcription repression and gene silencing .
the balance between hats and hdacs is critical for regulating the expression of a variety of genes that are involved in cell proliferation , survival , angiogenesis , and immunity [ 68 ] . to date , 18 hdacs have been identified in humans [ 9 , 10 ] .
hdacs are grouped in two major categories : zinc - dependent hdacs and nad - dependent hdacs .
furthermore , hdacs are classified into four major classes : class i includes hdac 1 , 2 , 3 , 8 , and 11 ; class ii includes hdac 4 , 5 , 6 , 7 , 9 , and 10 ; class iii includes homologues of yeast sirt 17 , and class iv , which currently includes only hdac 11 ( fig . 1 ) .
class iii is nad - dependent , whereas classes i , ii , and iv are zinc dependent .
in addition to histones , hats mediate post - translational protein acetylation on lysine residues in a variety of nonhistone proteins , including transcription factors ( p53 , stat3 , myc , gata-1 , gata-2 , e2f , nf-b , nuclear receptors , hif-1 , and tel ) , -tubulin , and heat shock protein-90 ( hsp90 ) [ 3 , 1113 ] .
because hdacs target histones and a variety of nonhistone proteins that control cell survival , proliferation , angiogenesis , and immunity , they became attractive targets for cancer therapy . at the present time , clinical grade pharmacologic inhibitors of the zinc - dependent hdacs
are available for clinical trials , and two inhibitors , vorinostat and romidepsin , have already been approved by the fda for the treatment of patients with relapsed cutaneous t - cell lymphoma [ 14 , 15 ] .
vorinostat inhibits a wide range of hdacs , including classes i and ii , and therefore is frequently referred to as pan - dac inhibitor . although romidepsin is considered a pan - dac inhibitor as well , it shows stronger activity against class i than against the class ii hdacs .
in contrast , entinostat ( sndx-275 ) and mocetinostat ( mgcd0103 ) are selective inhibitors that preferentially inhibit class i hdacs and to a less extent class iv ( fig . 2 ) .
2a schematic grouping of hdac inhibitors and their mechanisms of action a schematic grouping of hdac inhibitors and their mechanisms of action hdacis mediate antitumor activity by modulating a variety of survival pathways [ 3 , 9 , 13 , 17 ] .
hdacis induce cell cycle arrest by regulating the expression of p21 and p27 cell - cycle proteins , in addition to inducing cell death by activating the intrinsic and extrinsic apoptosis pathways .
in fact , a variety of hdaci have been shown to transcriptionally regulate several components of the death pathways , including upregulation of proapoptotic proteins ( bak , bax , apaf-1 , bad , bim , bid , caspase3 , and 9 ) , and repression of antiapoptotic proteins ( bcl2 , bcl - xl , mcl-1 , xiap , cflip ) .
furthermore , hdaci have been reported to upregulate the transcription of trail and its death receptors and fas ligand ( c95l ) and fas [ 1820 ] .
several hdacis are currently being evaluated for the treatment of cancer , and there is accumulating evidence that this treatment strategy is effective for the treatment of hematologic malignancies in general , and lymphoid malignancies in particular [ 13 , 19 , 21 , 22 ] .
in fact , several hdacis have recently reported promising clinical activity , including clinical remissions , in patients with hodgkin lymphoma ( hl ) and non - hodgkin lymphoma ( nhl ) [ 2329 ] .
hrs cells are b cells with epigentically - silenced b - cell genes molecular analysis demonstrated that the malignant hodgkin and reed - sternberg ( hrs ) cells of hodgkin lymphoma ( hl ) are of b - cell origin .
this loss of b - cell phenotype has been reported to be epigenetically regulated and is thought to enable hrs cells to evade immunosurveillance .
for example , gene expression profiles of hrs cells revealed decreased mrna levels for nearly all b - cell lineage - specific genes . the decrease in mrna expression
was associated with downregulation of protein expression in primary hl cases , as determined by immunohistochemistry , including cd20 , cd19 , syk , lck , blk , oct-2 , and b - cell receptor .
importantly , the expression of these silenced genes could be re - induced by the dna hypomethylating agent 5-aza - deoxycytidine [ 31 , 32 ] .
a variety of hdac inhibitors have demonstrated promising pre - clinical activity in hl cell lines by inducing cell cycle arrest and apoptosis . at the molecular level ,
hdacis upregulated p21 , activated the caspase pathway , downregulated stat6 expression , and altered cytokine and chemokine secretion [ 3335 ] .
in fact , we have recently demonstrated that vorinostat decreased the expression and secretion of th2-type cytokines and chemokines , including thymus and activation - regulated chemokine ( tarc / ccl17 ) and interleukin ( il)-5 , and increased th1-type cytokines / chemokines , including a profound increase in ip-10 levels , suggesting that hdaci may alter the microenvironment surrounding hrs cells to favor cell death .
epigenetic therapy induces the expression of epstein - barr virus antigens and tumor - associated antigens in hrs cells , which are potential targets for cytotoxic t cells epstein - barr virus ( ebv)-associated antigens are known to be expressed by a proportion of hrs cells , however , only the subdominant ebv antigens lmp1 and lmp2 are expressed and provide t - cell target antigens .
ebv - specific t cells that are enriched for lmp - specific t - cell clones have been expanded from patients and administered to patients with relapsed disease [ 36 , 37 ] . unfortunately , t cells specific for the viral latent nuclear antigens , ebnas 3a , 3b and 3 c and the immediate early lytic cycle antigen , bzlf1 , brlf1 and bmlf1 dominate the human immune response to ebv .
if these proteins could be reactivated in ebv - positive hrs cells , then their susceptibility to ebv - specific t - cell lines would be increased . both hdacis and hypomethylating agents induce ebv gene expression .
hdacs reactivate ebv from latency to replication , so that the immunogenic lytic cycle antigens are expressed [ 3840 ] , while azacytidine can switch latently infected cells from a minimal form of latency ( lmp1,lmp2 and ebna1 ) to an unrestricted latency in which all ebnas ( 2 , 3 s and - lp ) and lmps are expressed .
thus , these drugs may transform poorly antigenic tumors into highly immunogenic tumors and increase the number of available target antigens for t - cell recognition immunodominant viral proteins .
in addition , combining different agents like chemotherapy and hdac inhibitors results in synergistic effects on activating lytic viral replication .
since only about 20% of patients with relapsed hl have ebv - positive tumors , it is also important to determine if other tumor - associated , t - cell target antigens are upregulated by epigenetic therapy .
cancer testis antigens ( ctas ) are known to comprise at least 44 families of genes or isoforms . among these ,
19 families are purely testis - restricted , while others are expressed in one or more somatic tissues .
some ctas are expressed in lymphoma [ 41 , 42 ] , and provide potential t - cell targets .
cta - specific t cells from melanoma patients have demonstrated therapeutic benefits , including complete remissions .
recently , hypomethylating agents have been shown to induce the expression of several ctas in cultured tumor cell lines ( including lymphomas ) .
demethylation proved necessary and sufficient for mage - a1 gene expression in melanoma cell lines , suggesting that methylation is a primary mechanism of transcription control .
histone deacetylase ( hdac ) inhibitors , on their own , or in combination with hypomethylating agents , can also induce cta expression , including mage , ssx , and ny - eso-1 family members .
recent data suggested that hdaci may upregulate the expression of cta in hrs , suggesting hdacis may enhance the activity of ebv - directed cellular therapy in hl .
since epigenetically enhanced cta expression may activate endogenous cta - specific t cells in vivo , this may induce epitope spreading and facilitate the ex vivo activation and expansion of these normally low frequency cta - specific t cells .
a variety of hdacis are currently being evaluated in patients with relapsed hl ( table 1 ) .
the following is a brief update on the current status of hdacis in patients with relapsed hl .
consistent with their ubiquitous distribution in normal tissues , class i enzymes were highly expressed in all hodgkin and non - hodgkin lymphoma cell lines and primary tumors studied , including the non - malignant reactive cells in hl microenvironment .
in contrast , the class ii enzyme hdac6 was infrequently expressed , compared with hdacs 5 , 8 , and 10 .
these results suggest that hdac6 may not be an important therapeutic target in selected lymphoid malignancies .
table 1summary results of hdaci in patients with relapsed chldrugtargetroutephasenumber of evaluable patientsprcrpr + cr ( orr)mgcd0103 hdacsoralii21628 ( 38%)itf2357 hdacsoralii13000panobinostat hdacsorali20808 ( 40%)panobinostat hdacsoralii5310111 ( 21%)vorinostat hdacsoralii25101 ( 4% ) summary results of hdaci in patients with relapsed chl vorinostat ( saha ) vorinostat , which inhibits hdac classes i and ii , has a potent antiproliferative activity in hl cell lines , as it induces cell cycle arrest and apoptosis , in addition to synergizing with chemotherapy .
furthemore , vorinostat inhibited stat6 phosphorylation and transcription in hl cell lines , an effect that was associated with decreased expression and secretion of th2-type cytokines and chemokines ( tarc and il-5 ) .
this effect was associated with and increases of in th1-type cytokines / chemokines ( ip-10 ) .
the in vivo activity of vorinostat was recently evaluated in a phase ii study that was conducted by the southwest oncology group ( swog ) in patients with relapsed hl .
twenty - five patients were treated with 200 mg vorinostat given orally twice per day for 14 days every 21-day cycle .
vorinostat produced modest clinical activity , as only 1 patient ( 4% ) achieved a partial remission .
mocetinostat ( mgcd-0103 ) mecetinostat is a novel oral hdac inhibitor that selectively inhibits hdac 1 , 2 , 3 ( class i ) and 11 ( class iv ) isoforms , with no effect on class - ii hdacs .
the safety and efficacy of mgcd-0103 given orally 3 times per week ( 85 to 110 mg starting doses ) has been evaluated in a phase ii study in patients with relapsed and refractory hl .
patients were allowed to continue therapy up to 1 year in absence of heavy toxicity or disease progression .
of the 20 patients who were treated with 110-mg dose level , 7 ( 35% ) patients achieved partial or complete remissions .
however , this dose level was poorly tolerated resulting in dose interruptions and reductions , and discontinuation of therapy after a median of 4.5 months . subsequently
, the study was revised to allow a lower starting dose of 85 mg at the same schedule .
three of the 10 ( 30% ) patients enrolled on the reduced dose achieved partial remissions .
furthermore , grade 3 and 4 toxicity ( mainly fatigue , with no significant hematologic toxicity ) was reduced to 20% .
overall , 80% of the 30 evaluable patients had some decrease in their tumor measurements .
although none of the patients developed significant ekg abnormalities , two patients developed significant pericardial effusions requiring discontinuation of therapy .
importantly , serum tarc levels were determined in 15 patients before and 1 week after initiation of therapy .
after 1 week of therapy serum tarc levels were decreased by at least 40% in five patients , and all achieved major clinical responses .
collectively , this data indicate that class i hdac inhibitors have a potential therapeutic value in patients with hl .
panobinostat ( lbh 589 ) panobinostat is one of the most potent hdacis in hl in vitro , with an ic50 in the nanomolar range .
recently panobinostat was evaluated in a phase i trial in patients with hematologic malignancies that also included patients with relapsed hl .
the most common side effects were fatigue , thrombocytopenia , nausea , and diarrhea . based on this promising clinical activity , a large international phase ii study of panobinostat in relapsed hl , with an early report demonstrating a promising clinical activity in a large multicenter settings . in a most recent update of the study , 61 patients have been enrolled and treated : median age 31 years ( range 1870 ) , and 48% had stage iii / iv at initial diagnosis .
patients were heavily pretreated with a median number of prior regimens of 4 ( range 26 regimens ) .
of 61 patients , 53 have completed two cycles of therapy and have had one post baseline imaging studies to evaluate response to theapy .
sixty - nine percent had tumor reduction , including one complete response and 10 partial responses the most common grade 3/4 adverse events were thrombocytopenia ( 77% ) , anemia ( 20% ) , and neutropenia ( 16% ) .
based on this promising data , panobinostat - based combination studies are currently being conducted , including a phase i / ii trial of panobinostat plus everolimus in patients with relapsed hl and non - hodgkin lymphoma .
entinostat ( sndx-275 ) entinostat is an oral , class i isoform selective hdac inhibitor .
phase 1 studies in leukemia demonstrated the agent has a long half - life and that weekly or every other week dosing is sufficient for antitumor activity . in vitro experiments demonstrated a potent activity of entinostat in hl - derived cell lines , with an ic50 of 0.4 m . at the molecular level , entinostat induced its antiproliferative effect in hl cell lines by multiple mechanisms : it increased h3 acetylation , up - regulated p21 protein expression , and activated the intrinsic apoptosis pathway by down - regulating the anti - apoptotic x - linked inhibitor or apoptosis ( xiap ) protein , which was associated with activation of caspase 9 and 3 .
furthermore , entinostat had synergistic effects when combined with gemcitabine and bortezomib . moreover , entinostat increased il12 p40 - 70 , ip10 , and rantes , and decreased the level of il13 and il4 , in hl cell lines supernatants , thus favoring th1-type cytokines / chemokines . finally , entinostat induced the expression of a variety of tumor - associated antigens , including ssx2 and mage - a .
based on this encouraging in vitro data , the safety and efficacy of entinostat is currently being evaluated in a phase 2 multi - center study in patient with relapsed or refractory hl . in this study entinostat
is given 10 mg ( two 5 mg tablets ) orally , once every two weeks ( days 1 and 15 ) in a 28 day cycle increasing to 15 mg in absence of treatment related toxicity .
the efficacy , safety , and tolerability of daily 100-mg doses of itf 2357 in relapsed / refractory hl were investigated in a phase ii clinical trial at the national tumors institute of milan .
the preliminary results were presented at the 2008 asco meeting : 15 patients were enrolled and 13 were evaluable for response .
seven patients ( 54% ) had a stable disease , with a reduction in fdg - pet uptake in six patients ( 46% ) lasting at least 3 months ; six patients had disease progression .
interestingly , a correlation was found between a decrease in serum tarc levels and the response to treatment in this study . on the basis of the single - agent activity of itf 2357 and the documented synergistic activity of itf 2357 and mechlorethamine in hl cell lines ,
a phase ii trial of itf 2357 combined with mechlorethamine was conducted by the same group .
preliminary data from 17 evaluable patients demonstrated that two patients ( 12% ) achieved complete and three ( 18% ) partial remissions .
as expected , the major toxicity was hematologic , with seven patients experiencing grade iii / iv neutropenia and eight having thrombocytopenia ; four patients experienced infections during treatment . taken together , these data suggest that itf 2357 has encouraging clinical activity in relapsed / refractory hl .
class i and pan - dac inhibitors are promising new agents for the treatment of patients with relapsed hl .
future studies should examine predictive biomarkers , such as tarc levels and the pattern of hdac enzyme expression in hl patients treated with hdacis to determine whether a certain pattern of expression may predict response to therapy .
furthermore , because hdac inhibitors can modulate a variety of survival proteins , combination regimens with hdac inhibitors should be investigated .
ultimately , these studies will hopefully improve our treatment strategies for patients with relapsed and refractory hl . | summaryalthough hodgkin lymphoma ( hl ) is considered one of the most curable human cancers , the treatment of patients with relapsed and refractory disease , especially those who relapse after autologous stem cell transplantation , remains challenging . furthermore , because of the young age of these patients , the impact of early mortality on the number of years lost from productive life is remarkable .
patients with relapsed hl post stem cell transplantation currently have no curative therapy , and are in need for new drugs and novel treatment strategies .
while no new drugs have been approved for the treatment of patients with hl in more than three decades , several new agents are demonstrating promising results in early clinical trials .
this review will focus on the emerging role of histone deacetylase inhibitors in patients with relapsed hl . |
we describe a unique case of a plexiform neurofibroma involving the celiac and hepatic plexus in a healthy child without stigmata of neurofibromatosis type 1 ( nf-1 ) . given both the anatomic and functional details , magnetic resonance imaging ( mri ) has proven to be the modality of choice for both diagnosis and follow - up in this case .
a previously healthy 9-year - old boy presented with a 3-year history of nonspecific intermittent low back pain , for which a renal ultrasound was performed .
the kidneys were unremarkable , but the ultrasound examination did reveal a heterogeneous hyperechogenic ill - defined infiltrative periportal mass lesion .
the lesion was centered in the region of the porta hepatis with extension along the portal triads without biliary tract or vascular obstruction ( fig .
a computed tomographic ( ct ) scan was performed that demonstrated a hypoattenuating mass lesion encasing the periportal vasculature and celiac axis without compression or obstruction ( fig .
the diagnosis was not conclusive on the basis of ultrasound or ct findings , therefore mri was performed .
the mri scan showed a t2-hyperintense , t1-hypointense , mildly enhancing polylobulated , serpiginous mass lesion within the retroperitoneal space that wrapped around the celiac trunk and extended via the porta hepatis into the liver and along the periportal spaces without obstruction of the vasculature .
the differential diagnosis included the following : lymphatic malformation , lymphoma , lipoma , liposarcoma , and plexiform neurofibroma .
in addition , lymphoma and lymphatic malformation were unlikely because of the diffusion - weighted imaging ( dwi ) characteristics .
( a ) ultrasound of the liver demonstrates an infiltrative hyperechoic mass within the liver .
axial postcontrast computed tomographic scan at the level of the celiac axis demonstrates an infiltrative homogeneous low attenuation lesion with a periportal distribution ( black arrow ) .
the mass extends outside the liver and encases the pancreas ( white arrow ) and celiac axis ( dashed white arrow ) .
fat saturated ( a ) pre- and ( b ) postcontrast t1-axial images and ( c ) non fat saturated and ( d ) fat saturated t2-weighted magnetic resonance images of the liver demonstrate a mildly enhancing polylobulated lesion , which is predominately t2 hyperintense with central areas of t2 hypointensity with a target appearance ( white arrows ) .
open liver biopsy revealed an infiltrative lesion within the liver that initially appeared cystic ; however , it was determined to actually be a solid mass .
additional genetic counseling after this diagnosis revealed a negative family history ; and on further clinical evaluation , the patient did not meet the minimal criteria for nf-1 .
treatment varies on the basis of the location of the lesion , clinical symptoms , and evidence of malignant degeneration .
because our patient was asymptomatic and there was no evidence of malignant degeneration , conservative treatment was opted .
the dwi and the corresponding apparent diffusion coefficient ( adc ) maps were crucial in the diagnostic pathway .
the entire lesion had adc value of approximately 2,097 mm / s , confirming its benign nature .
ultrasound and ct findings for plexiform neurofibromas of the celiac and hepatic axis are often nonspecific .
most neurofibromas tend to be hypoattenuating with ill - defined borders on ct,1 which is thought to be the result of the myxoid and mucinous stroma of these tumors on pathological evaluation . the ropelike appearance , which represents the thickened , tortuous nerve sheath on cross section , is usually not appreciated on ct
although these features may suggest the diagnosis , ct can not definitively distinguish neurofibromas from other malignancies .
in contrast to ultrasound and ct , neurofibromas have characteristic imaging features on mri , and adding this modality to the diagnostic pathway can often be very helpful.2
3
4
neurofibromas are typically heterogeneous on t2-weighted images , with high t2 signal reflecting areas of myxoid matrix and cystic degeneration and the low t2 signal reflecting areas of collagen and fibrous tissue .
rather , the tumor follows the nerve fibers of the celiac and hepatic plexus , therefore extending along the periportal space .
only a few other pediatric cases have been described in which there is diffuse extension of the lesion toward the retroperitoneum and mesentery.5 because they often go unrecognized , these widespread lesions can mimic a malignancy .
the availability of dwi and the corresponding adc maps are of great diagnostic value in allowing differentiation from solid high density tumors like lymphoma and neuroblastoma and cystic lesions such as lymphatic malformations .
it is important to realize that plexiform neurofibromas can undergo malignant transformation and approximately 5% of nf-1 patients will develop malignant peripheral nerve sheath tumors.6 imaging findings suggesting malignant degeneration on mri include an increased largest dimension of the mass , a peripheral enhancement pattern , presence of a perilesional edema - like zone , and presence of intratumoral cystic lesions.7 dwi is also a promising adjunct to evaluate for malignant potential . in the study by niwa
et al,6 dwi was used to differentiate between the benign and malignant areas of a retroperitoneal plexiform neurofibroma .
the study showed that the malignant areas displayed high signal intensity on dwi and low signal intensity on the corresponding adc map , likely because of high cellularity in areas of malignancy , with reducing the ability of water protons to move freely .
the adc of the malignant portion was approximately 1,200 mm / s , whereas the benign portion was 2,000 mm / s .
similar results were seen in our patient because the entire lesion had adc values of approximately 2,097 mm / s ( fig .
4 ) . consequently , dwi and adc analysis allows us to limit the diagnosis and helps differentiate between malignant and benign plexiform neurofibromas .
the optimal treatment management would be resection but extensive intra - abdominal plexiform neurofibromas are difficult to remove completely without causing significant harm .
the use of dwi and adc analysis could aid in recognizing areas of malignant transformation , and therefore , both should be included as diagnostic tools to follow progression of the disease .
( a ) axial diffusion - weighted magnetic resonance image and ( b d ) axial apparent diffusion coefficient ( adc ) maps demonstrate no evidence of restricted diffusion .
( c ) this was elevated compared with the liver ( 1,095 mm / s ) ; ( d ) however , significantly lower than fluid containing structures like the gallbladder ( 3,008 mm / s ) .
plexiform neurofibromas mainly occur in the setting of nf-1 , an autosomal dominant neurocutaneous syndrome .
our patient , however , did not meet the minimal clinical criteria for this syndrome .
although sometimes patients may develop stigmata of nf-1 later in life , there is also literature supporting that plexiform neurofibromas are highly suggestive but not exclusive for nf-1.8
to summarize , the use of mri is extremely helpful in both diagnosis and management of patients with plexiform neurofibromas , especially because surgical resection is not always a viable option .
in addition , it is important to remember that plexiform neurofibromas are not pathognomonic for nf-1 and this entity should be included in the differential diagnosis even in patients without clinical features of nf-1 . | plexiform neurofibroma with involvement of the gastrointestinal tract is a very rare entity in children . here
, we present a rather unique case of a 9-year - old boy with no clinical signs or features of neurofibromatosis type 1 . a periportal mass lesion was incidentally found after performing an ultrasound in this previously healthy child .
computed tomographic scan was subsequently performed which showed a low - density mass in a periportal distribution with extension along the celiac axis . because the findings were nonspecific , a pre- and postcontrast magnetic resonance imaging of the abdomen was performed which included diffusion - weighted imaging .
the lesion was then confirmed to be a plexiform neurofibroma with open biopsy .
management of plexiform neurofibromas varies widely .
given the extensive nature of the lesion , managing the patient with follow - up rather than surgical excision was favored . |
antiepileptic drugs ( aeds ) are used in the treatment of epilepsy , pain , and psychiatric disorders , .
hepatic and renal status may impact the efficacy and toxicity associated with aeds which requires awareness by clinicians in multiple specialties and appropriate dose adjustments .
gabapentin is an aed approved by the fda for the adjunctive treatment of partial seizures with / without secondary generalization in children , adolescents , and adults and for the treatment of postherpetic neuralgia ; the prodrug gabapentin enacarbil is approved by the fda for the treatment of restless leg syndrome .
gabapentin is frequently used off - label for the treatment of both psychiatric and pain disorders , .
gabapentin is not hepatically metabolized and is cleared solely by renal excretion ; dosing requires consideration of the patient 's renal function , especially in the context of end - stage renal disease ( esrd ) , , .
we report 2 cases of myoclonic activity associated with gabapentin toxicity in the setting of renal disease and address treatment with hemodialysis ( hd ) and peritoneal dialysis ( pd ) .
a 78-year - old woman with congestive heart failure , history of thromboembolism , hypertension , diabetes mellitus , hyperlipidemia , asthma , diabetic peripheral neuropathy , and depression presented with tremors involving her upper extremities for 3 days prior to admission .
her admission medications included simvastatin , metformin , citalopram , gabapentin , fluticasone proprionate inhaler , inhaled albuterol , lisinopril , furosemide , and metolazone .
physical examination noted severe bilateral upper extremity myoclonus with normal mental status and without other neurological symptoms .
meq / l , a blood urea nitrogen ( bun ) level of 91 mg / dl , a creatinine ( cr ) level of 3.2 mg / dl , an elevated bun / cr ratio of 28.44 , and an estimated glomerular filtration rate of 13 ml / min/1.73 m. evaluation revealed acute kidney injury ( aki ) secondary to a recently increased furosemide total dose of 60 mg daily and lisinopril 5 mg daily with hyperkalemia and azotemia .
prior to admission , the patient had been chronically treated with gabapentin 900 mg total daily dose for neuropathic pain .
with discontinuation of gabapentin and initiation of hd , marked improvement in her myoclonus occurred .
the patient received 2 sessions of hd and was discharged with normal renal function , a bun level of 20 mg / dl and a cr level of 1.1 mg / dl , and resolved myoclonus .
. a 55-year - old man with esrd on pd , anemia , diabetes mellitus , hypertension , neuropathic pain , hyperlipidemia , hepatitis c , peripheral vascular disease with recently amputated gangrenous toe on long - term vancomycin and piperacillin / tazobactam , and acute pain syndrome presented for evaluation of bilateral upper extremity tremors , altered mental status , hypotension , and worsening leg infection .
following initiation of gabapentin 600 mg total daily dose for neuropathic pain 3 days prior to admission , the patient developed severe arm tremors with the inability to hold objects , lethargy , and intermittent episodes of confusion .
his other admission medications included clopidogrel , amlodipine , hydralazine , metoprolol , clonidine , atorvastatin , oxycodone , hydromorphone , sevelamer , lanthanum , epoetin , and insulin glargine .
abnormal renal function on admission , with a bun level of 49 mg / dl and a cr level of 12.7 mg / dl , was comparable to outpatient renal function during the prior three months , with a bun level of 5052 mg / dl and a cr level of 10.411.1 mg / dl , when the patient did not have myoclonus .
the patient 's pd treatment was increased from 4 to 6 exchanges daily . with increased dialysis and discontinuation of gabapentin , myoclonus and altered mental status resolved within four days .
a 78-year - old woman with congestive heart failure , history of thromboembolism , hypertension , diabetes mellitus , hyperlipidemia , asthma , diabetic peripheral neuropathy , and depression presented with tremors involving her upper extremities for 3 days prior to admission .
her admission medications included simvastatin , metformin , citalopram , gabapentin , fluticasone proprionate inhaler , inhaled albuterol , lisinopril , furosemide , and metolazone .
physical examination noted severe bilateral upper extremity myoclonus with normal mental status and without other neurological symptoms .
meq / l , a blood urea nitrogen ( bun ) level of 91 mg / dl , a creatinine ( cr ) level of 3.2 mg / dl , an elevated bun / cr ratio of 28.44 , and an estimated glomerular filtration rate of 13 ml / min/1.73 m. evaluation revealed acute kidney injury ( aki ) secondary to a recently increased furosemide total dose of 60 mg daily and lisinopril 5 mg daily with hyperkalemia and azotemia .
prior to admission , the patient had been chronically treated with gabapentin 900 mg total daily dose for neuropathic pain .
with discontinuation of gabapentin and initiation of hd , marked improvement in her myoclonus occurred .
the patient received 2 sessions of hd and was discharged with normal renal function , a bun level of 20 mg / dl and a cr level of 1.1 mg / dl , and resolved myoclonus .
a 55-year - old man with esrd on pd , anemia , diabetes mellitus , hypertension , neuropathic pain , hyperlipidemia , hepatitis c , peripheral vascular disease with recently amputated gangrenous toe on long - term vancomycin and piperacillin / tazobactam , and acute pain syndrome presented for evaluation of bilateral upper extremity tremors , altered mental status , hypotension , and worsening leg infection .
following initiation of gabapentin 600 mg total daily dose for neuropathic pain 3 days prior to admission , the patient developed severe arm tremors with the inability to hold objects , lethargy , and intermittent episodes of confusion .
his other admission medications included clopidogrel , amlodipine , hydralazine , metoprolol , clonidine , atorvastatin , oxycodone , hydromorphone , sevelamer , lanthanum , epoetin , and insulin glargine .
abnormal renal function on admission , with a bun level of 49 mg / dl and a cr level of 12.7 mg / dl , was comparable to outpatient renal function during the prior three months , with a bun level of 5052 mg / dl and a cr level of 10.411.1 mg / dl , when the patient did not have myoclonus .
the patient 's pd treatment was increased from 4 to 6 exchanges daily . with increased dialysis and discontinuation of gabapentin , myoclonus and altered mental status resolved within four days .
these unique cases raise a series of important points specifically related to these patients that should be considered in the general treatment of patients with gabapentin and other aeds that require renal clearance .
first , a recent study of off - label prescriptions found the overall rate to be 21% ; however , aeds as a class are higher at 46% , and gabapentin , in particular , has the highest rate of all drugs with 83% prescribed off - label , , .
research supports the off - label use of gabapentin to treat diabetic peripheral neuropathic pain , .
one mechanism suggested for the broad spectrum effects of gabapentin is voltage - sensitive calcium channel modulation .
second , with normal renal function , the half - life of gabapentin ranges from 5 to 7 h . in patients with esrd on non - hd days ,
that study noted that 35% of a single gabapentin dose was removed after three sessions of hd , supporting the use of hd in the treatment of gabapentin toxicity .
third , myoclonus is a known adverse effect associated with gabapentin with a prevalence as high as 12.5% which , depending on severity , may not interfere with aed therapy . in the context of renal failure , myoclonus and neurotoxicity
most reports demonstrate that serum concentrations greater than 15 g / ml are associated with symptomatic toxicity . in patients who have intact renal function ,
toxicities are uncommon as gabapentin is rapidly cleared based on its short half - life ; however , gabapentin concentrations are significantly increased in patients with renal dysfunction , and in cases of symptomatic toxicity , dialysis should be instituted .
both intermittent and continuous forms of renal replacement therapy have been effectively utilized to treat gabapentin - induced neurotoxicity and myoclonic activity , , .
although this report suggests the efficacy of pd in treating gabapentin - induced myoclonus and neurotoxicity , there are no published data regarding clearance of gabapentin with pd .
fourth , pregabalin is an alternative aed with fda / ema approval for pain syndromes ( central neuropathic pain , diabetic peripheral neuropathic pain , postherpetic neuralgia , and fibromyalgia ) .
pregabalin , which , like gabapentin , is a branched - chain amino acid aed that is cleared solely by renal excretion , is also noted to induce myoclonus , . as with gabapentin
fifth , a threshold effect for the development of myoclonic activity with gabapentin and pregabalin has been previously reported , . this is consistent with the concept that adverse events for specific aeds may require an individual threshold concentration which may be within the reference range , , .
azotemia was present in both cases ; however , in case 2 , the patient had chronic stable renal function with the development of myoclonus only after initiation of gabapentin . case reports implicate metformin , citalopram , albuterol , amlodipine , oxycodone , and hydromorphone as potential etiologies for myoclonus , , , , , , . in both cases ,
the patients had been stable on these and other admission medications prior to the aki in case 1 and the addition of gabapentin in case 2 .
seventh , potentially inappropriate medications ( pims ) are frequently prescribed in both geriatric patients and patients with chronic kidney disease , , . in patients at risk for aki or with esrd on dialysis , these cases suggest that gabapentin is a relative pim unless there is renal dosing with close monitoring of efficacy and adverse effects .
in such instances , duloxetine , a serotonin norepinephrine reuptake inhibitor antidepressant with fda approval for diabetic peripheral neuropathic pain and fibromyalgia , may be an effective treatment option .
eighth , each case had multiple potential risk factors for the development of myoclonus . in the context of additive risk factors ,
the probability of gabapentin inducing myoclonus was determined by naranjo 's adverse reaction probability scale in case 1 as possible and in case 2 as probable .
there are specific limitations to this paper . as a case report ( n = 2 )
both cases were complicated by the presence of medical comorbid conditions and specific medications that have been implicated as potential etiologies for myoclonus .
gabapentin drug monitoring was not performed such that the threshold concentration associated with developing and resolving myoclonus in each case could not be specified .
neither patient was rechallenged ( case 1 maintaining gabapentin with increased total daily dose of furosemide and addition of lisinopril ; case 2 addition of gabapentin ) ; further , the impact of renal dosing was not assessed in either case .
myoclonic activity may occur as a complication of gabapentin toxicity , especially in the setting of renal dysfunction . in the cases reported , both hd and pd were effective in treating myoclonic activity in acute and chronic renal dysfunction .
gabapentin requires renal dosing in patients with chronic kidney disease and in patients at risk for developing aki . in cases of symptomatic gabapentin - induced toxicity , dialysis
as gabapentin has multiple indications and off - label uses , an understanding of myoclonus , neurotoxicity , and renal dosing is important to clinicians in multiple specialties .
| gabapentin , an aed approved for the adjunctive treatment of partial seizures with / without secondary generalization and for the treatment of postherpetic neuralgia , is frequently used off - label for the treatment of both psychiatric and pain disorders .
since gabapentin is cleared solely by renal excretion , dosing requires consideration of the patient 's renal function .
myoclonic activity may occur as a complication of gabapentin toxicity , especially with acute kidney injury or end - stage renal disease .
we report 2 cases of myoclonic activity associated with gabapentin toxicity in the setting of renal disease which resolved with discontinuation of gabapentin and treatment with hemodialysis and peritoneal dialysis .
as gabapentin has multiple indications and off - label uses , an understanding of myoclonus , neurotoxicity , and renal dosing is important to clinicians in multiple specialties . |
in early 1954 mead
has defined an impacted tooth as a tooth that is prevented from
erupting into position because of malposition , lack of space , or
other impediments .
later peterson ,
characterized impacted teeth as those teeth that fails to erupt
into the dental arch within the expected time . in 2004 farman
wrote that impacted teeth are those teeth that prevented from eruption
due to a physical barrier within the path of eruption . according to elsey and rock
impaction of the third molar is occurring in up to 73% of young
adults in europe .
generally , third molars have been found to erupt
between the ages of 17 and 21 years .
furthermore , third molar eruption time have been reported to vary
with races [ 5 - 8 ] .
for example , mandibular third molars may erupt as early as 14 years
of age in nigerians ,
and up to the age of 26 years in europeans .
the average age for the eruption of mandibular third molars in male
is approximately 3 to 6 months ahead of females .
most authors claim that the incidence of mandibular third molar
impaction is higher in females .
third molar eruption and continuous
positional changes after eruption can be related not only with race
but also with nature of the diet , the intensity of the use of the
masticatory apparatus and possibly due to genetic background .
impaction of mandibular third molars
is a common condition related with different difficulty degree of
extraction operation and risk of complications , including iatrogenic
trigeminal nerve injury .
the purpose of present article was to review
impacted mandibular third molar aetiology , clinical anatomy , radiologic
examination , surgical treatment and possible complications , as well
as to create new mandibular third molar impaction and extraction
difficulty degree classification based on anatomical and radiologic
findings and literature review results .
literature was selected through a
search of pubmed , embase and cochrane electronic databases .
the
keywords used for search were mandibular third molar , impacted mandibular
third molar , inferior alveolar nerve injury third molar , lingual
nerve injury third molar .
the search was restricted to english language
articles , published from 1976 to april 2013 .
additionally , a manual
search in the major anatomy and oral surgery journals and books
was performed .
many theories have been proposed
owing to high incidence of mandibular third molar impaction .
one
of the most popular theory is insufficient development of the retromolar
space .
mandibular ramus growth is related to resorption at its anterior
surface and deposition at its posterior surface , but in case of
disbalance of this process , the mandibular third molars do nt get
enough space to erupt .
for example , if the tooth bud is medially angulated
during the initial stages of calcification and root development
the path of eruption will be unfavourable .
however , impaction of mandibular third molars can develop due to
a decrease in the angulation of the mandible and an increase in
the angulation of the mandibular plane .
yamaoka et al .
found the relation between the root angulation and impaction : angulated
roots were more common in impacted mandibular third molars as compared
to erupted mandibular third molars .
some authors indicates other
important third molar impaction causes : malposition of the tooth
germ , hereditary factors ,
lack of sufficient eruption force for third molars , and the theory
of phylogenetic regression of the jaw size - insufficient mesial
movement of the dentition of modern human due to lack of interproximal
attrition .
mandibular third molar is situated
at the distal end of the body of the mandible where is connection
with relatively thin ramus .
there is the region of weakness and
the fracture can occur if excessive force will be applied during
impacted wisdom tooth elevation without preliminary and adequate
removing of surrounding bone .
there is high risk of lingual nerve damage using lingual
split technique or elevating third molar flap medially to the distoangular
recess .
rood and shehab
showed on panoramic radiographs that in most cases the roots of
third molars are in close proximity to the mandibular canal .
furthermore ,
in some cases third molar roots can contact or penetrate into mandibular
canal or they can be deflected .
close relationship of the canal
with the roots can evoke inferior alveolar nerve damage during the
surgery .
radiologic examination the location and configuration of
impacted third molar , surrounding bone , mandibular canal and adjacent
tooth are important in imaging diagnosis for the proper surgical
operation planning .
periapical radiographs have been used for many
years to assess the jaws during impacted teeth surgery .
long cone
paralleling technique for taking periapical x - ray is the technique
of choice for the following reasons : reduction of radiation dose ;
less magnification ; a true relationship between the bone height
and adjacent teeth is demonstrated .
since the film is highly flexible , literally and figuratively , its
processing can be suboptimal and it often leads poor image .
during the last decade , many dental practices replaced the film
with digital imaging systems .
nevertheless , the biggest concern
of periapical radiographs is that mandibular canal could not be
clearly identified in the third molar region .
furthermore , the angulation
of the periapical film can affect the perceived location of the
canal with respect to the bone crest .
when a specific region that is too
large to be seen on a periapical view , panoramic radiograph can
be the method of choice .
the major advantages of panoramic images
are the broad coverage of oral structures , low radiation exposure
( about 10% of a full - mouth radiographs ) , and relatively inexpensive
of the equipment .
the major drawbacks of panoramic imaging are :
lower image resolution , high distortion , and presence of phantom
images .
these can artificially produce apparent changes thus may
hide some of important vital structures .
furthermore , it depicts a two - dimensional view of an intricate
three - dimensional anatomic relationship and also fails to accurately
project the buccolingual relation between the tooth and the inferior
alveolar canal .
cone beam computed tomography ( cbct )
have been advocated as method of choice than there is need to have
a three dimensional view of the mandibular third molar and adjacent
anatomical structures .
ghaeminia et al .
in prospective study evaluated the role of cbct in the treatment
of patients with impacted mandibular third molars ( n = 53 ) at increased
risk of inferior alveolar nerve injury . after reviewing the cbct
images ,
significantly more subjects were reclassified to a lower
risk for ian injury compared with the panoramic radiograph assessments .
this change in risk assessment also resulted in a significantly
different surgical approach ( p < 0.03 ) .
authors concluded that cbct
contributes to optimal risk assessment and , as a consequence , to
more adequate surgical planning , compared with panoramic radiography .
it was reconfirmed by study of matzen et al . ,
where cbct influenced the treatment plan for 12% of cases .
direct
contact in combination with narrowing of the canal lumen and canal
positioned in a bending or a groove in the root complex observed
in cbct images were significant factors for deciding to change treatment
plan .
indications for mandibular third molar extraction according to the recommendations
of national institute of health ( nih )
both impacted and erupted mandibular third molars with evidence
of follicular enlargement should be removed electively and that
the associated soft tissue should be submitted for microscopic examination .
impacted teeth with pericoronitis
should also be extracted electively
because of their known potential for repetitive infection and morbidity .
furthermore , third molars with non - restorable carious lesions and
third molars contributing to resorption of adjacent teeth should
be also extracted . following indications for mandibular
third molar extraction
were highlighted by koerner :
existing pathology or pain due to pericoronitis , periodontitis ,
periapical abscess , cysts or neoplasms , resorption of adjacent roots ,
and inflammation of the opposing soft tissue ; aberrant positions
in which the tooth is oriented buccally or lingually ; preceding
dental work with fixed or removable appliances ; arch length discrepancy
in cases when the impacted third molars are affecting the stability
of orthodontic treatment .
lytle
added infection around the impaction ; loss of bone around the impacted
teeth ; dental caries and damage of adjacent teeth ; crowding of the
dental arch ; cysts and tumours associated with impacted teeth ; pre - irradiation
removal of impacted teeth ; for prosthodontic reasons ; and for chronic
facial pain .
the national institute of clinical excellence ( nice )
of england introduced guidelines relating to third molars surgery .
these recommended against the prophylactic removal of third molars
and listed specific clinical indications for surgery .
surgical extraction of impacted mandibular third molar and possible complications there are two main intraoral approaches
for surgical removal of impacted mandibular third molars : one through
the sublingual space and the other buccally through the entire mandibular
thickness .
there is also extraoral method from the submandibular
space [ 36 - 38 ] .
sublingual access requires incision and elevation of a wide mucoperiosteal
intrasulcular flap on the lingual side of the mandible , in the molar
and premolar regions .
the buccal approach requires
the elevation of a wide mucoperiosteal flap localized around the
molar premolar region .
however there are many
modifications of flap techniques , including envelope flap , two sided
flap , and coma shaped flap .
in every case
after mucoperiosteal flap elevation
excessive bone must be excised using bur before third molar extraction .
in most cases there will be necessary to remove buccal and distal
bone borders . in difficult cases
the tooth should be sectioned with
a fissure bur in a high - speed handpiece .
after tooth extraction
using elevator or forceps it is necessary to clean operation area
and to suture the wound without tension .
the frequency and severity of untoward
events associated with surgical procedures are influenced by multiple
factors that may be related to the procedure , patient , and/or surgeon
.
complications related to mandibular third molar extraction can be
classified to intraoperative and postoperative .
intraoperative complications are as follows : mandibular fracture ,
damage of adjacent teeth , tooth or tooth fragments displacement
into soft tissues and bleeding . in cases
if the excessive intraoral
force was applied or / and part of bone was removed , risk of mandibular
fracture or damage of adjacent teeth is increased .
tooth or tooth fragments displacement into soft tissues can occur
in case of wrong operation technique .
the most serious and unpleasant iatrogenic
complication that arise from third molar surgery is inferior alveolar
and/or lingual nerve injury and neurosensory function disturbance .
the incidence of inferior alveolar nerve injury according to different
authors varies from 0.81% to 22% of cases [ 42 - 47 ] .
lingual nerve injury incidence was reported between 0.4% and 25%
[ 49 - 53 ] .
inferior alveolar nerve injury can cause paresthesia to complete
numbness and/or pain
in the region of the skin of the mental area , the lower lip , mucous
membranes , and the gingiva as far posteriorly as the second premolar
.
furthermore this commonly interferes with speech , eating , kissing ,
make - up application , shaving and drinking .
the injury of the lingual nerve leads to numbness of the ipsilateral
anterior two thirds of the tongue and taste disturbance .
typical postoperative complications
are pain , swelling , bruising , trismus ,
osteitis and surgical site infection .
classifications and risk factors identification in order to minimise number of complications
during mandibular third molar extraction several classifications
have been developed that are assessing the difficulty of surgical
procedure and helping to create an optimal treatment plan .
the most
popular are winter s
and pell and gregory s
systems who are classifying the inclinations and positions of the
third molars based on the relation among the dental longitudinal
axis , occlusal plane and ascending mandibular ramus .
however some authors claim that these scales have little value for
predicting the degree of extraction difficulty ,
mainly because these systems of classification introduce error of
interpretation by the observer .
later peterson
proposed a modification of the pell and gregory scale that included
a third factor , the angulation of the molar ( mesio - angular , horizontal ,
vertical or disto - angular ) .
clinical studies showed that there is
no doubt about the importance of individual parameters of mentioned
above classifications .
chuang et al .
demonstrated in their study that the level of impaction is associated
with an increased risk of inflammatory complications following third
molar surgery .
carvalho and vasconcelos
extracted 473 mandibular third molars for 285 patients and concluded
that root number ( p < 0.004 and morphology ( p < 0.031 ) , tooth position
( p = 0.001 ) , periodontal space ( p <
0.004 ) and second molar relation
( p = 0.001 ) were significant predictors of surgical difficulty .
authors mentioned that not all significant predictors of surgical
difficulty should be considered indicators of complications .
akadiri
and obiechina
demonstrated in their study wisdom tooth depth angulation and root
morphology as the most consistent determinants of extraction difficulty .
eruption status of the lower third
molar is important risk factor for inferior alveolar nerve injury .
incidences of inferior alveolar nerve injury in fully erupted , partially
erupted and unerupted lower wisdom teeth were 0.3% , 0.7% and 3.0% ,
respectively .
the risk of nerve injury is increasing with the depth of the impacted
mandibular wisdom teeth .
it was demonstrated the relationship between pattern of impaction
and inferior alveolar nerve injury .
the incidence of nerve injury
was highest in horizontally impacted lower wisdom teeth ( 1.7% ) ,
followed by distal impaction ( 1.4% ) , mesial impaction ( 1.3% ) and
vertical impaction ( 1.1% ) .
in general the proximity of the mandibular
third molar to the mandibular canal is considered a risk factor
for damage to the inferior alveolar nerve . this fact inspirited
further studies for the predictive radiographic parameters identification .
rood and shehab
distinguished four radiographic indicators observed in the tooth
root ( darkening , deflection and narrowing of the root , and a bifid
root apex ) , and the other three in the canal ( diversion , narrowing ,
and interruption in the white line of the canal ) .
studies demonstrated
that the most important parameters for inferior alveolar nerve injury
prediction are third molar root apices inside or in contact with
the mandibular canal [ 46,67 - 69 ] .
furthermore , the prevalence of post - extraction complications correlated
with the absence of cortication around the mandibular canal .
( 179 patients and 259 teeth )
where the overall prevalence of paresthesia was 4.2% .
in contrast ,
the prevalence of paresthesia in group involving an interrupted
mandibular canal cortex was 11.8% .
ueda et al .
performed similar study ( 99 patients and 145 teeth ) and showed that
inferior alveolar nerve injury was observed in 7 of 145 cases ( 4.8% ) .
all 7 cases exhibited absence of cortication . leung and cheung
in literature review demonstrated that 16.2% of the surgery with
the inferior alveolar nerve exposed developed postoperative inferior
alveolar nerve deficit , whilst only 1.1% of the surgeries without
nerve exposure developed inferior alveolar nerve deficit ( p < 0.0001 ) .
the risk ratio of inferior alveolar nerve injury from intraoperative
nerve exposure is 14.9 times more likely than if the nerve is not
exposed .
iatrogenic injury to the lingual
nerve may happen during third molar surgery due to the anatomical
proximity of the cortex region of the molar to the nerve , being
separated from it by the periosteum alone .
surgery on unerupted mandibular third molars was at higher risk
( 5.8% ) of lingual nerve injury compared with erupted ( 0.3% ) or partially
erupted ( 2.0% ) teeth ( p < 0.0001 ) .
the incidence of lingual nerve injury was highest in distally impacted
lower wisdom teeth ( 4.0% , p < 0.01 ) , followed by horizontal impaction
( 2.8% ) , mesial impaction ( 2.4% ) and vertical impaction ( 1.9% ) .
the risk ratio of lingual nerve injury was 1.94 times more likely
to occur if the lingual flap was raised than if it was not and 4.1
times more likely to occur if the lingual split technique was used
in comparison with the buccal approach .
mandibular third molar impaction classification based on anatomical and radiologic features new mandibular third molar impaction
and extraction difficulty degree classification based on anatomical
and radiologic findings and literature review results is suggested
( table 1 ) .
mandibular third molar impaction classification ian = inferior alveolar nerve ; ln = lingual nerve .
classification of mandibular third
molar impaction and extraction difficulty degree enables the clinician
to determine the difficulty in removal of the impacted tooth , to
choose optimal treatment and to avoid the majority of possible complications .
this classification describes wisdom tooth relation to the adjacent
anatomical structures : mandibular ramus , second molar , alveolar
crest , mandibular canal , and the spatial position of the tooth .
wisdom tooth position assessment should be performed clinically
and using cbct and panoramic radiographic images .
the tooth position
according to the all aforementioned landmarks has been not completely
classified yet .
proposed classification is determining mandibular
third molar mesiodistal position ( in relation to the second molar
- m and the mandibular ramus - r ) , apicocoronal position ( in relation
to the alveolar crest - a , and the mandibular canal - c ) , buccolingual
position ( in relation to mandibular lingual and buccal walls - b )
and spatial tooth position - s. risk degree of presumptive intervention is scored as follows : conventional extraction is determined ,
when all parameters are equal to score 0 ; simple , when at least one parameter
is equal to score 1 and surgical extraction with coronectomy
and/or sectioning of roots is determined ; moderate , when at least one parameter
is equal to score 2 and surgical extraction with coronectomy
and/or sectioning of roots is determined ; complicated , when at least one
parameter is equal to score 3 and surgical extraction with coronectomy
and/or sectioning of roots is determined .
extraoral approach
can be indicated . to make the classification more informative ,
each component of the indices ( m , r ,
for example , position , extraction difficulty degree of tooth 48
and risk of trigeminal nerve damage during surgery is described
as following : m1,r1,a2,c2,b1,s3 ( figure
1a , b ) .
detailed explanation :
crown is in contact below the equator to the coronal third of the
second molar ( m1 ) , partially impacted in the ramus ( r1 ) , widest
part of the crown ( equator ) is below the bone ( a2 ) , roots are contacting
or penetrating the mandibular canal , wall of the mandibular canal
is unidentified ( c2 ) , tooth is located in the middle between lingual
and buccal walls ( b1 ) ; horizontal spatial position ( s3 ) .
complicated
extraction is anticipated and c2 value presumes moderate risk of
inferior alveolar nerve damage . a = tooth no .
b = impaction in horizontal spatial position index ( s3 )
predicts complicated surgical extraction .
there are some new approaches in
assessing different anatomical and radiological parameters in the
present classification .
for example , the depth of tooth impaction
in pell and gregory s
classification was assessed according to the occlusal plane , but
in some cases the crown of wisdom tooth is small in size and located
below occlusal plane .
the assessment of tooth impaction ( coronal
position ) should be evaluated from the alveolar crest , because the
extraction difficulty is determined predominantly by the depth of
impaction in the bone .
furthermore , it is necessary to highlight
the lower landmark of the possible apicocoronal wisdom tooth position
which is determined by mandibular canal .
it was mentioned above
that the proximity of the mandibular third molar to the mandibular
canal is considered a risk factor for damage to the inferior alveolar
nerve . in contrast , some previous classifications recommended assessing
too many radiological parameters determining wisdom tooth roots
relationship with mandibular canal .
for example , rood and shehab
distinguished four radiographic indicators observed in the tooth
root ( darkening , deflection and narrowing of the root , and a bifid
root apex ) , and the other three in the canal ( diversion , narrowing ,
and interruption in the white line of the canal ) .
latest clinical
studies demonstrated that the most important parameters for inferior
alveolar nerve injury prediction are third molar root apices inside
or in contact with the inferior alveolar canal [ 46,67 - 69 ]
and absence of cortication around the inferior alveolar canal [ 70 - 72 ] ,
this is why mentioned above parameters were included into inferior
alveolar nerve injury risk evaluation assessment . in such cases
clinicians should avoid apical pressure during root elevation or
even to perform multiple sectioning of the tooth to reduce any stress
to a root on elevation .
cbct scan should be also accomplished for
detailed surgery planning in cases when c2 or c3 relation to the
mandibular canal is expected on two - dimensional radiographs ( figure 2a , b ) .
some authors are recommending to perform coronectomy
of impacted wisdom tooth if roots are surrounding the mandibular
canal because there is high risk or inferior alveolar nerve injury
.
in contrast , it was considered that in cases when wisdom tooth position
is 3 mm away from the mandibular canal , there is no risk to damage
mandibular canal during surgical extraction ( figure 3 ) .
a = on orthopantomograph close contact between impacted
right mandibular third molar and mandibular canal is suspected .
b = more detailed view on the cbct images reveals tooth
penetration through the mandibular canal wall ( c2 ) and moderate
risk of inferior alveolar nerve damage . roots of tooth no .
48 are 3 mm away from the mandibular
canal ( c0 ) on the orthopantomograph .
mesiodistal position is defined in
relation to the second molar and the mandibular ramus .
it is important
to assess impacted tooth relationship to the second molar in order
to avoid iatrogenic tooth traumatisation .
the impaction degree of
mandibular third molar in the ramus of mandible is associated with
extraction operation difficulty score and postoperative complications
manifestation .
for example , high risk degree is registered when
tooth is completely impacted in the mandibular ramus in distoangular
or horizontal position ( figure 4 ) .
38 , completely impacted in the mandibular ramus
in distoangular position and classified as a3 and r3 according
to the relation to alveolar crest and mandibular ramus ,
is noticed on the orthopantomograph .
buccolingual third molar position
in relation to mandibular lingual and buccal walls is reflecting
risk of lingual nerve injury .
it was discussed previously that iatrogenic
injury to the lingual nerve may happen during third molar surgery
due to the anatomical proximity of the cortex region of the molar
to the nerve .
surgery on unerupted mandibular third molars was at higher risk
( 5.8% ) of lingual nerve injury compared with erupted ( 0.3% ) or partially
erupted ( 2.0% ) teeth ( p < 0.0001 ) .
thus the highest risk of lingual nerve injury was scored in case
when the tooth is partially impacted or completely encased in the
bone ( a2 or a3 ) and is located closer to the lingual wall .
spatial mandibular third molar position
is reflecting extraction difficulty degree especially in combination
with other indices .
for example distoangular or horizontal impacted
tooth position in combination with deep impaction in the mandibular
ramus , can be complicated case even for experienced clinician .
there are selected only the most
informative parameters in presented herein classification , because
it is impossible to reflect all important parameters , such as periodontal
ligament width , soft tissue condition , patient characteristic , clinician s
experience , and et cetera in one classification which should be
useful in daily practice .
the classification proposed here based
on anatomical and radiological impacted mandibular third molar features
is promising to be a helpful tool for impacted tooth assessment
as well as for planning for surgical operation .
further clinical
studies should be conducted for new classification validation and
reliability evaluation .
| abstractobjectivesthe purpose of present
article was to review impacted mandibular third molar aetiology , clinical
anatomy , radiologic examination , surgical treatment and possible complications ,
as well as to create new mandibular third molar impaction and extraction
difficulty degree classification based on anatomical and radiologic findings
and literature review results.material and methodsliterature
was selected through a search of pubmed , embase and cochrane electronic
databases .
the keywords used for search were mandibular third molar , impacted
mandibular third molar , inferior alveolar nerve injury third molar , lingual
nerve injury third molar .
the search was restricted to english language
articles , published from 1976 to april 2013 . additionally , a manual search
in the major anatomy and oral surgery journals and books was performed .
the publications there selected by including clinical and human anatomy
studies.resultsin total 75 literature
sources were obtained and reviewed .
impacted mandibular third molar aetiology ,
clinical anatomy , radiographic examination , surgical extraction of and possible
complications , classifications and risk factors were discussed .
new mandibular
third molar impaction and extraction difficulty degree classification based
on anatomical and radiologic findings and literature review results was
proposed.conclusionsthe classification
proposed here based on anatomical and radiological impacted mandibular third
molar features is promising to be a helpful tool for impacted tooth assessment
as well as for planning for surgical operation .
further clinical studies
should be conducted for new classification validation and reliability evaluation . |
type 2 diabetes mellitus is characterized by hyperglycemia , insulin resistance , and an impairment in insulin secretion . in the late nineteenth century , william osler described diabetes as a rare disorder more likely to develop in obese people and patients with gout .
he estimated its prevalence as approximately two to nine cases per 100,000 population in the usa and europe being more common in the latter .
diabetes , one of the leading causes of death in the united states , affects over 200 million people worldwide .
the estimated prevalence of diabetes among adults in the united states ranges from 4.4 to 17.9 percent .
the community - based framingham heart study , in a predominantly non - hispanic white population , found a doubling in the incidence of type 2 diabetes over the last 30 years . identifying the etiology of type 2 diabetes is a key to its prevention .
studies have documented high rates of type 2 diabetes in the absence of classic obesity .
this suggests that other risk factors besides obesity might play a role in the epidemic of type 2 diabetes .
fructose is a simple sugar present in fruits and honey and is responsible for their sweet taste .
however , the major source of fructose worldwide is sucrose or table sugar , which is derived from sugar cane and sugar beets .
it is man - made , first developed in new guinea and in the indian subcontinent and was a rare and expensive commodity that was introduced into europe via venice , italy , and other trading ports during the middle ages .
after ingestion , sucrose is degraded in the gut by sucrase , releasing fructose and glucose that are then absorbed .
in addition to sucrose , the other major source of fructose is high fructose corn syrup ( hfcs ) , which was introduced in the early 1970s as an additional sweetener .
hfcs consists of fructose and glucose mixed in a variety of concentrations , but most commonly as 55% fructose and 45% glucose . in the united states ,
hfcs and sucrose are the major sources of fructose in the diet , and hfcs is a major ingredient in soft drinks , pastries , desserts , and various processed foods [ 6 , 7 ] . despite the similarity in their chemical structures , fructose and glucose
are metabolized in completely different ways and utilize different glut transporters . in the liver ,
fructose bypasses the two highly regulated steps of glycolysis , catalyzed by glucokinase / hexokinase and phosphofructokinase both of which are inhibited by increasing concentrations of their byproducts .
instead , fructose enters the pathway at a level that is not regulated and is metabolized to fructose-1-phosphate primarily by fructokinase or ketohexokinase ( khk ) ( figures 1 and 2 ) .
fructose may also be metabolized by hexokinase ; however , the km for fructose is much higher than glucose , and hence minimal amounts of fructose are metabolized via this pathway .
fructokinase has no negative feedback system , and atp is used for the phosphorylation process . as a result , continued fructose metabolism results in intracellular phosphate depletion , activation of amp deaminase , and uric acid generation which is harmful at the cellular level [ 6 , 9 , 10 ] .
fructose-1-phosphate is subsequently converted to dihydroxyacetone - phosphate and d - glyceraldehyde by the action of the aldolase b. d - glyceraldehyde is phosphorylated and continued downstream in the glycolysis pathway to form pyruvate .
two of the most energetic reactions of all organophosphates are in the pathway of fructose metabolism , catalyzed by phosphoglycerate and pyruvate kinases .
two atp molecules as well as free energy , approximately 12 kcal / mole , are released .
fructose controls the activity of glucokinase , the principle enzyme of glucose metabolism in the liver .
this improvement is primarily mediated by the activation of hepatic glucokinase resulting in improved liver glucose uptake .
uric acid , a byproduct of fructose degradation , stimulates khk expression through the activation of the transcription factor chrebp , which in turn results in the transcriptional activation of khk by the binding to a specific sequence within its promoter .
acid inhibits endothelial no both in vivo and in vitro , and directly induces adipocyte dysfunction .
serum uric acid increases rapidly after ingestion of fructose , resulting in increases as high as 2 mg / dl within 1 hour [ 1719 ] .
visceral adiposity contributes to hepatic triglyceride accumulation , protein kinase c activation , and hepatic insulin resistance by increasing the portal delivery of free fatty acids to the liver .
khk - c is primarily expressed in the liver , kidney , pancreas , and duodenum , while khk - a is expressed more widely including adipose tissue , heart , and the adrenal gland .
khk - a has a higher km for fructose ( 7 mmol / l ) than does khk - c ( 0.8
recently , it was found that adiposity and metabolic syndrome were prevented in mice lacking both khk isoforms but exacerbated in mice lacking khk - a .
it was also demonstrated that neither khk isoform is required for normal growth and development in rats .
serum leptin , triglycerides , and fasting blood glucose levels are higher in humans placed on a high fructose diet for four weeks compared with those on a starch - based diet .
deficiency of khk - c , an autosomal recessive inborn error of metabolism , results in essential fructosuria with an estimated incidence of 1 : 130,000 .
it is an anomaly rather than a disease , since it does not lead to any outward signs or symptoms .
it has no metabolic or morbid manifestations other than having transient fructosuria after meals containing either sucrose or fructose .
this condition used to be detected during routine medical examination when tests based on reducing properties of glucose like benedict 's solution and clini test were used to diagnose diabetes .
these tests have since been replaced by the more specific glucose oxidase method which does not react with fructose ; therefore , patients with essential fructosuria are no longer being identified .
moreover , the lack of treatment consideration and counseling for affected individuals and their family members , the absence of screening recommendations , and the lack of serum khk assay makes it difficult to identify subjects with this anomaly .
this is in contrast to hereditary fructose intolerance , a disease characterized by the deficiency of fructose-1-phosphate aldolase which has significant metabolic and developmental complications that manifest themselves as early as in the neonatal period . in a well - characterized family ,
in which three of eight siblings have fructosuria , all affected individuals are compound heterozygotes for the mutations gly40arg and ala43thr .
comparison of the 19771978 nfcs analysis with the analyses of nhanes for the period 19992004 indicated that , over the intervening period , mean individual intake of total fructose increased by 32% .
hfcs now represents nearly 50% of caloric sweeteners use in the united states [ 30 , 31 ] .
increased total fructose consumption has been implicated in the development of the obesity epidemic in the united states with the consumption of hfcs increased > 1000% between 1970 and 1990 , far exceeding the changes in intake of any other food or food group .
several reviews have concluded that intake of both fructose and hfcs by children and adults was associated with an increased risk of obesity and metabolic syndrome [ 3337 ] .
however , not all published meta - analyses have reported a statistically significant link [ 3841 ] .
recently , sievenpiper and colleagues concluded in a meta - analysis of controlled feeding trials that fructose does not cause weight gain when substituted for other carbohydrates in isocaloric trials .
this was criticized on the basis of fructose causing weight gain by altering appetite resulting in increased food intake , by inducing leptin resistance and by a direct effect on the brain indicating that isocaloric trials do not show a difference in weight gain between groups . moreover , the use of weight gain as a marker is subject to debate , since other fructose effects , beyond body weight as central fat accumulation and insulin resistance , can be more important . in children ,
a prospective cohort analyses conducted from 1991 to 1999 among nondiabetic women in the nurses ' health study ii concluded that higher consumption of sugar - sweetened beverages was associated with greater magnitude of weight gain and an increased risk for the development of type 2 diabetes . on the other hand ,
a recent analysis of nhanes 19992006 databases comparing data from > 25,000 subjects showed no relation between daily fructose intake and the indicators of metabolic syndrome , uric acid , and bmi .
thus , the intake data may not truly represent the long term , consistent consumption of food .
moreover , due to no fructose intake data being available in nhanes databases and a lack of fructose content data for many food items in the usda national nutrient database for standard reference , the fructose intake of individuals was indirectly estimated using several databases .
interpretation of cross - sectional studies examining the relationship of sugar intake to obesity can be misleading due to the fact that subjects who become obese may well reduce their sugar intake , since sugar is widely recognized to cause weight gain .
therefore , examining the relationship of sugar to obesity is best performed with well controlled , long - term longitudinal studies .
it is also important to recall that leptin resistance leading to liporegulatory failure , and , subsequently , insulin resistance can be perpetuated once obesity and intracellular lipid accumulation are manifest , especially in sites other than adipose tissue including pancreatic b cells and cardiomyocytes .
this further adds to the complexity and clouds the interpretation of these epidemiological data [ 6 , 47 , 48 ] .
the relationship between fructose intake and hypertension was also examined in several clinical studies . in a randomized controlled trial , high dose fructose ( 200 gm/24 h ) increased ambulatory blood pressure and elevated fasting insulin levels . in this study ,
allopurinol prevented the increase in mean arterial blood pressure . in an analysis of the nhanes
20032006 data , fructose intake , in the form of added sugar , was independently associated with higher blood pressure levels .
fructose consumption in the form of sugar - sweetened beverages was associated with hypertension and elevated uric acid level in usa adolescents but not in adults .
uric acid may raise systemic blood pressure by increasing inflammation , activating the renin - angiotensin system , and decreasing nitric oxide production contributing to renal vasoconstriction that results in salt insensitive hypertension .
persistent vasoconstriction may contribute to arteriosclerosis and the subsequent development of salt - sensitive hypertension , even if the hyperuricemia is corrected .
this may explain the different results in the preceding two studies that looked at two different age groups .
a recent meta - analysis of controlled feeding trials found that isocaloric substitution of fructose with other carbohydrates did not adversely affect blood pressure in humans suggesting that there is a need for long - term and large trials to clarify these findings .
few studies have examined the role of naturally occurring sugars , for example , 100% fruit juice , in the origin of obesity and related end points .
it is believed that fructose from natural sources can be less harmful because the presence of additional nutrients and antioxidants . on the other hand , crystalline fructose , as in table sugar , and hfcs are regarded as less safe , since glucose present in these sugars can accelerate fructose absorption .
100% fruit juice consumption among usa adults is associated with lower insulin resistance and lower odds of obesity and metabolic syndrome .
what is interesting in this study is that 100% juice consumers had significantly higher white milk intake which , on its own and among other dairy products , has been shown to enhance weight loss [ 43 , 56 , 57 ] .
there is mounting evidence from studies looking at the association between fructose and obesity and metabolic syndrome that primary fructose malabsorption in children was negatively associated with obesity . in obese african - americans ,
high rates of fructose malabsorption were associated with reduced liver fat thought to be protective against fatty liver disease . in conclusion , it is evident that there is a need for clinical trials with variable amounts of fructose intake to determine effects on metabolic outcomes rather than depending on meta - analyses of existing studies of mixed design and duration .
adipocyte development in mice and humans follows a well - defined pathway that begins with a common stem cell mediated adipocyte regeneration and is referred to as adipogenesis .
the first step of adipogenesis is the generation and commitment of mesenchymal stem cells ( mscs ) to adipocyte lineage .
the effect of fructose - mediated renin - ang ii activation is on the stages of cell differentiation ( commitment ) and involves local and systemic effects .
briefly , the stages of adipocyte differentiation are affected by increases in the levels of fructose and ang ii which cause msc - derived adipocyte growth arrest , clonal expansion , and early differentiation .
ang ii blockade can prevent terminal differentiation leading to the development of the mature adipocyte phenotype [ 62 , 63 ] .
normal diets result in the activation of ppar followed by adipose expansion through adipocyte hyperplasia , resulting in an increased number of new preadipocytes .
the resulting adipocytes are small in size and effectively store lipids , thereby reducing lipotoxicity in the liver and adipose tissue and release adiponectin [ 64 , 65 ] .
activation of these genes leads to repartitioning of lipids resulting in an increased triglyceride content of adipose tissue , a lowered free fatty acid content in circulation and availability for liver and muscle use , thereby improving insulin sensitivity .
methylisobutylxanthine ( mix ) , an phosphodiesterases inhibitor , increases intracellular camp , activating adipocyte differentiation in a pka - independent manner .
mix also increased the expression of c / ebp- , required for the subsequent expression of ppar- .
although , the mechanisms by which fructose controls adipogenesis in vivo are largely unknown , there are a number of candidates that mediate adipocyte differentiation in culture and are thought to control adipocyte accumulation and function in vivo .
two main factors fit this criterion : ( 1 ) high fructose diets , ( 2 ) increases in ros .
they have been implicated as the link between adipogenesis and metabolic diseases including t2 dm .
recent studies demonstrated that the induction of oxidative stress by high fructose or glucose increased nad(p)h oxidase and the mitochondrial respiratory chain which is associated with diabetic complications .
therefore , fructose diets may lead to adipocyte differentiation associated with adipocyte dysfunction and formation of adipocytes external to normal adipocyte depots , that is , muscle , liver , and pancreas leading to advanced diabetic complications .
the fructose - mediated increase in ros via activation of the adipocyte renin - ang ii system may lead to adipocyte dysfunction and insulin resistance .
adipose tissue is a key endocrine organ , the function of which , via interaction with the vascular endothelium system , regulates lipid uptake , storage , synthesis , and secretion of paracrine and autocrine factors that regulate insulin sensitivity .
however , fructose - mediated vascular dysfunction may have a negative effect on adipocyte function and the secretion of anti - inflammatory molecules such as adiponectin , il-1 , and il-10 .
the glucose or fructose - mediated decrease in vascular function increases adipocyte size resulting in decreased levels of adiponectin , but increased levels of mcp-1 , il-6 , and tnf- that have systemic effects on cells ( figure 4 ) . the adipocyte - mediated increase in adipokine
release plays a critical role in the regulation of blood pressure ( angiotensinogen ) , vascular haemostasis , and angiogenesis .
the release of these cytokines by adipocytes suggest that fructose - mediated diabetes may be related to systemic effects which include altered adiposity and insulin resistance .
adipocyte dysfunction occurs as a consequence of chronic overfeeding of fructose leading to adipocyte enlargement and inflammation and mitochondrial dysfunction ( figure 5 ) .
mitochondria play an important role in adipocyte differentiation and function . during the early stages of preadipocyte development ,
an increased number of mitochondria are required , resulting in small mature adipocytes , highly sensitive to insulin , and secreting high levels of adiponectin . by contrast
the results of impaired mitochondrial function include increased ffa levels resulting in the accumulation of mitochondrial products including fatty acyl coenzyme a ( coa ) and reduced insulin sensitivity . in summary ,
fructose diets , inactivity , and gluttony results in adipocyte expansion with the resultant detrimental perturbations in the renin - ang ii system and in mitochondria , both of which undergo cellular changes that result in the increased generation of ros and tnf- , il-1 , and il-6 and a decrease in adiponectin levels .
adiponectin is synthesized and released only by the adipocyte and has an essential role in vascular and renal function .
increases in adipocyte release of adiponectin inhibits both the expression of hepatic gluconeogenic enzymes and the rate of endogenous glucose production in diabetic mice . in adiponectin transgenic mice , adiponectin reduced the expression of phosphoenolpyruvate carboxylase and glucose-6-phosphatase , which are associated with elevated phosphorylation of hepatic ampk and decreased glucose production [ 70 , 71 ] .
hypertension , diabetes , and obesity were originally documented in england and france where sugar first became available to the public .
the rise in sugar intake in the united kingdom and the united states also correlated with the rise in obesity rates observed in these countries . in the early 1900s , blood pressure in over 140,000 healthy adults who applied for life insurance in the new york region suggested that a blood pressure of 140 ( systolic)/90 ( diastolic ) mm hg was abnormal because it reflected only 5 - 6% of the population in the united states .
subsequent studies over the past century showed a significant and dramatic rise in the prevalence of hypertension in the united states [ 7274 ] ( figure 6(a ) ) .
body mass index ( bmi ; in kg / m ) > 30 was observed in only 3.4% of 50- to 59-year - old male veterans in 1890 , compared with 30.4% in 19992002 ( figure 6(b ) ) .
this is paralleled by the epidemic of sugar consumption that has worsened over the past 300 years . during the last decade ,
emerging data have altered our perspective on the link between fructose , uric acid , and hypertension .
epidemiologic data , in the form of large , longitudinal studies strengthened the link between elevated uric acid and hypertension [ 50 , 7579 ] . in animals ,
mild hyperuricemia induced by the uricase inhibitor oxonic acid , mimicking levels in humans , increased blood pressure by crystal - independent mechanism resulting in stimulation of the renin - angiotensin system and inhibition of nitric oxide synthase .
two - thirds of adolescents with newly diagnosed essential hypertension and elevated uric acid normalized their blood pressure when treated with the xanthine oxidase inhibitor allopurinol .
this study could not exclude the possibility that some or all of the observed effect could have been mediated by a reduction in superoxide production , a byproduct of xanthine oxidase function . a more recent study done by the same group confirmed these results by using two differently acting urate lowering drugs , allopurinol , and probenecid .
the first is salt insensitive which is likely to be managed by urate lowering drugs , while the second phase is salt sensitive . due to a paucity of outcome data , recommendations on how to treat uric acid associate hypertension
can not be made at this time although the mechanism appears clear , especially in the early stages before the development of salt sensitivity .
future clinical trials are required to include different levels of hypertension and different age groups before recommending urate lowering agents especially as they have an inferior efficacy profile when compared with antihypertensive medications presently in clinical use .
consumption of high - fructose chow by mice produced nocturnal hypertension and autonomic imbalance which may be related to activation of the sympathetic and ras systems .
subsequent data suggested that changes in autonomic modulation may be an initiating mechanism underlying the cluster of symptoms associated with cardiometabolic disease .
the earliest recorded metabolic perturbation resulting from fructose consumption is postprandial hypertriglyceridemia , which increased visceral adipose deposition .
visceral adiposity contributes to hepatic triglyceride accumulation , protein kinase c activation , and hepatic insulin resistance by increasing the portal delivery of free fatty acids to the liver . with insulin resistance , vldl production is upregulated and this , along with systemic free fatty acids , increase lipid delivery to muscle .
it is also possible that fructose initiates hepatic insulin resistance independently of visceral adiposity and free fatty acid delivery .
splanchnic perfusion studies have shown that hepatic production of triglycerides is much greater with fructose compared with equimolar concentrations of glucose .
unlike glucose , fructose does not stimulate insulin secretion , due to its hepatic metabolism and the low level of expression of the fructose transporter glut5 in pancreatic -cells .
consumption of fructose - sweetened beverages with meals produced a rapid and prolonged elevation of plasma triglycerides compared with glucose - sweetened beverages . because insulin , leptin , and possibly ghrelin function as key signals to the central nervous system in the long - term regulation of energy balance , decreases of circulating insulin and leptin and increased ghrelin concentrations could lead to increased caloric intake and ultimately contribute to weight gain and obesity during chronic consumption of diets high in fructose .
apolipoprotein b levels were found to be higher following fructose consumption compared with isocaloric amount of glucose .
in fact , countries electing to use hfcs in their food supply have a 20% higher prevalence of diabetes compared to countries that did not use hfcs independent of obesity .
uric acid stimulates fructokinase and the development of nafl via an increase in fructose metabolism thereby increasing the development of type 2 diabetes in children .
this may be related to an increase in srebp-1c and reduced acyl - coa oxidase during pregnancy .
body size at birth is related to food intake and the content of fructose or an elevation of estrogen during pregnancy in women with a family history of type 2 diabetes [ 94 , 95 ] .
genetic morphism in the glucokinase regulatory protein , which binds to glucokinase and inhibits its activity in the presence of fructose-6-phosphate ( f6p ) is associated with ethnicity and may be responsible for different response rates to obesity and diabetes in different populations .
an increase of fructose-1,6-biophosphatase , or in angiotensin 17 decreases fructose - mediated diabetes by an increase in pancreatic islet metabolism .
based on the present knowledge of fructose and its detrimental metabolic effects when in excess and the unique nature of khk , it is clear that fructose is , at least , partially responsible for the pandemic of diabetes and the metabolic syndrome that is presently occurring .
a number of therapeutic approaches appear viable as a result of the data outlined previously .
( 1 ) assessing khk activity in human blood samples opens a new approach to the diagnosis as well as the treatment of type 2 diabetes .
we hypothesize that individuals either completely or partially deficient in khk activity are immune at variable levels from developing type 2 diabetes mellitus . in order to test this hypothesis , the following require clarification : ( a ) khk - c is primarily expressed in the liver and kidney .
thus , the in vivo handling of fructose to assess enzyme expression and efficiency would be difficult to monitor .
we propose that pmn expression will parallel hepatic expression , a series of pilot clinical studies in individuals selected across populations at varying risk for type 2 diabetes mellitus is required .
( b ) once this association has been demonstrated , measurement of khk - c activity in pmn 's in young adults with type 2 diabetes and age / sex matched controls is required .
if results support the hypothesis , new approach to the prediction as well as a therapeutic approach to treat type 2 diabetes mellitus is at hand .
( 2 ) interference with fructose transport at the glut transporter level diminishes the availability of fructose . due to their hydrophilic nature
several transporters have been identified , and these include the facilitative glucose transporter ( glut ) which primarily regulates the clearance of blood glucose along a concentration gradient and the sodium - coupled glucose cotransporter ( sglt ) family members that are distinct at both the primary and secondary structural levels from the glut proteins .
expression of sglt proteins is restricted to the gut and kidney , where their role is energy - dependent reabsorption of glucose from lumen .
the glut transporter family comprises 13 members that exhibit tissue distribution , substrate specificity , and transport kinetics that reflect their physiologic role .
fructose gains access to the circulation and hepatocytes via glut 5 , glut 7 , and glut 11 .
interference with fructose transport at the level of these transporters represents a therapeutic approach to prevent fructose - induced adiposity and insulin resistance .
( 3 ) interference at the level of khk is an attractive approach to modify the metabolism of fructose and , possibly , alleviating adiposity and vascular dysfunction .
( a ) an inhibitor is currently being developed ( richard johnson , patent number wo/2012/019188 , public knowledge ) .
it remains unclear how effective this approach will be , and if this intervention will be dependent on an individual 's khk activity profile as suggested previously .
( b ) the use of certain furanose sugars , which can also be substrates for khk and compete with fructose for its metabolism to hexose-1-phosphate .
d - tagatose , a furanose sugar metabolized by khk , when compared with fructose , caused markedly higher levels of serum uric acid and lower of pi .
this highlights the importance of the poorly regulated khk in this pathway and necessitates further study .
( 4 ) therapeutic agents that decrease the effect of high fructose diets on diabetes and insulin resistance include statins , metformin , and renin inhibitor attenuated diabetes and insulin resistance .
the latter is due to a decrease in lipid peroxidation . in support of these observations ,
candesartan cilexetil and losartan improve renal hemodynamic and insulin resistance and lower blood pressure in diabetic rodents fed a high fructose diet presumably by the activation of amp - activated protein kinase .
infusion of ang ii decreased adiponectin and potentiated fructose - mediated insulin resistance in fructose - fed rats .
the presence of renin - angiotensin aldosterone in adipose tissue has been described [ 110112 ] in which ang ii increased nadph oxidase activity and wat mediated inflammation and blocked raas thereby preventing the onset of diabetes . blocking ros via the inhibition of ang ii converting enzyme reduced obesity in rats and dysregulation of inflammatory cytokines released by adipocytes [ 114 , 115 ] .
( 5 ) uric acid can be a byproduct of uncontrolled fructose metabolism due to the rapid consumption of atp as noted previously .
in contrast , uric acid also functions as an antioxidant . treating hyperuricemia in the absence of gout
( 6 ) the ideal and logical , but most difficult , approach would be to modify fructose content in food .
the reason that khk is a poorly regulated enzyme may be due to the high levels of physical activity and limited fructose intake that was prevalent in humans before the introduction of refined cane sugar and high fructose corn syrup ( hfcs ) .
this does not exist today where calorie sweeteners , including hfcs , are part of the normal diet . moreover , and of grave concern , is the increased consumption of sugar - sweetened beverages , and fresh and processed juices which provide an easy vehicle for excessive sugar intake , over very a short period of time and have been directly linked to obesity [ 35 , 120 ] and type 2 diabetes [ 45 , 121 ] . at the end of the day , we might conclude that only drinking
the present choice is either to continue with the currently high rates of fructose consumption which lead to adipocyte expansion , obesity , and hypertension or to minimize fructose intake and alleviate health care cost and the cost of managing hypertension and its complications , thereby improving the lots of health care professional , patients , and their families and the well - being of millions of individuals .
the consequences of uncontrolled fructose metabolism can be harmful at the cellular level resulting in intracellular atp depletion , increased uric acid production , endothelial dysfunction , oxidative stress , and increased lipogenesis .
high fructose consumption induces insulin resistance and other manifestations of metabolic syndrome in a series of animal models .
human epidemiological data are generally of poor quality due to the lack of consistency in study design , methodology , and length .
it remains unclear if targeting fructose by interfering with its transport or metabolism can be of any clinical benefit . | diabetes mellitus and the metabolic syndrome are becoming leading causes of death in the world . identifying
the etiology of diabetes is key to prevention . despite the similarity in their structures , fructose and glucose
are metabolized in different ways .
uric acid , a byproduct of uncontrolled fructose metabolism is known risk factor for hypertension . in the liver ,
fructose bypasses the two highly regulated steps in glycolysis , glucokinase and phosphofructokinase , both of which are inhibited by increasing concentrations of their byproducts .
fructose is metabolized by fructokinase ( khk ) .
khk has no negative feedback system , and atp is used for phosphorylation .
this results in intracellular phosphate depletion and the rapid generation of uric acid due to activation of amp deaminase .
uric acid , a byproduct of this reaction , has been linked to endothelial dysfunction , insulin resistance , and hypertension .
we present possible mechanisms by which fructose causes insulin resistance and suggest actions based on this association that have therapeutic implications . |
we report the first autopsy findings of a personalized external aortic root support ( pears ) operation , which confirm the incorporation of the mesh into the aortic adventitia as was shown in sheep .
our patient had a typical marfan phenotype with a characteristic habitus , pectus excavatum and a dislocated lens .
his aortic root diameter was 42 mm at the level of coaptation of the leaflets when measured by magnetic resonance imaging ( mri ) prior to operation in september 2008 ( fig .
echocardiography showed a functioning bicuspid aortic valve and dilated cardiomyopathy with mildly impaired left ventricular function : left ventricular end - diastolic ( lvedd ) and left ventricular end - systolic dimensions ( lvesd ) were 5.4 and 3.8 cm , respectively . the left ventricular ejection fraction ( lvef ) was 49% .
figure 1:diastolic frames selected from complete cine acquisition in the long axis of the left ventricle and the short axis of the aortic valve acquired before ( a and c ) and 11 months after pears ( d and f ) .
the lv is dilated in both studies and the aortic valve is bicuspid with only mild aortic regurgitation and no stenosis . the thickened aortic wall at the site of pears can be seen ( arrow ) . the actual mesh support on the personalized former as used in this patient , derived by cad modeling ,
is shown above ( b ) and a depiction of it in position is shown below ( e ) .
cad : computer - aided design ; pears : personalized external aortic root support ; lv : left ventricle .
diastolic frames selected from complete cine acquisition in the long axis of the left ventricle and the short axis of the aortic valve acquired before ( a and c ) and 11 months after pears ( d and f ) .
the lv is dilated in both studies and the aortic valve is bicuspid with only mild aortic regurgitation and no stenosis . the thickened aortic wall at the site of pears can be seen ( arrow ) . the actual mesh support on the personalized former as used in this patient , derived by cad modeling ,
is shown above ( b ) and a depiction of it in position is shown below ( e ) .
cad : computer - aided design ; pears : personalized external aortic root support ; lv : left ventricle .
prophylactic aortic root surgery was advised and the patient requested pears . in december 2008 ,
aged 26 , he was the 16th patient to have this operation . a model of his aorta was made by computer - assisted design and rapid prototyping , commonly known as 3d printing . a supporting sleeve from a soft and pliable macroporous mesh
( john pepper ) , the ascending aorta was mobilized to the aortoventricular junction and the mesh , extending proximal to the coronary arteries , was tethered to the left ventricle ( fig .
intraoperative echocardiography showed his lvef and his right ventricular ejection fraction to be unchanged from baseline as was his mild aortic regurgitation .
he made an uncomplicated recovery and left hospital on the 6th postoperative day taking bisoprolol and lisinopril .
, he was sailing in cold sea water ( 5c ) when his dinghy capsized in strong winds .
ng / l ( normal < 0.1 ) and had returned to normal ( 0.09 ) 24 h later .
the electrocardiogram showed st depression in ii , iii , v4 and v5 , which resolved after 24 h. cardiac catheterization showed severe dilatation of the left ventricle with ballooning of the apex .
the wall motion abnormality of the lv was not in a coronary distribution and selective coronary arteriography showed normal coronary ostia and unobstructed coronaries .
left ventricular dimensions measured 5 days later after he had been transferred to the royal brompton hospital were lvedd 5.9 cm and lvesd 4.4 cm .
an mri scan performed eight days after the acute event on 2 november 2009 showed a return to the preoperative appearances of a moderately dilated left ventricle with mild global impairment of systolic function : left ventricular end - diastolic volume 250 ml ; left ventricular end - systolic volume 130 ml .
comparison with before the operation ( september 2008 ) showed that the aortic root dimensions were 42 and 41 mm ( before and after ) at the level of coaptation of the leaflets , and 35 mm at the sinotubular junction at both times ( fig.1a and d ) .
he rapidly improved , returned to work and was well when reviewed at 6 weeks and again 1 year later . in may 2013 , 4.5 years after his pears operation , he was found dead in bed . at autopsy ,
the external aortic mesh was fully incorporated in the adventitia and could not be separated from it .
there was no impingement on the coronary arteries or their orifices by the external support .
there was good coaptation of the valve leaflets and , on testing with water , there was minimal leak in keeping with the mild degree of aortic regurgitation known to have been present through his bicuspid aortic valve . the examining pathologist ( martin goddard ) found no reason to suspect that the mesh support had contributed to death .
a cross section from the unsupported arch of the aorta showed focal fragmentation of the elastic lamellae consistent with the mfs . in the supported portion ,
examination of the heart confirmed a dilated cardiomyopathy presumed to be related to mfs , as was the cause of death in his mother .
figure 2:(a ) sections from the unsupported aortic arch shows focal fragmentation of elastic fibres and a mild increase in mucopolysaccharides ( mag .
there is no root in contrast to ( c ) and the adventitia is not clearly defined as it is in the ascending aorta .
( b ) a high - power view of the media of the unsupported aortic arch ( mag .
( c ) section of the aortic root of a total thickness of 4.5 mm .
collagen fibres ( red staining ) pass through the interstices between the filaments of the root ( blue arrows ) embedding it in the adventitia .
foreign body - type giant cells and a few scattered chronic inflammatory cells are present ( mag .
( d ) high - power view of the protected aortic root wall ( mag .
the underlying media shows well - preserved elastic lamellae with no fragmentation , loss or pooling of mucopolysaccharides .
( a ) sections from the unsupported aortic arch shows focal fragmentation of elastic fibres and a mild increase in mucopolysaccharides ( mag .
2.5 ) . there is no root in contrast to ( c ) and the adventitia is not clearly defined as it is in the ascending aorta .
( b ) a high - power view of the media of the unsupported aortic arch ( mag .
( c ) section of the aortic root of a total thickness of 4.5 mm .
collagen fibres ( red staining ) pass through the interstices between the filaments of the root ( blue arrows ) embedding it in the adventitia . foreign body - type giant cells and a few scattered chronic inflammatory cells are present ( mag .
( d ) high - power view of the protected aortic root wall ( mag .
the underlying media shows well - preserved elastic lamellae with no fragmentation , loss or pooling of mucopolysaccharides .
( n.b . there has been minor image size adjustment to create the montage ) .
elective root replacement in mfs has greatly improved life expectancy in affected patients . as a result ,
three forms of surgery are now available : total root replacement with a valved conduit , valve sparing root replacement ( vsrr ) and pears with a macroporous mesh sleeve , manufactured to the patient 's own aortic dimensions .
pears is intended to prevent further expansion of root aneurysms and to preserve the architecture of the aortic valve support .
the operation is intended to be prophylactic , performed at an earlier stage in the natural history of mfs .
the patient 's own aortic images are used to create a faithful copy of the aorta by computer - aided design , which is then made into a physical copy by
3d printing. on this is made a macroporous fabric sleeve to be placed around the aorta , including the segment proximal to the coronary arteries , down to the aortoventricular junction .
the incorporation of the mesh in the outer layers of the aortic wall in this case is in accordance with the histological appearance previously shown by cohen et al . , who used a limited ad hoc mesh support with a similar material in 102 patients from 1984 to 2003 .
the distensibilty and therefore the afterload are similar to that with any tube graft aortic replacement and so there is no additional stress on the left ventricle
. one might expect any degenerative changes to be worse in the root than in the arch but the protected aortic root in this patient had normal histology .
this raises the intriguing possibility that the support enabled the aorta to heal by sparing it from the repetitive stress of systolic distension in an inelastic marfan aorta , but we have no histology prior to pears on which to base a firm conclusion . because the aorta is not allowed to dilate further , the major factor predisposing to dissection is obviated and this is likely to diminish the risk of dissection . in the now less likely event of dissection , the strong root / aorta composite would prevent intrapericardial rupture , the proximate cause of death in acute aortic dissection .
it will also provide a technically more secure aortic wall than native marfan aortic tissue if any further surgery were to be performed .
although this is an isolated case report , it shows that the external supporting root strengthens the aortic wall by becoming fully incorporated .
pears seeks to achieve the benefits of vsrr but , as a non - ablative procedure , it can be offered earlier . because it preserves the size and configuration of the aortic root , pears optimizes the chance of maintaining aortic valve function
funding to pay the open access publication charges for this article was provided by university college london . | in 2008 , a 26-year old man had personalized external aortic root support ( pears ) with a macroporous mesh .
he was the 16th of 46 patients to have this operation .
he had a typical marfan habitus .
his mother died of this disease as did his brother , with an aortic dissection .
the patient himself died suddenly 4.5 years after his pears operation . at autopsy
, there was no blood in the pericardium .
the coronary orifices and proximal arteries were normal .
his bicuspid aortic valve was minimally regurgitant as it was prior to operation and remained throughout follow - up .
macroscopically the implanted mesh was embedded in the adventitia and not separable from the aortic wall .
microscopically it was fully incorporated with collagen fibres as has been seen in our animal studies .
the unsupported aortic arch showed some focal fragmentation of elastic fibres and a mild increase in mucopolysaccharides consistent with marfan syndrome .
these appearances were not present in the supported aortic root , which had the histological appearance of a normal aorta .
he was the first patient to die with an implant .
the histological appearances suggest the possibility that the incorporated support of the aortic root allowed recovery of the microstructure of the media . |
the designs of the health professionals follow - up study ( hpfs ) and nurses health study ( nhs ) have been described ( 10,11 ) .
men , all health professionals , who were 4075 years of age in 1986 ; and the nhs enrolled 121,700 u.s .
women , all registered nurses , who were 3055 years of age in 1976 . in both cohorts , participants have been followed with biennial questionnaires on medical history , risk factors , lifestyle , and disease incidence . for the present analysis
, we used prospectively collected data on toenail mercury concentrations from prior nested case - control studies of incident cardiovascular disease in both cohorts ( 12,13 ) ( see supplementary data online for details ) .
the study was designed by the authors and approved by the human subjects committees of all author institutions .
funding for the present investigation was provided by the national institute of environmental health sciences , national institutes of health . from among nhs and hpfs participants who had stored toenail samples and were free of cardiovascular disease at the time of toenail sampling ( 19821983 in nhs
; 1987 in hpfs ) , we measured toenail mercury concentrations in 9,308 men and women , including 4,654 participants who went on to develop coronary heart disease ( chd ) or stroke during follow - up and 4,654 participants who did not develop chd or stroke during equivalent follow - up ( matched on month of toenail sample return , age , sex , race , and smoking status ) .
we excluded dentists from these measurements , as their toenail mercury also reflects occupational exposure to inorganic ( rather than methyl ) mercury ( 14 ) .
for the present analysis , we excluded 41 participants with prevalent diabetes at the time of toenail sampling , leaving a total of 9,267 individuals with measured toenail mercury concentrations who were free of prevalent diabetes at baseline .
toenail concentrations of mercury and selenium , which in some animal models mitigate toxicity of mercury ( 15 ) , were measured using neutron activation analysis by personnel unaware of the participants clinical information ( 12 ) .
see supplementary data for details on analytic methods , validity , and reproducibility of these measures
. potential cases of incident diabetes were identified from biennial questionnaires asking about any new physician - diagnosed diabetes .
these potential cases were then reviewed and confirmed using a validated supplementary questionnaire that obtained data on symptoms , diagnostic tests , and medical therapy .
incident diabetes was diagnosed according to original national diabetes data group criteria , based on the presence of one or more of the following : 1 ) classic symptoms plus a plasma glucose concentration 140 mg / dl ( 7.8
mmol / l ) in the fasting state or a randomly measured plasma glucose concentration 200 mg / dl ( 11.1
mmol / l ) ; 2 ) in the absence of symptoms , at least two elevated plasma glucose concentrations on different occasions ( 140 mg / dl fasting , 200 mg / dl randomly measured , or 200 mg / dl 2-h postoral glucose challenge ) ; or 3 ) drug treatment with hypoglycemic medication ( insulin or an oral hypoglycemic agent ) .
these diagnostic criteria were modified after 1998 to account for the new lower diagnostic cutoff for fasting glucose concentration of 126 mg / dl .
the validity of these supplementary questionnaire criteria for diagnosing diabetes in these cohorts has been demonstrated . in subsets of participants in whom
diabetes was diagnosed based on these criteria and then compared with a full review of medical records , 98% in nhs and 97% in hpfs had a confirmed diagnosis of diabetes ( 16,17 ) .
data on demographics , risk factors , and lifestyle habits were collected via validated self - administered questionnaires , using the closest report preceding toenail sample collection from each participant ( 18 ) .
information on weight and height was obtained ; self - reported weight was validated against technician - measured weight ( r = 0.96 ) ( 19 ) .
physical activity was assessed as metabolic equivalent tasks ( mets ) using validated questionnaires ( 20 ) .
usual dietary habits were assessed using validated semiquantitative food frequency questionnaires that inquired about usual consumption of foods , beverages , and supplements over the prior year ( 21,22 ) .
associations of toenail mercury concentrations with incident diabetes were evaluated using cox proportional hazards , with time at risk from the time of toenail sampling until the first event , death , or censoring at the date of return of the last questionnaire in 2008 .
mercury concentrations were evaluated as indicator categories in quintiles and also in deciles to evaluate a broader range of dose response . because toenail mercury measures were measured as part of a prior nested case - control study of incident cardiovascular disease , participants were weighted according to their inverse probability of being sampled from the overall cohorts ( sas proc phreg , normalize option ) .
individuals selected as future cardiovascular case subjects were given a weight of 1 , and control subjects received a weight of 17 in the nhs ( women ) and 24 in the hpfs ( men ) . with such weighting
tests for trend were performed by assigning participants the median value in their quintile of exposure and evaluating this as a continuous variable . statistical evaluation for interaction
was performed by multiplying this variable by the effect modifier of interest and evaluating the wald test for the multiplicative interaction term .
potential confounding was assessed using multivariable models adjusted for demographics , major diabetes risk factors , and lifestyle and dietary habits including fish and omega-3 fatty acid consumption .
multivariable modeling was guided by parsimony , clinical relevance of covariates , observed strength of association between covariates and exposure or outcome , and percent change in risk estimate when covariates were included .
we performed sensitivity analyses to minimize potential reverse causation due to the presence of potential unrecognized diabetes at baseline by excluding cases occurring during the first 2 years of follow - up , and to minimize potential misclassification due to exposure changes over time by restricting analyses to events within 10 years of toenail sampling .
we also performed sensitivity analyses restricting to symptomatic cases who reported at least one classic symptom at the time of diagnosis .
all analyses were performed using sas version 9.1 ( sas institute ) with two - tailed = 0.05 .
the designs of the health professionals follow - up study ( hpfs ) and nurses health study ( nhs ) have been described ( 10,11 ) .
men , all health professionals , who were 4075 years of age in 1986 ; and the nhs enrolled 121,700 u.s .
women , all registered nurses , who were 3055 years of age in 1976 . in both cohorts , participants have been followed with biennial questionnaires on medical history , risk factors , lifestyle , and disease incidence . for the present analysis
, we used prospectively collected data on toenail mercury concentrations from prior nested case - control studies of incident cardiovascular disease in both cohorts ( 12,13 ) ( see supplementary data online for details ) .
the study was designed by the authors and approved by the human subjects committees of all author institutions .
funding for the present investigation was provided by the national institute of environmental health sciences , national institutes of health . from among nhs and hpfs participants who had stored toenail samples and were free of cardiovascular disease at the time of toenail sampling ( 19821983 in nhs
; 1987 in hpfs ) , we measured toenail mercury concentrations in 9,308 men and women , including 4,654 participants who went on to develop coronary heart disease ( chd ) or stroke during follow - up and 4,654 participants who did not develop chd or stroke during equivalent follow - up ( matched on month of toenail sample return , age , sex , race , and smoking status ) .
we excluded dentists from these measurements , as their toenail mercury also reflects occupational exposure to inorganic ( rather than methyl ) mercury ( 14 ) .
for the present analysis , we excluded 41 participants with prevalent diabetes at the time of toenail sampling , leaving a total of 9,267 individuals with measured toenail mercury concentrations who were free of prevalent diabetes at baseline .
toenail concentrations of mercury and selenium , which in some animal models mitigate toxicity of mercury ( 15 ) , were measured using neutron activation analysis by personnel unaware of the participants clinical information ( 12 ) .
see supplementary data for details on analytic methods , validity , and reproducibility of these measures .
potential cases of incident diabetes were identified from biennial questionnaires asking about any new physician - diagnosed diabetes .
these potential cases were then reviewed and confirmed using a validated supplementary questionnaire that obtained data on symptoms , diagnostic tests , and medical therapy .
incident diabetes was diagnosed according to original national diabetes data group criteria , based on the presence of one or more of the following : 1 ) classic symptoms plus a plasma glucose concentration 140 mg / dl ( 7.8 mmol / l ) in the fasting state or a randomly measured plasma glucose concentration 200 mg / dl ( 11.1 mmol / l ) ; 2 ) in the absence of symptoms , at least two elevated plasma glucose concentrations on different occasions ( 140 mg / dl fasting , 200 mg / dl randomly measured , or 200 mg / dl 2-h postoral glucose challenge ) ; or 3 ) drug treatment with hypoglycemic medication ( insulin or an oral hypoglycemic agent ) .
these diagnostic criteria were modified after 1998 to account for the new lower diagnostic cutoff for fasting glucose concentration of 126 mg / dl .
the validity of these supplementary questionnaire criteria for diagnosing diabetes in these cohorts has been demonstrated . in subsets of participants in whom
diabetes was diagnosed based on these criteria and then compared with a full review of medical records , 98% in nhs and 97% in hpfs had a confirmed diagnosis of diabetes ( 16,17 ) .
data on demographics , risk factors , and lifestyle habits were collected via validated self - administered questionnaires , using the closest report preceding toenail sample collection from each participant ( 18 ) .
information on weight and height was obtained ; self - reported weight was validated against technician - measured weight ( r = 0.96 ) ( 19 ) .
physical activity was assessed as metabolic equivalent tasks ( mets ) using validated questionnaires ( 20 ) .
usual dietary habits were assessed using validated semiquantitative food frequency questionnaires that inquired about usual consumption of foods , beverages , and supplements over the prior year ( 21,22 ) .
associations of toenail mercury concentrations with incident diabetes were evaluated using cox proportional hazards , with time at risk from the time of toenail sampling until the first event , death , or censoring at the date of return of the last questionnaire in 2008 . the proportional hazards assumption was tested and not rejected based on schoenfeld residuals .
mercury concentrations were evaluated as indicator categories in quintiles and also in deciles to evaluate a broader range of dose response . because toenail mercury measures were measured as part of a prior nested case - control study of incident cardiovascular disease , participants were weighted according to their inverse probability of being sampled from the overall cohorts ( sas proc phreg , normalize option ) .
individuals selected as future cardiovascular case subjects were given a weight of 1 , and control subjects received a weight of 17 in the nhs ( women ) and 24 in the hpfs ( men ) . with such weighting , results can be interpreted as generalizable to the full cohorts .
tests for trend were performed by assigning participants the median value in their quintile of exposure and evaluating this as a continuous variable . statistical evaluation for interaction was performed by multiplying this variable by the effect modifier of interest and evaluating the wald test for the multiplicative interaction term .
potential confounding was assessed using multivariable models adjusted for demographics , major diabetes risk factors , and lifestyle and dietary habits including fish and omega-3 fatty acid consumption .
multivariable modeling was guided by parsimony , clinical relevance of covariates , observed strength of association between covariates and exposure or outcome , and percent change in risk estimate when covariates were included . missing covariates ( < 1% ) were imputed using multiple imputation ( 24 ) .
we performed sensitivity analyses to minimize potential reverse causation due to the presence of potential unrecognized diabetes at baseline by excluding cases occurring during the first 2 years of follow - up , and to minimize potential misclassification due to exposure changes over time by restricting analyses to events within 10 years of toenail sampling .
we also performed sensitivity analyses restricting to symptomatic cases who reported at least one classic symptom at the time of diagnosis .
all analyses were performed using sas version 9.1 ( sas institute ) with two - tailed = 0.05 .
at baseline , mean sd age was 61.2 8.9 years among men and 53.7 6.1 years among women ( table 1 ) .
the 95th percentile of toenail mercury concentration was 1.32 g / g in men and 0.76 g / g in women , corresponding to concentrations in hair of 3.67 and 2.11 g / g ( 12 ) , respectively , or 3.5- and 2-fold higher than the u.s .
environmental protection agency ( epa ) reference dose exposure corresponding to 1.0 g / g in hair ( 25 ) .
unadjusted ( bivariate ) associations of baseline characteristics of participants in these cohorts according to toenail mercury concentrations have been previously reported ( 12,13 ) . in brief , higher mercury concentrations were associated with more frequent hypercholesterolemia , slightly lower bmi , modestly higher physical activity , and greater alcohol use .
mercury concentrations were also positively associated with fish consumption ( spearman r = 0.39 ) and dietary factors related to fish consumption , including slightly lower intakes of saturated fat , monounsaturated fat , trans fat , and dietary cholesterol and slightly higher intakes of protein and polyunsaturated fat .
mercury concentrations were not significantly associated with age , smoking , family history of diabetes , or presence of hypertension .
* during 19.7 7.0 years of follow - up , 1,010 new cases of diabetes were diagnosed .
the median duration of follow - up from time of toenail sampling to diagnosis of diabetes was 12.7 years ( interquartile range 7.518.1 ) . in both age- and
sex - adjusted as well as multivariable - adjusted analyses , toenail mercury concentrations were not associated with higher incidence of diabetes in women , men , or overall ( table 2 ) . comparing the highest to the lowest quintile of mercury exposure ,
the fully adjusted hazard ratio ( hr ) ( 95% ci ) for incident diabetes was 0.86 ( 0.661.11 ) ( p trend = 0.21 ) in women , 0.69 ( 0.421.15 ) ( p trend = 0.12 ) in men , and 0.77 ( 0.610.98 ) ( p trend = 0.04 ) in both cohorts combined .
men and women in two separate prospective cohorts * findings were not altered with further adjustment for total energy intake or toenail selenium concentrations or if we adjusted for estimated dietary long - chain omega-3 fatty acids rather than fish consumption ( data not shown ) .
higher risk of diabetes with higher mercury exposure was also not seen in analyses stratified by fish consumption , bmi , or age ( table 3 ) .
indeed , trends toward lower incidence of diabetes with higher mercury exposure were seen when fish consumption was low among overweight or obese subjects and among older subjects .
men and women in two separate prospective cohorts , by subgroup * findings were also similar across a broader dose response of deciles of toenail mercury ( supplementary table 1 ) , and in sensitivity analyses excluding early cases of diabetes ( within the first 2 years ) or late cases ( after the first 10 years ) ( supplementary table 2 ) .
results were also similar or even more strongly inverse when restricted to symptomatic cases ( 48.7% of all cases ) reporting at least one classic symptom at the time of diagnosis ( supplementary table 3 ) . in prior analyses in these cohorts , self - reported fish and dietary long - chain omega-3 consumption
for example , compared with participants consuming fish less than once a month , the pooled multivariate relative risk ( 95% ci ) among those consuming fish five or more times per week was 1.22 ( 1.081.39 ) .
similarly , across quintiles of long - chain omega-3 fatty acids , the pooled multivariate relative risk ( 95% ci ) was 1.24 ( 1.091.40 ) .
when we reevaluated these associations in the present subset of cohort participants who also had mercury measures , the findings were very similar : both self - reported fish and omega-3 consumption were associated with a higher incidence of diabetes ( data not shown ) . in exploratory analyses
, we evaluated joint associations with incident diabetes of toenail mercury concentrations and self - reported fish consumption ( supplementary table 4 ) or estimated long - chain omega-3 consumption ( supplementary table 5 ) . compared with the reference group of individuals having both low toenail mercury and low fish ( or low omega-3 ) consumption , no clear pattern of risk was evident
indeed , participants having both higher toenail mercury and lower fish or omega-3 consumption tended to have lowest incidence of diabetes , whereas participants having both higher toenail mercury and higher fish or omega-3 consumption did not have a significantly different incidence of diabetes .
prospective cohort studies , we found no evidence that methylmercury exposure was associated with higher risk of diabetes .
these results provide evidence that at usual levels of exposure seen in these men and women , methylmercury exposure is unlikely to increase the risk of diabetes . when the cohorts were combined and in some population subgroups , higher mercury levels were associated with trends toward lower incidence of diabetes . because methylmercury does not induce biological effects that would plausibly protect against diabetes , this observed relationship is likely due to confounding ; i.e. , links between higher methylmercury exposure and other factors that lower risk of diabetes .
consistent with the major source of exposure , higher toenail mercury was associated with factors linked to higher fish consumption , including slightly lower bmi , modestly higher physical activity , and greater alcohol use . although we adjusted for these factors and self - reported fish consumption in multivariable models , residual confounding due to imperfect measurements or other unmeasured factors can not be excluded .
these two cohorts also comprised relatively racially and socioeconomically uniform populations , greatly minimizing confounding by race , education , and income .
interestingly , overall fish consumption in these cohorts has been linked to a modestly higher incidence of diabetes ( 26 ) , whereas toenail mercury concentrations were associated with trends toward lower risk .
these differences may partly relate to choices of fish species consumed ; associations for overall fish consumption reflect the average intakes of all types of fish consumed , whereas associations for toenail mercury reflect intakes of a limited set of large , predatory fish species .
the varying associations could also partly relate to differences in validity or bias of self - reported fish consumption , compared with toenail mercury as an objective biomarker .
when we evaluated joint associations of fish or omega-3 consumption and mercury levels , we did not find any clear pattern of joint associations that would suggest offsetting benefits versus risks .
overall , the results provide little evidence that higher methylmercury exposure from fish consumption increases risk of diabetes .
the cardia cohort recently reported on the association between toenail mercury concentrations and incident diabetes ( 9 ) .
after adjustment for age , sex , race , bmi , education , smoking , alcohol use , physical activity , and family history , mercury exposure was not significantly associated with incident diabetes . however , after further adjustment for dietary omega-3 intake , dietary magnesium , and toenail selenium , a positive association was seen ( p trend = 0.02 ) .
these findings suggested that , only after accounting for other beneficial components in fish , the remaining methylmercury exposure might increase risk of diabetes .
interestingly , this positive association also appeared potentially weaker among participants consuming higher amounts of dietary omega-3 fatty acids or magnesium .
the reasons for the divergent results between our findings and those in cardia are unclear .
biomarker laboratory , adjusted for major confounders including other beneficial components in fish , and included participants with similar overall absolute risk ( in cardia , 7.2% of subjects developed diabetes during 18 years of follow - up ; in our cohorts , 10.9% developed diabetes during 25 years of follow - up ) and similar ranges of mercury exposure . among
both our cohorts combined , geometric mean mercury exposure in the top quintile was twofold higher than the u.s .
epa reference dose ( 25 ) , and in the top decile , nearly threefold higher .
these ranges of mercury exposure were also similar to exposure levels documented in european populations ( 27,28 ) and in nationally representative u.s .
surveys ( 29 ) . pooling our results with those reported by cardia , no significant association was evident ( fig .
cardia included fewer total participants ( 3,875 vs. 9,267 ) and fewer cases of incident diabetes ( 280 vs. 1,010 ) . in comparison with our participants who were largely middle age at baseline ,
the cardia cohort was younger ( 2032 years of age at baseline ) , raising the possibility that biological effects of methylmercury could influence diabetes risk in youth but not middle age .
conversely , we did not find statistically significant effect modification by age ( p interaction = 0.71 ) or evidence for higher risk at age < 50 years even with a similar number of cases in this age stratum as in the entire cardia cohort .
cohort - specific hrs in the top quintile of toenail mercury concentrations , compared with the lowest quintile , are shown and were combined using random - effects meta - analysis with inverse - variance weights . using fixed - effects meta - analysis ,
we found inverse associations between mercury exposure and incident diabetes among people with low omega-3 intake or who were overweight or obese .
whether the results in these groups represent benefits of fish consumption , for which toenail mercury concentration is a marker , or chance findings requires further study . given absence of a priori hypotheses about interaction in these subgroups , these findings require confirmation and should be interpreted cautiously . for sensitive subpopulations ,
specific guidance exists to balance benefits and risks of fish consumption versus methylmercury exposure to optimize brain development during gestation and infancy ( 4,30 ) .
no corresponding guidelines exist for the general adult population , owing to insufficient evidence for any significant long - term effects of chronic low - level methylmercury exposure in adults .
some early studies suggested that methylmercury exposure may be linked to higher chd ( 27,28 ) , but our recent work in these two large prospective cohort studies demonstrated no evidence for increased risk of either chd or stroke at typical population ranges of chronic methylmercury exposure ( 12 ) .
dietary guidelines , and the world health organization have each concluded that regular fish consumption is a recommended part of a healthy diet and that among adults , health benefits far outweigh potential risks ( 3133 ) .
our findings provide further credence to these conclusions , providing little evidence that methylmercury exposure from fish consumption increases diabetes risk .
| objectiveemerging in vitro and animal evidence suggests that methylmercury could increase type 2 diabetes , but little evidence exists in humans .
we aimed to prospectively determine associations of mercury exposure , as assessed by biomarker measurement , with incident diabetes.research design and methodswe used neutron activation analysis to measure toenail mercury , an objective biomarker of methylmercury exposure , in 9,267 adults free of diabetes at baseline in two separate u.s .
prospective cohorts .
incident diabetes was identified from biennial questionnaires and confirmed by validated supplementary questionnaire using symptoms , diagnostic tests , and medical therapy .
associations of mercury exposure with incident diabetes were assessed using cox proportional hazards.resultsduring mean sd follow - up of 19.7 7.0 years , 1,010 new cases of diabetes were diagnosed .
the 95th percentile of toenail mercury was 1.32 g / g in men and 0.76 g / g in women , corresponding to exposures 3.5-fold and 2-fold higher than the u.s .
environmental protection agency reference dose . in multivariable analyses ,
toenail mercury concentrations were not associated with higher incidence of diabetes in women , men , or both cohorts combined . comparing the highest to lowest quintile of exposure , the hazard ratio ( 95% ci ) for incident diabetes was 0.86 ( 0.661.11 ) in women , 0.69 ( 0.421.15 ) in men , and 0.77 ( 0.610.98 ) in the combined cohorts .
findings were similar when more extreme categories ( deciles ) of mercury were compared , and in analyses stratified by fish or omega-3 consumption , bmi , and age.conclusionsthese findings from two separate large prospective cohorts do not support adverse effects of methylmercury on development of diabetes in men or women at usual levels of exposure seen in these populations . |
inherited metabolic disorders ( imds ) are a complex group of genetic conditions in which an enzyme deficiency prevents normal metabolism . when the enzyme deficiency prevents normal use of nutrients , effective treatment of the associated imd depends primarily on dietary restriction because use of off - the - shelf foods containing substances that can not be metabolized by the affected person results in toxic effects .
typically , a health - care provider prescribes a specialized diet restricting the food component that can not be metabolized .
for example , individuals affected with phenylketonuria are unable to properly metabolize the amino acid phenylalanine , which must be selectively limited in their diet to prevent severe intellectual disability . for some disorders ,
food products restricted in the offending material are available without modification of conventional products , e.g. , galactosemia is treated in infancy by conventional formulas using sucrose as a sugar source rather than the galactose - containing disaccharide , lactose . for others , particularly disorders of amino acid metabolism , conventional products that restrict the offending amino acid(s ) are not available .
treatment of disorders of amino acid metabolism requires products specially manufactured to exclude an offending amino acid or amino acids .
these are typically supplied in powdered formula or in other product forms such as bars , capsules , or ready - to - consume liquids . beyond powdered formulas , bars , and capsules
, physicians can also prescribe modified low - protein foods : products specially manufactured to provide versions of common foods made with restricted protein content .
medical foods , defined by the us food and drug administration in section 5(b ) of the orphan drug act ( 21 u.s.c .
360ee ( b ) ( 3 ) ) as food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for specific dietary management of the disease or condition for which distinctive nutritional requirements , based on recognized scientific principles , are established by medical evaluation .
in addition to medical foods ( formulas , bars , and modified low - protein foods ) , other specialized nutritional products , including high doses of vitamins and amino acids , are used in the management of imds .
the use of medical foods , modified low - protein foods , amino acid supplements , and high doses of vitamins for individuals with imds is not merely an optional , alternative food choice but rather a medical necessity .
although each metabolic disorder has its own pathological profile if left untreated , children who can not consistently obtain specialized nutritional products may experience serious adverse health effects , including severe acidosis and/or coma requiring complex hospital care , intellectual impairment , behavioral dysfunction , inadequate growth , abnormal development , nutrient deficiencies , and even death .
the prepared formulas that comprise the major part of the medical foods used for imd treatment are more expensive than standard infant formulas .
for example , a case of standard powdered infant formula costs the state of mississippi women , infants and children ( wic ) program $ 17.66 , whereas a case of a specialized medical food formula for infants costs $ 202.32 .
a box of low - protein chocolate cake mix costs $ 10 as compared with $ 3.25 for a generic store brand of cake mix .
a box of low - protein pasta costs $ 9.25 , nearly four times the price of a regular store brand ( $ 2.49 ) .
overall , modified low - protein foods cost two to eight times more than their unmodified counterparts .
some states pay for and provide medical foods as a part of their overall newborn screening ( nbs ) program .
most states that do not directly provide medical foods have enacted legislation that requires insurers to provide coverage for medical foods .
however , policies regarding coverage vary from state to state ; even when legislation has been enacted , there are exceptions and exclusions , such as the employee retirement income security act exemptions to state legislative mandates . because levels of insurance coverage for medical foods are not uniform across states , three of the regional genetic and newborn screening service collaboratives surveyed families with a child affected with an imd requiring medical foods , modified low - protein foods , high doses of vitamins , and/or amino acid supplements .
the purpose of the survey was to ascertain use of these products , sources and methods of payment , limitations in insurance coverage for purchase of the products , and the amount of out - of - pocket
( oop ) expenditures required of the surveyed families to make these medically necessary purchases .
working jointly , three health resources and services administration maternal and child health bureau regional genetic and newborn screening services collaboratives ( http://www.nccrcg.org)region 2 new york
mid - atlantic consortium for genetic and newborn screening services ( includes delaware , district of columbia , maryland , new jersey , new york , pennsylvania , virginia , and west virginia ) ; region 3 southeast newborn screening and genetics collaborative ( includes alabama , florida , georgia , louisiana , mississippi , north carolina , puerto rico , south carolina , tennessee , and the virgin islands ) ; and region 4 genetics collaborative ( comprising illinois , indiana , kentucky , michigan , minnesota , ohio , and wisconsin)developed a survey instrument and conducted the survey .
the survey was piloted in two centers in different regions and two family focus groups provided feedback regarding the instrument .
questions were refined on the basis of feedback from the focus groups and results of the survey pilot .
materials for consent were prepared , and institutional review board approvals obtained for administration of a pen - and - paper survey based on a convenience sample of parents of children with an imd .
recruitment methods varied by clinic site , but in general , all parents of children with conditions requiring the use of medical foods who had clinic appointments during the summer of 2009 were asked to participate .
parents of 305 children < 18 years of age completed the anonymous survey during regular clinic visits .
twenty - two of the respondents lived in one of the nine states where medical foods are supplied as part of their state 's nbs program ( north carolina ) . medical diagnosis and medical food reference lists
were included with the survey document as aids for parents in responding to survey questions .
the final paper survey included questions about health insurance coverage for the child 's health care ( i.e. , public or private insurance , wic , military / veterans program , indian health service , or other source ) ; the medical foods , modified low - protein foods ( e.g. , low - protein pastas , breads , and cereals ) , prescribed dietary supplements ( i.e. , amino acids , vitamins , carnitine , or other supplements ) , and medical feeding supplies / equipment ( e.g. , pumps , port - o - caths , syringes , tubing , and mic - key buttons ) used by the child ; an estimate of how much families had to pay oop for their child 's dietary treatment ( asked by ranges : $ 0/month , > $ 0<$100/month , $ 100<$500/month , $ 500$1,000/month , and > $ 1,000/month ) ; the extent to which these items were covered by their health plan ; and the annual cap or limit on the amount covered by the family 's policy .
demographic information was limited to age , diagnosis , and state in which the child resided .
data on annual caps are not reported here due to the number of null responses .
the data were first analyzed using basic descriptive statistics such as frequencies and percentage statistics for the survey items described above .
tests were used to determine if there were statistically significant differences in the distribution of ( i ) reported payment sources used by parents purchasing medical foods , modified low - protein foods , dietary supplements , and medical feeding supplies and ( ii ) the level of oop expenses families paid for the four different product types .
bonferonni - adjusted post hoc tests ( p = 0.005 ) were performed following the finding of significant results for each test .
working jointly , three health resources and services administration maternal and child health bureau regional genetic and newborn screening services collaboratives ( http://www.nccrcg.org)region 2 new york
mid - atlantic consortium for genetic and newborn screening services ( includes delaware , district of columbia , maryland , new jersey , new york , pennsylvania , virginia , and west virginia ) ; region 3 southeast newborn screening and genetics collaborative ( includes alabama , florida , georgia , louisiana , mississippi , north carolina , puerto rico , south carolina , tennessee , and the virgin islands ) ; and region 4 genetics collaborative ( comprising illinois , indiana , kentucky , michigan , minnesota , ohio , and wisconsin)developed a survey instrument and conducted the survey .
the survey was piloted in two centers in different regions and two family focus groups provided feedback regarding the instrument .
questions were refined on the basis of feedback from the focus groups and results of the survey pilot .
materials for consent were prepared , and institutional review board approvals obtained for administration of a pen - and - paper survey based on a convenience sample of parents of children with an imd .
recruitment methods varied by clinic site , but in general , all parents of children with conditions requiring the use of medical foods who had clinic appointments during the summer of 2009 were asked to participate .
parents of 305 children < 18 years of age completed the anonymous survey during regular clinic visits .
twenty - two of the respondents lived in one of the nine states where medical foods are supplied as part of their state 's nbs program ( north carolina ) . medical diagnosis and medical food reference lists
were included with the survey document as aids for parents in responding to survey questions .
the final paper survey included questions about health insurance coverage for the child 's health care ( i.e. , public or private insurance , wic , military / veterans program , indian health service , or other source ) ; the medical foods , modified low - protein foods ( e.g. , low - protein pastas , breads , and cereals ) , prescribed dietary supplements ( i.e. , amino acids , vitamins , carnitine , or other supplements ) , and medical feeding supplies / equipment ( e.g. , pumps , port - o - caths , syringes , tubing , and mic - key buttons ) used by the child ; an estimate of how much families had to pay oop for their child 's dietary treatment ( asked by ranges : $ 0/month , > $ 0<$100/month , $ 100<$500/month , $ 500$1,000/month , and > $ 1,000/month ) ; the extent to which these items were covered by their health plan ; and the annual
demographic information was limited to age , diagnosis , and state in which the child resided .
data on annual caps are not reported here due to the number of null responses .
the data were first analyzed using basic descriptive statistics such as frequencies and percentage statistics for the survey items described above .
tests were used to determine if there were statistically significant differences in the distribution of ( i ) reported payment sources used by parents purchasing medical foods , modified low - protein foods , dietary supplements , and medical feeding supplies and ( ii ) the level of oop expenses families paid for the four different product types .
bonferonni - adjusted post hoc tests ( p = 0.005 ) were performed following the finding of significant results for each test .
of the 305 children of families that responded to the survey , 150 ( 49% ) were newborn to 5 years of age , 91 ( 30% ) were between 6 and 11 years , and 64 ( 21% ) were between 12 and 17 years .
fifty percent of the children had phenylketonuria , 10% had other amino acidemias , 12% had organic acidemias , 6% had urea - cycle disorders , 12% had fatty acid oxidation disorders , and 10% had other conditions or an unspecified diagnosis .
the distribution of disorders reflects the general prevalence of the disorders within the us population and the nature of therapies for the conditions .
the survey asked families to describe all sources of insurance coverage ( private , public , and none ) for health care or payment through wic for medical foods and formula available to them . in this sample
medicaid ( 55% , 167 families ) and private insurance ( 52% , 159 families ) were common sources of partial or full coverage reported by families .
wic was the only other entity reported as a source of coverage by more than 10% ( 33 ) of families .
of the subset of 22 patients from a state where medical foods are provided by the nbs program , multiple sources were still employed , and only one family indicated they received their medical foods from the state program .
families reported obtaining their dietary products , supplements , and supplies from a variety of sources , depending on the nature of the product being used .
families requiring medical foods reported purchasing them primarily from pharmacies ( 34% ) , hospital / clinics ( 18% ) , health departments ( 14% ) , and medical supply companies ( 12% ) .
more than two - thirds ( 69% ) of families requiring dietary supplements reported pharmacies as the source from which they obtained these products . by contrast , families requiring modified low - protein foods largely obtained them from internet sources ( 41% ) , health food stores ( 16% ) , and directly from manufacturers ( 14% ) . among families purchasing feeding supplies , pharmacies ( 32% ) , medical supply companies ( 17% ) , and clinics ( 18% ) were the principal sources .
for each product type , however , families often obtained the products from multiple sources .
affected children often required items from multiple categories for nutritional support of their conditions : 257 families ( 84% ) reported using medical foods , 181 families ( 59% ) reported using modified low - protein foods , 152 families ( 50% ) reported using one or more dietary supplements , and 152 families ( 50% ) reported using feeding supplies .
overall , the parents surveyed indicated that 80% of children used at least two of the surveyed product types and 48% used three or more .
families relied on multiple payment sources for individual products and/or may have used multiple payment sources for multiple product types .
test results indicated that across the product types , there were significant differences in the distribution of reported payment sources used to purchase the different types of products ( = ( 12 , n = 753 ) 159.53 , p < 0.005 ) .
medicaid often covered medical foods , dietary supplements , and feeding ( 40 , 35 , and 31% of reported sources , respectively ) .
private insurance also frequently contributed to the coverage of medical foods ( 30% ) and dietary supplements ( 32% ) .
of note was the frequency of oop expenses parents were required to pay for nutritional treatment of their affected child due to a lack of coverage from one of the other sources included in the survey ( i.e. , medicaid , private insurance , wic , or other source ) .
families paid oop for all types of products , most notably for modified low - protein foods .
oop spending accounted for 11% of medical foods payment sources , 26% of supplement sources , 33% of feeding supplies payment sources , and ~60% of payment sources for modified low - protein foods .
bonferroni - adjusted post hoc tests indicated that modified low - protein foods were the only product type for which the percentage of total mentions of outside coverage was not more frequent than oop purchasing ( medical foods : ( 1 , n = 281 ) = 170.08 , p < 0.005 ; modified low - protein food : ( 1 , n = 174 ) = 5.89 , p > 0.005 ) ; dietary supplements : ( 1 , n = 160 ) = 36.1 , p < 0.005 ; and medical feeding supplies ( 1 ,
test results indicated that across product types there were statistically significant differences in the distribution of oop expenditure levels for the different types of products ( ( 9 , n = 632 ) = 141.27 , p < 0.005 ) .
a total of 21% of parents purchasing medical foods paid more than $ 100 per month ( beyond the costs for an unaffected child ) , as did 48% of parents purchasing modified low - protein foods , 11% purchasing dietary supplements , and 6% requiring feeding supplies .
some families reported paying $ 500 or more for medical foods ( 4% ) , modified low - protein foods ( 5% ) , dietary supplements ( 2% ) , and feeding supplies ( 3% ) .
bonferroni - adjusted post hoc tests indicated that modified low - protein foods were the only product type for which parents overall reported paying in excess of $ 100/month more often than they paid < $ 100/month ( medical foods : ( 1 , n = 224 ) = 59.93 , p < 0.005 ; modified low - protein food : ( 1 , n = 165 ) = 0.30 , p > 0.005 ) ; dietary supplements : ( 1 , n = 123 ) = 73.37 , p < 0.005 ; and medical feeding supplies ( 1 ,
although the majority of parents purchased multiple product types to fully meet the medical needs of their children , we were not able to determine the additive oop costs for those parents due to the categorical nature of the response options . overall , the survey results found that 41% of families had oop expenses for some of the products that were purchased to maintain imd treatment , with some families paying for the cost of multiple products .
medical foods were more frequently covered by insurance and obtained from medically based sources , whereas families more often paid oop for modified low - protein foods and obtained these from the internet or directly from the manufacturer .
this is the first survey to obtain specific information about insurance coverage available to families who must use medical foods and related products .
the populations of the three regions in this collaboration represent 57% of the us population , based on us 2010 census figures .
each state has a different regulatory environment for payment for these products , but when we summarized the data from these centers , our findings revealed , at best , uneven coverage for these needed treatments , with an associated potential for financial burdens for these families managing their child 's chronic metabolic disorder .
we found that some families experienced additional expenditures to cover the costs of their child 's required medical food treatment .
many had oop expenses ; a few families had expenses amounting to more than $ 500 per month .
although 99% percent of the children in our survey had some type of health - care coverage , the insurance often did not provide complete coverage for medical foods , modified low - protein foods , supplements , or feeding supplies necessary for management of an imd . of note ,
the level of health insurance coverage observed in this study is not typical of the general population , for which ~10% of children remain uncovered . some of this discrepancy in coverage rates may have resulted from a selection bias from our convenience survey of individuals who already had access to health services due to their child 's diagnosis .
consequently , we suggest that the families in this sample represent a best - case scenario relative to insurance coverage relative to medical foods .
the families as respondents had a great deal of difficulty identifying all of their oop costs .
although this underscores the complexities in understanding insurance coverage for families even when they are frequent users of this service , it also limited our ability to gather detailed quantitative information about the magnitude of expenses related to the use of medical foods and associated products . despite these shortcomings , our observations
show that families had significant oop expenses for treatments that are essential for these complex disorders .
we also found that families depended on need - based resources , such as medicaid or wic , for purchasing medical foods .
in several states surveyed here , wic was an important source of support for many families .
wic programs typically provide families with formulas , including medical foods for infants and very young children .
when assessed by age group , wic provided a notable portion of required nutritional treatments for infants and younger children .
however , because such programs typically terminate coverage at age 5 , older children were unlikely to have access to this resource .
as originally conceived , the study was undertaken with funding specific to assessment of children .
however , in our observation , limitations on coverage for adults are substantially worse than those on coverage for children , particularly because resources such as wic are not available to older children or adults .
medicare part d , a common resource for adults , for example , excludes coverage of amino acid supplements . because the care for these conditions does not end with childhood , these exceptions and terminations of coverage are particularly disturbing .
we asked families about the sources of their products with the expectation that if they needed to acquire products directly from the manufacturer or from web - based sources on the internet that they were likely purchasing their products directly and paying themselves .
we surmised that products obtained from a pharmacy or directly from a department of health source were likely covered , with less financial impact on the family .
the reported sources of products reflected these assumptions , with most medical foods , supplements , and supplies being obtained from medically based resources ( pharmacies , hospital / clinic , medical supply , and home health company or health department ) whereas modified low - protein foods indeed were often obtained from external sources ( internet and manufacturer ) .
the relation of cost of product and source of product was not ascertained by the survey and should be the subject of further investigation . as a society , we have accepted the utility of diagnosis and management of imds by initiating nbs programs . the rationale for nbs for imds
is based on overwhelming evidence showing that initiation of prompt and effective therapies offers children with these metabolic conditions improved outcomes . for imds requiring special foods or formulas ,
we found that insurance or other resources do not consistently cover costs of medical foods used to treat imds , potentially resulting in inequities in access to these essential products .
greater awareness by health - care providers and policy makers of the challenges due to the lack of uniform coverage might help to close these gaps .
as a society , we have accepted the utility of diagnosis and management of imds by initiating nbs programs . the rationale for nbs for imds is based on overwhelming evidence showing that initiation of prompt and effective therapies offers children with these metabolic conditions improved outcomes . for imds requiring special foods or formulas ,
we found that insurance or other resources do not consistently cover costs of medical foods used to treat imds , potentially resulting in inequities in access to these essential products .
greater awareness by health - care providers and policy makers of the challenges due to the lack of uniform coverage might help to close these gaps . | purposetreatment of inherited metabolic disorders is accomplished by use of specialized diets employing medical foods and medically necessary supplements .
families seeking insurance coverage for these products express concern that coverage is often limited ; the extent of this challenge is not well defined.methodsto learn about limitations in insurance coverage , parents of 305 children with inherited metabolic disorders completed a paper survey providing information about their use of medical foods , modified low - protein foods , prescribed dietary supplements , and medical feeding equipment and supplies for treatment of their child 's disorder as well as details about payment sources for these products.resultsalthough nearly all children with inherited metabolic dis orders had medical coverage of some type , families paid out of pocket for all types of products .
uncovered spending was reported for 11% of families purchasing medical foods , 26% purchasing supplements , 33% of those needing medical feeding supplies , and 59% of families requiring modified low - protein foods .
forty - two percent of families using modified low - protein foods and 21% of families using medical foods reported additional treatment - related expenses of $ 100 or more per month for these products.conclusioncosts of medical foods used to treat inherited metabolic disorders are not completely covered by insurance or other resources . |
angiostrongyliasis , gnathostomiasis , toxocariasis , lymphoma , and drugs can produce eosinophils in the cerebrospinal fluid ( csf ) .
eosinophilic meningitis is defined as the presence of 10 eosinophils / ml in the cerebrospinal fluid ( csf ) or at least 10% eosinophils in total csf leukocyte count .
we describe a rare case of eosinophilic meningitis caused due to angiostrongylus cantonensis ( rat lungworm ) following ingestion of raw monitor lizard meat .
this case report presents unique magnetic resonance imaging ( mri ) findings in a case of eosinophilic meningitis and identification of the larva in the csf . to the best of our knowledge ,
this is a very rare case from india where the larva was identified on the csf wet mount .
he had fever , headache , and vomiting for 7 days , prior to hospital admission .
central nervous examination showed bilateral rectus palsy , left third cranial nerve palsy , and stiffness of the neck .
laboratory investigations gave the following results : hemoglobin 14.4 g / dl ( normal range : 1115 g / dl ) ; total white blood cell count 10,000 cells / mm ( normal range : 400010,000 cells / mm ) ; differential count neutrophils : 91% , lymphocytes : 7% , monocytes : 2% ; platelet count 335,000 cells / mm ( normal range : 150,000400,000 cells / mm ) ; and erythrocyte sedimentation rate 8 mm/1 hour ( normal range : 020 mm ) .
serum electrolytes , renal and liver function tests were within normal limits . chest x - ray did not reveal any abnormality .
mri of the brain showed subtle hyperintensities in the basal ganglia and periventricular regions as seen on the t2 and fluid - attenuated inversion recovery ( flair ) images , which were isointense on t1-weighted images [ figure 1 ] .
the foci were more distinct on the diffusion - weighted images ( dwi ) as foci of restricted diffusion appearing bright on the dwi series and hypointense on the apparent diffusion coefficient ( adc ) series [ figure 2 ] .
these lesions showed subtle enhancement and associated meningeal enhancement on contrast administration [ figure 3 ] .
csf analysis showed the following results : total cell count 620/l ( normal range 05 cells/ll ) ; differential count eosinophils : 70% , lymphocytes : 30% ; protein 62 mg / dl ( normal range 1560 mg / dl ) ; and glucose 92 mg / dl ( normal range 4080 mg / dl ) .
16-year - old boy presented with altered sensorium of 1 day duration diagnosed with eosinophilic meningitis caused by a. cantonensis .
mr images , ( a and b ) axial t2 ( c and d ) axial flair images , at the basal ganglia level demonstrate subtle scattered hyperintensities in the periventricular regions ( white arrows ) .
16-year - old boy presented with altered sensorium of 1 day duration diagnosed with eosinophilic meningitis caused by a. cantonensis .
( a d ) mr diffusion - weighted images show foci of restricted diffusion appearing bright ( white arrows ) . ( e and f )
16-year - old boy presented with altered sensorium of 1 day duration diagnosed with eosinophilic meningitis caused by
a. cantonensis .
( a d ) mr post - gadolinium contrast - enhanced t1wi images show subtle meningeal enhancement ( double black arrows ) and faint enhancement of the lesions in the basal ganglia ( single black arrow ) . gram stain and ziehl neelsen stain did not reveal any organism , and csf culture was negative .
the csf wet mount showed a larva which matched the larval form of a. cantonensis [ figure 4 ] .
16-year - old boy presented with altered sensorium of 1 day duration diagnosed with eosinophilic meningitis caused by
a. cantonensis .
( a d ) images of the microscopic examination of wet mount of csf 45 show third - stage larva of angiostrongylus , knob - like tip ( black arrow ) , rod - like structure ( red arrow ) , intestine ( asterisk ) , and terminal projection on the tip of the tail ( blue arrow ) .
the patient gave history of consumption of raw meat of monitor lizard 14 days before the onset of symptoms .
an immunologic study was not done , as it is not available in our state .
the patient was treated with oral albendazole and prednisolone for 2 weeks , and there was gradual improvement of symptoms with complete resolution of cranial nerve palsies .
cases of angiostrongyliasis have been reported from australia , the caribbean , america , and asia .
the first - stage larvae migrate to the pharynx , are swallowed and passed in the feces .
snails or slugs ( intermediate hosts ) ingest the first - stage larvae and these larvae develop into the infective , third - stage larvae .
humans are incidental hosts and become infected by ingesting raw or undercooked snails or paratenic hosts such as freshwater shrimp / crabs / frogs which have the third - stage larvae .
prawns , crabs , and toads may also act as sources of infection to humans .
third - stage larvae penetrate the intestinal tissues of humans and are carried to the central nervous system ( cns ) . while in the cns , the larvae continue to develop to the fourth and fifth stages .
monitor lizards are eaten in parts of southern india as their meat according to local belief is supposed to have aphrodisiac properties . in a case
series of eosinophilic meningitis published from south india , all male patients developed the illness after consumption of uncooked meat of monitor lizard .
our patient had also consumed raw meat of monitor lizard for its presumed aphrodisiac properties .
the acute illness is characterized by headache , fever , meningitis , focal neurologic deficits , ocular lesions , radiculomyelitis , and palsies of the sixth and seventh cranial nerves .
the most common clinical manifestations mentioned in literature are headache , neck pain / stiffness , fever , and sensory symptoms . our patient presented with fever , headache , and vomiting .
a presumptive diagnosis of angiostrongyliasis can be made in a patient presenting with eosinophilic meningitis with a history of exposure to an intermediate host .
worms were only recovered from 28 ( 11% ) of 259 human cases in taiwan and only 10 ( 2% ) of 484 human cases in thailand .
dwi is superior to conventional mri for the detection of early signal abnormalities in viral encephalitis and meningitis .
our case showed scattered t2 hyperintensities and unique finding of dwi hyperintensities which represent the foci of microabscesses or developing infarcts .
this finding of multiple foci of dwi hyperintensities representing microabscesses has been described in fungal infections and bacterial meningitis , but has not yet been reported in angiostrongyliasis .
the final diagnosis of angiostrongyliasis is not based on the mri findings , but on serology , csf cell counts and microscopic examination , and blood counts .
treatment options consist of serial lumbar punctures , anti - helminthic therapy , steroid therapy , or combination of these .
in the present case , the patient developed signs and symptoms of meningitis after eating monitor lizard , had csf eosinophilia , and demonstrable larva in the csf .
food - borne zoonotic disease can be prevented or reduced in endemic areas by increasing awareness and public knowledge about the dangers of eating raw or undercooked meat .
physicians must consider a diagnosis of eosinophilic meningitis in patients who present with headache and neurological deficits after eating monitor lizard . | eosinophilic meningitis may be caused by non - infectious and infectious agents .
angiostrongylus cantonensis is the commonest causative agent of eosinophilic meningitis .
rats are the primary hosts of this parasite .
humans get infected by ingestion of raw or inadequately cooked hosts ( snails or monitor lizard ) or food contaminated with the infective third - stage larvae .
a 16-year - old boy was admitted to our hospital with history of fever , headache , and altered sensorium .
magnetic resonance imaging of the brain showed unique findings .
cerebrospinal fluid ( csf ) examination showed eosinophilia and the csf wet mount identified a larva .
patient history revealed ingestion of monitor lizard 2 weeks prior to onset of symptoms .
hence , a diagnosis of eosinophilic meningitis caused by a. cantonensis was made .
he was treated with oral albendazole and steroids , resulting in gradual improvement .
a. cantonensis as a cause of eosinophilic meningitis is a possibility in patients who present with headache and vomiting after eating raw meat ( monitor lizard ) . to the best of our knowledge ,
this is a very rare case being reported from india where the larva was identified during the microscopic examination of the csf . |
multiple sclerosis ( ms ) is a chronic auto immune disease that affects all aspects of patient 's lives .
physical , emotional , cognitive , intellectual , sexual , and social feature of their lives are impaired .
health - related quality of life ( qol ) is defined as individual 's perception of function and health further than the contemporary condition .
nearly , 85% of the patients present with relapsing - remitting ( rr ) form of the disease . in relapse phase , new clinical signs ,
relapses are not predictable and have negative impact on qol . in a previous study ,
different scales have been developed for qol assessment in ms cases , but none of these scales focus on relapse phase .
recently , the perception de la sclerose en plaques et de ses poussees ( persepp ) scale was designed specifically for rr ms patients to assess ms - related relapse on qol .
different aspects such as social support , difficulties with social relationships , fatigue , state of mind and sleep , time perspective , coping , symptoms , and perception of treatment are included in the persepp scale .
the aim of this study was to evaluate validity and reliability of persian version of persepp scale in iranian patients with ms .
in this cross - sectional study , 250 patients with rr type of ms who referred to ms clinic of sina hospital ( affiliated to tehran university of medical sciences ) were enrolled .
the inclusion criteria were definite ms due to mcdonald criteria and rr type of the disease .
two groups of patients enrolled : patients in relapse and those who were not in relapse at the time of study .
demographic data ( sex and age ) , duration of the disease , marital status , and types of medications were recorded .
the kurtzke expanded disability status scale ( edss ) was recorded after examination by an expert neurologist . using the forward - backward translation method ,
persepp questionnaire was translated into the persian by two medical physicians in the field of research , and then persian version was translated again into english .
two independent neurologists compared the translated english version with the original english version ( content validity ) .
the persepp questionnaire includes 66 questions ; each question contains six response types where 0 was strongly disagree and 5 was strongly agree according to a likert scale .
it includes scales such as social support , relationship difficulties , satisfaction with care , fatigue , state of mind and sleep disorders , time perspective , and coping ( module ) as symptoms ( module ) and treatment ( module ) .
the scores should be transformed to a range of 0100 , where 100 was the best qol possible .
participants were asked to answer the valid and reliable persian version of the msqol-54 and sf-36 questionnaires ( convergent validity ) .
msqol-54 is a structured , self - report questionnaire containing 14 subscales ( physical function , role limitations - physical , role limitations - emotional , pain , emotional well - being , energy , health perceptions , social function , cognitive function , health distress , overall qol , sexual function , satisfaction with sexual function , and change in health ) .
sum of subtotals such as physical function , health perceptions , energy / fatigue , role limitation - physical , pain , sexual function , social function , and health distress makes the physical health composite and sum of subtotals such as health distress , overall qol , emotional well - being , role limitation - emotional , and cognitive function makes the mental health composite .
the sf-36 questionnaire consists of 36 questions in eight aspects and is an instrument for the evaluation of the qol .
all questions are scored on a scale of 0100 , with 100 representing the highest level of functioning possible .
the questionnaire consists of eight subscales such as physical functioning , role limitations due to physical health , role limitations due to emotional problems , energy / fatigue , emotional well - being , social functioning , pain , and general health . in order to perform test - retest assessment , 50 patients filled the questionnaire 2 weeks after the first completion .
data were presented as mean standard deviation for continuous or frequencies for categorical variables .
the intraclass correlation coefficient ( icc ) was measured for repeatability evaluation , and icc coefficient over 0.70 was considered acceptable .
cronbach 's alpha was calculated in each three - factor of the questionnaire to assess the internal consistency reliability .
correlation coefficient along with the partial correlation coefficient was calculated for assessing the relationship between persepp questionnaire scores and other variables .
mean age and mean duration of disease were 32.2 8.4 years and 6.5 2.5 years , respectively .
one hundred sixty - seven ( 79.1% ) were female and 44 ( 20.9% ) were male .
eighty - five ( 40.3% ) were single , 121 ( 57.3% ) were married , and 5 ( 2.4% ) were divorced .
one hundred twenty - three ( 58.3% ) were unemployed and 88 ( 41.7% ) were employed .
icc score of different factors cronbach 's alpha of all items was above 0.8 [ table 2 ] .
cronbach 's alpha of all items pearson correlation score for qol54 and persep calculated as r = 0.44 , p < 0.001 and r = 0.66 , p < 0.001 between sf36 and persep . except four items ,
mean scores of different items were significantly different between cases with and without relapse [ table 3 ] .
mean scores of different items in cases with and without relapse coping and relationship difficulties scores were significantly different between different edss groups [ table 4 ] .
the aim of this study was to determine validity and reliability of the persian version of percepp questionnaire in a sample of iranian ms patients .
the high icc values are indicative of the high reliability of the persian version of percepp questionnaire .
we also found that cronbach 's alpha of each factor is high and indicative of acceptable internal consistency . in a previous study conducted by baroin et al .
, cronbach 's of all items except coping were more than or equal to 0.7 .
their results also showed that icc score of all items was above 0.7 , which are consistent with our results .
the results show that the mean scores of four items ( relationship difficulties , time perspective , and symptoms ) were significantly different between cases in relapse and none relapse ones that could show discriminant validity of these items . in baroin
et al . study , time perspective , treatment , and symptoms were significantly different between relapse and none relapse ones .
the different between symptom scale could show clinical differences between relapse and none relapse cases . on the other hand , disturbances and concerns met in the relapse period could explain the difference of time perspective scale .
total score of percepp questionnaire was significantly correlated with sf36 and qol54 questionnaires that could show convergent validity that is consistent with baroin et al . ,
symptoms and treatment subscales were significantly different between cases with different edss scores while in this study , mean relationship difficulties and coping were significantly different .
this could be indicative of impact of advanced disease on managing the personal and inter personal relations .
nowadays , considering qol in patients with ms is an important issue in the field of neurology .
different questionnaires such as sf36 and ms - qol54 have been applied for this purpose , but they do not focus on relapse phase of the disease .
the relapse phase has negative effect on patient 's qol and it has especial effect on psychological and social dimensions .
so , the persepp scale that is designed for rr ms patients is valuable to assess qol in ms patients and impact of relapse phase on their qol .
this scale helps physicians to consider aspects of qol of patients in relapse and nonrelapse phases .
persian version of percepp questionnaire provides a valid and reliable instrument to assess ms - related qol . | background : multiple sclerosis ( ms ) affects all aspects of patients .
recently , the perception de la sclerose en plaques et de ses poussees ( persepp ) scale was designed to assess ms - related relapse on quality of life ( qol ) .
the aim of this study was to evaluate validity and reliability of persian version of persepp scale in iranian patients with ms.methods:two-hundred eleven patients with relapsing - remitting form of the disease asked to fill the persepp scale , msqol-54 , and sf-36 questionnaires .
fifty cases filed the questionnaire 2 weeks later to assess reliability .
the intraclass correlation coefficient ( icc ) and cronbach 's alpha analysis were used.results:mean age and mean duration of disease were 32.2 8.4 years and 6.5 2.5 years , respectively .
one hundred sixty - seven ( 79.1% ) were female and 44 ( 20.9% ) were male .
forty - one ( 19.4% ) were in relapse phase of the disease .
icc score of all items was above 0.8 .
cronbach 's alpha of all items was above 0.8 .
the results show that the mean scores of four items ( relationship difficulties , time perspective , and symptoms ) were significantly different between cases in relapse and none relapse . coping and relationship difficulties scores were significantly different between different expanded disability status scale groups .
pearson correlation score for qol 54 and persep calculated as r = 0.44 , p < 0.001 and r = 0.66 , p < 0.001 between sf36 and persep.conclusions:persian version of percepp questionnaire provides valid and reliable instrument to assess ms - related qol . |
a large amount of norepinephrine ( ne ) is released from sympathetic nerves during acute myocardial ischemia .
ne participates in ischemic preconditioning ( ipc ) during short - term myocardial ischemia , which can reduce myocardial injury induced by long - term myocardial ischemia and the incidence of arrhythmias .
however , a large amount of ne released during long - term myocardial ischemia has cardiac and neurological toxicity , resulting in a significant increase in the incidence of arrhythmias [ 13 ] . the effect of excessive release of ne on sympathetic nerves and the electrophysiology of myocardial cells after ischemia is not clear
it is known that gap43 , a protein expressed in the growth cones of sprouting axons , is a marker of nerve sprouting .
furthermore , th is the rate - limiting enzyme of ne synthesis , which serves as not only a marker of sympathetic nerve terminals but also an indirect indicator of sympathetic activity .
in addition , sympathetic sprouting and sympathetic remodeling reach a peak at one week after myocardial infarction [ 4 , 5 ] .
the findings of sympathetic remodeling and its electrophysiological implications suggest that drugs that affect the release or storage of ne may have important effects on infarct size and arrhythmias .
therefore , the purpose of this study was to use desipramine , a norepinephrine transporter ( net ) inhibitor , to increase the concentration of ne before myocardial ischemia and reduce the excessive release of ne by an net - mediated mechanism during acute myocardial ischemia .
one week after myocardial ischemia - reperfusion , the vft and infarct size were measured and the distribution of nerve fibers in ventricular muscle was determined .
the experiment protocol conformed to the guideline for the care and use of laboratory animals published by the us national institutes of health and was approved by the institutional animal care and use committee .
eighty sd male rats , weighing 200250 g , were supplied by the animal experiment center of wuhan university ( no .
all rats were randomly divided into one of four groups with 20 rats in each group : a control ischemic group , a preischemic treatment group , a delayed application group , and a sham - operated group .
after intraperitoneal anesthesia , the pericardium was incised and the anterior wall of the left ventricle was exposed . the left anterior descending ( lad )
coronary artery was isolated under the left atrial appendage and carefully clamped for 30 minutes followed by reperfusion .
myocardial ischemia was confirmed by elevation of the st segment by more than 0.2 mv in leads i , ii , and avl . in the preischemic treatment group , desipramine (
0.8 mg / kg iv ) was administered five minutes before ischemia , and then the lad was clamped for 30 minutes followed by reperfusion . in the delayed application group ,
desipramine ( 0.8 mg / kg iv ) was given by the tail vein after clamping the lad for ten minutes .
reperfusion was performed after thirty minutes of lad clamping . in the sham - operated group ,
postoperatively , each rat received 200,000 iu penicillin intramuscularly twice daily for 3 days and was fed a standard diet for one week .
a bipolar needle pacing electrode was inserted in the infracted border zone about 1 mm deep .
the pacing electrode in the sham - operated group was placed on the corresponding part of the heart .
a train of rectangular 10 ms pacing pulses was administered for 30 seconds at a frequency of 60 hz with a df-5a type electrophysiological stimulation apparatus ( suzhou dongfang electronic instrument factory , china ) .
the pacing voltage of the first pulse train started at 4 v and was increased in 1 v increments .
the hearts of the remaining rats in the four groups were removed and sectioned into 2 mm thick slices and incubated in phosphate buffer ( ph 7.4 ) containing 1% triphenyltetrazolium chloride for 20 min to visualize infarcted tissue .
the infarct size in each heart was expressed as the weight of infarcted area divided by the total weight of the heart .
these hearts were cut into two parts along the long axis and infarcted area , one part was placed in a refrigerator at 70c , and another part was immediately placed in 4% paraformaldehyde for 1824 hours .
these parts were then embedded in paraffin , sliced perpendicular to the epicardium , and then subjected to h&e or immunohistochemical staining .
antibodies including anti - gap43 antibody and anti - th antibody ( rabbit anti - gap43 and anti - th , abd serotec , 1 : 450 , resp . ) , were used for immunohistochemical staining . details of the staining techniques have been published elsewhere .
nerve densities were determined by a computer - assisted image analysis system ( image - pro plus 3.0 , media cybernetics , usa ) .
the nerve density was the area occupied by the nerves divided by the actual tissue area examined ( um / mm ) .
the mean density of nerves in these three selected fields was used to represent the nerve density of that slide . th and
the total rna from the cardiac muscle samples was extracted and purified using a trizol reagent kit ( invitrogen , usa ) .
total rna was reverse transcribed into complementary dna ( cdna ) strands using a cdna synthesis kit , according to the manufacturer 's protocol . the opticon-2 real - time pcr reactor ( mj research , usa ) and real - time pcr kit were employed based on the manufacturer 's instruction .
the rt - pcr conditions were 70c/5 min , 42c/90 min for reverse transcription ; the polymerase chain reaction condition included predenaturing at 94c for 5 min , then 35 cycles of 94c for 30 s , and 60c for 30 s , 72c for 45 s , and 72c for 10 min . in this experiment ,
gene products ( 4 ul of th , gap43 , and gapdh ) were mixed with 1 ul of sample buffer .
the sample was placed on a 1.5% agarose gel , and 120 v electrophoresis was performed for 25 min .
the th , gap43 , and gapdh band areas and absorbance ( a ) were calculated by the computer image analysis system . semiquantitative analysis was performed using with the band area and a. all values were expressed as the mean the standard deviation .
linear correlation analysis was used to determine the correlation of the indices between sympathetic nerve density and vft .
in the animal model preparation , two rats died after surgery , one in the control ischemic group and one in the delayed application group .
there was a clear distinction between the infarcted and non - infarcted areas ( figure 1 ) .
h&e staining also showed a clear demarcation between necrotic and healthy tissue ( figure 2 ) .
the vft of the preischemic treatment group was 11.0 2.65 v and was higher than that of the control ischemic group ( 7.2 1.30 v , p < 0.05 ) .
infarct size in the preischemic treatment group was 23.3 2.4% and significantly lower than that in the control ischemic group ( 30.8 1.3% , p < 0.05 ) and the delayed application group ( 27.1 2.6% , p < 0.05 ) ( table 2 ) .
th- and gap43-positive nerve fibers was particularly evident in myocardial tissue sections after staining ( figures 3 and 4 ) . compared with sham - operated group , th- and gap43-positive nerve fibers in ventricular muscle in control ischemia group increased significantly , thickened , or gathered into a network structure , distributed disorderly . compared with control ischemia group , nerve fibers after treatment with desipramine
th- and gap43-positive nerve fiber density in control ischemia group was significantly higher than that of sham - operated group ( p < 0.05 ) .
the ration of th / gap43 in control ischemia group was 82.8% and was higher than sham - operated group 68.8% ( p < 0.05 , table 3 ) .
there was a significant negative correlation between the vft and both the th- and gap43-positive nerve fiber density in the infarct border zone ( p < 0.01 ) .
the correlation coefficient ( r ) was 0.91 and 0.87 , respectively , and the r of linear correlation was 0.83 and 0.76 , respectively , ( figure 5 ) .
the rt - pcr products of th , gap43 , and gapdh were consistent with the expected amplified fragments .
the electrophoretic bands were clear and nonhybrid ( figures 6 and 7 ) . th and gap43 mrna expression among the four groups was highest in the control ischemic group . in this group ,
the ratios of band area a to gapdh for th and gap42 were 2.05 0.60 and 1.96 0.83 , respectively .
these values were significantly higher compared with the other three groups ( p < 0.05 ) . th and gap43 mrna expression in the preischemic and delayed application groups were slightly higher than that in the sham - operated group , but the differences were not significant ( p > 0.05 ) .
its main functions are to reuptake ne from the synaptic cleft back into the presynaptic membrane , regulate the ne level in the synaptic cleft , terminate nerve impulse signals , and maintain the sensitivity of receptors to neurotransmitter . however , when myocardial ischemia is longer than 10 min , the release of ne becomes nonexocytotic and is thought to involve net - mediated efflux of ne in reverse of its normal transport direction . a brief episode of myocardial
many studies concluded that ipc can reduce myocardial infarct size , reduce apoptosis , attenuate myocardial stunning , and reduce arrhythmias .
several possible triggers for ipc have been postulated , including adenosine , ne , and bradykinin [ 11 , 12 ] .
previous studies on the effect of ipc have focused primarily on infarct size limitation , but its effect on cardiac sympathetic nerve injury is not known . during myocardial ischemia , massive myocardial ne release into the interstitial space was observed , caused by a nonexocytotic mechanism reflecting sympathetic nerve injury . a dramatic increase in ne in the synaptic cleft binds to -adrenergic receptors , producing positive inotropic and chronotropic effects .
this increases myocardial oxygen demand and may cause coronary artery constriction that can further aggravate myocardial ischemia .
recently , the myocardial release of ne after prolonged ischemia was attenuated by a preceding period of transient ischemia in rat and rabbit hearts , suggesting a beneficial effect of ipc on sympathetic nerve injury .
, we showed that the administration of the net inhibitor , desipramine , before myocardial ischemia improved myocardial ischemia - reperfusion injury tolerance and reduced myocardial infarct size .
the reason is that desipramine pretreatment can increase the concentration of ne in the synaptic cleft and blood , and this may cause preconditioning . at the same time , desipramine reduces net - mediated ne release and toxic products and free radicals produced from ne when myocardial ischemia is sustained .
this can ultimately reduce the damage of myocardial cells and sympathetic nerves . in this study , we observed that th- and gap43-positive nerve fibers in infarct border zone in the control ischemic group increased significantly compared with the sham - operated group .
furthermore , the ratio of th / gap43 was significantly higher in the control ischemic group .
these results indicate that there was hyperplasia of sympathetic nerves , and this is consistent with vracko et al .
desipramine can improve the distribution of th- and gap43-positive nerve fibers in infarct border zone and reduces the density of nerve fibers .
the reason may be that desipramine decreases sympathetic nerve injury during acute myocardial ischemia and reduces nerve regeneration after myocardial ischemia , thus normalizing the distribution of nerve fibers in ventricular muscle .
this study also found that th and gap43 mrna expression levels in the preischemic treatment group were significantly lower than that in the control ischemic group , which is consistent with the changes of th and gap43 protein levels .
we suggest that desipramine may also influence nerve growth after myocardial ischemia at the gene level , although the specific mechanism remains to be elucidated .
we observed that the vft in the control ischemic group with sympathetic hyperplasia was significantly reduced .
statistical analysis showed that there was a negative correlation between sympathetic nerve density and the vft .
it is known that sympathetic remodeling after myocardial ischemia changes the electrophysiological properties of the ischemic heart , which lowers the vft and increases the occurrence of arrhythmias [ 17 , 18 ] . in the present study ,
sympathetic remodeling increased nerve hyperplasia in the infarct border zone and presumably the concentration of ne .
the concentration gradient of ne in the normally innervated region and in the region with nerve hyperplasia may cause a dispersion of myocardial repolarization and excitability leading to arrhythmias .
exposure of myocardial cells in the infarct border zone to chronically elevated concentrations of catecholamines changes the signal transduction pathway of the receptor , reduces the delayed rectifier potassium current ( iks ) and transient outward potassium current ( ito ) , and extends the calcium current ( ica ) and myocardial action potential duration ( apd ) [ 17 , 19 ] .
iks and ito increase and apd shortens in the normal myocardial cells when sympathetic nerve are activated , and this increases myocardial repolarization heterogeneity and electrical instability [ 17 , 19 , 20 ] .
desipramine reduces sympathetic nerve injury , improves the distribution of nerves , and reduces myocardial infarct size .
this increases the heart 's electrical stability and vft , which should reduce the likelihood of arrhythmias .
the incidence of ventricular arrhythmia and sudden cardiac death increases in the first week after myocardial infarction .
the current study found that the decrease in vft was closely related to sympathetic remodeling after myocardial infarction .
ipc is an important self - protection mechanism in the heart and provides a novel approach to prevent myocardial ischemia in the clinical setting .
it reduced the acute release of ne and improved sympathetic remodeling and vft one week after myocardial ischemia - reperfusion .
however , further studies are necessary since there are no trials of desipramine in patients with acute myocardial infarction .
in addition , we were not able to measure the concentration of ne in the synaptic cleft .
desipramine treatment before acute myocardial ischemia can decrease infarct size , improve sympathetic remodeling , and increase vft and electrical stability of ischemic hearts . | abnormal increase in sympathetic nerve sprouting was responsible for the ventricular arrhythmogenesis after myocardial infarction .
this study investigated whether the norepinephrine transporter inhibitor , desipramine , can modulate sympathetic remodeling and ventricular fibrillation threshold ( vft ) after myocardial ischemia - reperfusion .
rats were administered desipramine ( 0.8 mg / kg , iv ) before or after myocardial ischemia .
vft , infarct size , tyrosine hydroxylase ( th ) and growth - associated protein 43 ( gap43)-positive nerve fibers were measured after one week .
the vft of preischemic treatment group was 11.0 2.65 v and significantly higher than that of control ischemic group ( 7.2 1.30 v , p < 0.05 ) .
infarct size in the preischemic treatment group ( 23.3 2.4% ) was significantly lower than that in the control ischemic group ( 30.8 1.3% , p < 0.05 ) and the delayed application group ( 27.1 2.6% , p < 0.05 ) .
the density of th and gap43-positive nerve fibers in the control ischemic group was significantly higher than that in the other three groups ( p < 0.05 ) .
the density of nerve fibers improved after desipramine treatment .
moreover , there was a negative correlation between the vft and both th and gap43-positive nerve fiber density in the infarct border zone ( p < 0.05 ) .
desipramine treatment before acute myocardial ischemia can decrease infarct size , improve sympathetic remodeling , and increase vft and electrical stability of ischemic hearts .
desipramine appears to cause myocardial ischemic preconditioning . |
this is a video describing shidham s method for cell block preparation from liquid based cytology ( lbc ) specimen using histogeltm ( thermo scientific ) ( hg ) .
as compared to other random approaches , the following are the two critical features of this protocol for preparing cell blocks from relatively hypocellular specimens with singly scattered loose cells ( 1 - 5 ) .
this protocol involves steps to concentrate the cells along the plane parallel to the cutting surface of the cell block .
it also includes a beacon - like dark inclusion of av - marker ( figure 1b ) , which serves two of the following purposes : to visualize the level at which the cells are concentrated .
the area of the cell block with the cells of interest now could be visualized by the histotechnologist when the dark colored beacon is exposed during cutting .
this ability to monitor would prevent one from cutting through the level with most of the cells , or not cutting too superficial into the level with highest concentration of sample cells.to serve as a locator reference point in serial cell block section on different slides .
this reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the scip approach ( 6,7 ) . to visualize the level at which the cells are concentrated .
the area of the cell block with the cells of interest now could be visualized by the histotechnologist when the dark colored beacon is exposed during cutting .
this ability to monitor would prevent one from cutting through the level with most of the cells , or not cutting too superficial into the level with highest concentration of sample cells . to serve as a locator reference point in serial cell block section on different slides .
this reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the scip approach ( 6,7 ) .
transfer the residual lbc cervical cytology specimen to a flat bottom glass tube ( 15 mm diameter x 45 mm ) ( figure 2.1 through 2.4 ) .
place the glass tube into a larger plastic carrier tube ( 28 x 85 mm ) and centrifuge . remove the glass bottom tube from the carrier tube and pour off the supernatant .
the glass tube is then capped ( to prevent spillage of heating water in the next step ) and placed back inside a larger flat bottom carrier plastic tube the carrier plastic tube containing the glass tube is then capped , placed in a centrifuge ( with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube ) , and spun at 1805 g ( 3000 rpms , rotor radius- 17 cm ) for five minutes ( figure 2.5 ) .
the tubes are then removed vertically from the centrifuge and the smaller glass tube is removed with forceps from the larger carrier plastic tube without disturbing the sedimented pellet with cells .
the glass tube with specimen is uncapped and the supernatant is poured off taking care not to disturb the flat layer of sediment cells at the bottom ( figure 2.6 ) .
a dark beacon av - marker ( about 2 mm x 2 mm size , flat surfaced , fragment of dark colored , sectionable material ) ( figure 3 ) is added as a signpost to the glass tube ( figure 2.7 ) .
liquefy an aliquot of hg by melting it in microwave for 10 seconds at medium power .
add 0.5 ml of molten hg to the tube , mix with the sediment quickly , and recap it ( figure 2.8 ) ( proceed to the next step quickly without allowing the hg to begin solidifying ) .
add about 2.5 ml of warm ( 45 c ) water to the carrier plastic tube ( figure 2.9 ) .
( this step is necessary to keep the hg from solidifying during the next steps ) ( figure 2.9 ) .
the carrier plastic tube is placed in the centrifuge ( with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube ) , and spun at 1805 g ( 3000 rpms , rotor radius- 17 cm ) for five minutes .
the purpose of this centrifugation step is to push the av - marker and to concentrate the cells into a layer closer to the cutting surface of the final paraffin embedded cell block ( figure 1d ) .
the tubes are then removed gently and vertically from the centrifuge taking care not to disturb the sedimented thin layer with sample cells at the bottom .
the larger plastic tube is uncapped and the smaller glass tube is removed vertically by a forceps without disturbing the sediment layer of specimen cells .
the small glass tube is refrigerated in vertical position for 15 minutes to cool and solidify the hg ( figure 2.11 ) .
the solidified hg disk , with the layer of concentrated / sediment specimen at the bottom is dislodged from the flat bottom glass tube by squirting 10% formalin through a 23 gauge needle with the syringe ( figure 2.12 ) .
the needle is inserted along the side of the tube at the periphery of solidified hg disc with specimen ( figure 2.12 ) .
the needle is rotated along the side of the tube while formalin is slowly pushed through the syringe .
this results in the separation of the hg button along with dark colored beacon av - marker and the concentrated specimen in it from the flat bottom of the glass tube ( figure 2.12 ) . the cell block ( gel button with specimen cells ) is then placed in a labeled cassette and submitted for tissue processing to prepare paraffin embedded cell blocks ( figure 2.13 ) . the disk is embedded in paraffin with the dark beacon marker side down as cutting surface ( figure 1 ) . the block is sectioned until the dark colored av - marker as a beacon is exposed and clearly visible .
three to four micron sections are cut from this level which should contain most of the singly scattered cells from the specimen .
the sections are collected on the glass slide for further staining , immunohistochemical staining , or other tests as indicated .
the protocols for these tests including the type of slides to use for mounting the sections may vary . generally for immunostaining , coated slides are used to prevent floating and loss of sections from the slides during the immunostaining steps .
abbreviations used ( in alphabetic order ) : cish , chromogenic in - situ hybridization tests ; fish , fluorescent in - situ hybridization tests ; ffpe , formalin - fixed paraffin - embedded ; fna , fine needle aspiration ; hg , histogel ( thermo scientific ) ; lbc , liquid based cytology ; pcr polymerase chain reaction ; figure 1 .
the summary of different steps for preparing cell block from lbc specimen by shidham s protocol .
this is a video describing shidham s method for cell block preparation from liquid based cytology ( lbc ) specimen using histogeltm ( thermo scientific ) ( hg ) .
as compared to other random approaches , the following are the two critical features of this protocol for preparing cell blocks from relatively hypocellular specimens with singly scattered loose cells ( 1 - 5 ) .
this protocol involves steps to concentrate the cells along the plane parallel to the cutting surface of the cell block .
it also includes a beacon - like dark inclusion of av - marker ( figure 1b ) , which serves two of the following purposes : to visualize the level at which the cells are concentrated .
the area of the cell block with the cells of interest now could be visualized by the histotechnologist when the dark colored beacon is exposed during cutting .
this ability to monitor would prevent one from cutting through the level with most of the cells , or not cutting too superficial into the level with highest concentration of sample cells.to serve as a locator reference point in serial cell block section on different slides .
this reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the scip approach ( 6,7 ) . to visualize the level at which the cells are concentrated .
the area of the cell block with the cells of interest now could be visualized by the histotechnologist when the dark colored beacon is exposed during cutting .
this ability to monitor would prevent one from cutting through the level with most of the cells , or not cutting too superficial into the level with highest concentration of sample cells . to serve as a locator reference point in serial cell block section on different slides .
this reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the scip approach ( 6,7 ) .
transfer the residual lbc cervical cytology specimen to a flat bottom glass tube ( 15 mm diameter x 45 mm ) ( figure 2.1 through 2.4 ) .
place the glass tube into a larger plastic carrier tube ( 28 x 85 mm ) and centrifuge . remove the glass bottom tube from the carrier tube and pour off the supernatant .
the glass tube is then capped ( to prevent spillage of heating water in the next step ) and placed back inside a larger flat bottom carrier plastic tube the carrier plastic tube containing the glass tube is then capped , placed in a centrifuge ( with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube ) , and spun at 1805 g ( 3000 rpms , rotor radius- 17 cm ) for five minutes ( figure 2.5 ) .
the tubes are then removed vertically from the centrifuge and the smaller glass tube is removed with forceps from the larger carrier plastic tube without disturbing the sedimented pellet with cells .
the glass tube with specimen is uncapped and the supernatant is poured off taking care not to disturb the flat layer of sediment cells at the bottom ( figure 2.6 ) .
a dark beacon av - marker ( about 2 mm x 2 mm size , flat surfaced , fragment of dark colored , sectionable material ) ( figure 3 ) is added as a signpost to the glass tube ( figure 2.7 ) .
liquefy an aliquot of hg by melting it in microwave for 10 seconds at medium power .
add 0.5 ml of molten hg to the tube , mix with the sediment quickly , and recap it ( figure 2.8 ) ( proceed to the next step quickly without allowing the hg to begin solidifying ) .
add about 2.5 ml of warm ( 45 c ) water to the carrier plastic tube ( figure 2.9 ) .
( this step is necessary to keep the hg from solidifying during the next steps ) ( figure 2.9 ) .
the carrier plastic tube is placed in the centrifuge ( with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube ) , and spun at 1805 g ( 3000 rpms , rotor radius- 17 cm ) for five minutes .
the purpose of this centrifugation step is to push the av - marker and to concentrate the cells into a layer closer to the cutting surface of the final paraffin embedded cell block ( figure 1d ) .
the tubes are then removed gently and vertically from the centrifuge taking care not to disturb the sedimented thin layer with sample cells at the bottom .
the larger plastic tube is uncapped and the smaller glass tube is removed vertically by a forceps without disturbing the sediment layer of specimen cells .
the small glass tube is refrigerated in vertical position for 15 minutes to cool and solidify the hg ( figure 2.11 ) .
the solidified hg disk , with the layer of concentrated / sediment specimen at the bottom is dislodged from the flat bottom glass tube by squirting 10% formalin through a 23 gauge needle with the syringe ( figure 2.12 ) .
the needle is inserted along the side of the tube at the periphery of solidified hg disc with specimen ( figure 2.12 ) .
the needle is rotated along the side of the tube while formalin is slowly pushed through the syringe .
this results in the separation of the hg button along with dark colored beacon av - marker and the concentrated specimen in it from the flat bottom of the glass tube ( figure 2.12 ) . the cell block ( gel button with specimen cells ) is then placed in a labeled cassette and submitted for tissue processing to prepare paraffin embedded cell blocks ( figure 2.13 ) .
the disk is embedded in paraffin with the dark beacon marker side down as cutting surface ( figure 1 ) . the block is sectioned until the dark colored av - marker as a beacon is exposed and clearly visible .
three to four micron sections are cut from this level which should contain most of the singly scattered cells from the specimen .
the sections are collected on the glass slide for further staining , immunohistochemical staining , or other tests as indicated .
the protocols for these tests including the type of slides to use for mounting the sections may vary . generally for immunostaining , coated slides are used to prevent floating and loss of sections from the slides during the immunostaining steps .
abbreviations used ( in alphabetic order ) : cish , chromogenic in - situ hybridization tests ; fish , fluorescent in - situ hybridization tests ; ffpe , formalin - fixed paraffin - embedded ; fna , fine needle aspiration ; hg , histogel ( thermo scientific ) ; lbc , liquid based cytology ; pcr polymerase chain reaction ; figure 1 .
the summary of different steps for preparing cell block from lbc specimen by shidham s protocol .
cell blocks are a valuable tool for evaluation of various cytology specimens ( 1 ) .
most importantly , in addition to the architectural details of the specimen , cell blocks allow for evaluation of ancillary studies such as immunocytochemistry , fluorescent / chromogenic in - situ hybridization tests ( fish / cish ) and in - situ pcr . a variety of methods for preparation of cell block are described . however , most of these are suitable for non - gynecologic cytology specimens which contain relatively many cells and with tissue microfragments such as in fna aspirates and some serous fluids such as effusions ( 1,3,4,5 ) .
because cell blocks primarily provide the opportunity for immunocytochemical evaluation , their processing should preferably be similar to that of the formalin - fixed paraffin - embedded ( ffpe ) tissues .
ultimately the results obtained after immunocytochemical evaluation of cell block sections are compared to those with the published literature performed predominantly on ffpe tissue sections .
any alterations in the protocol potentially compromise and nullify the validity of the results obtained on cell blocks processed through different fixatives and reagent sequences other than that used for routine ffpe .
some commercial techniques may have the drawback of processing through a protocol of exposure to other fixative - reagent exposure .
various methods of cell block preparation are available for specimens with a significant quantity of sediment and tissue fragments .
the maneuverable button is then embedded in paraffin after processing like the surgical pathology specimens / biopsies .
the gels used include gelatin , agar , fibrinogen / plasma - thrombin , and other commercial gels such as hg .
the methods of concentration vary from simple pelleting of the sediment by centrifugation to concentration of cells along various types of membranes .
examples include : milipore , collodin ( celloidin ) bags or scraping the cells from the cytology smears on glass slides ( 1 ) .
we also evaluated a variety of gels by trying different combinations of agar and gelatin .
none of the combinations achieved the a firm enough consistency to obtain an easily maneuverable disc of solidified gel with embedded cells from the specimen in one piece .
hg showed appropriate consistency and in our experience the immunostaining results on hg cell block sections have been excellent .
liquid based cytology ( lbc ) specimens for cervicovaginal cytology are generally less cellular than non - gynecologic specimens as mentioned above .
in addition , the gynecologic lbc specimens predominantly contain individual scattered exfoliated superficial cells from cervicovaginal mucosa . due to this
, appropriate cellularity within the cell block sections may not be achieved without a special approach .
as these singly scattered cellular components in the the block can not be seen by the histotechnologist during section cutting , the level at which the cells start appearing in the sections can not be appreciated and may be missed , either by cutting past the level with most cells or not cutting deep enough into the level with highest concentration of sample cells ( figure 4 ) .
shidham s protocol addresses both of these issues using hg as embedding medium and conventional lab equipments ( 2 ) ( figure 2 ) .
this protocol involves following two major features ( figure 1 ) : concentration and alignment of singly scattered cells in a narrow plane adjacent and parallel to the cutting surface of the cell block ( figure 5 ) .
this is critical for achieving following features : identifying and monitoring the level at which the cells are concentrated in the cell block .
the exposure of the dark colored signpost highlights the level at which most of the singly scattered cells in the cell block are expected to be located ( figure 4 ) .
this prevents the overcutting ( cutting past the level with most cells ) or undercutting ( not cutting deep enough into the level with highest concentration of sample cells ) in to the cell block and allow selection of the sections from the level in the cell block corresponding with highest concentration of cells.the dark colored signpost in the sections also serves as a reference point to survey and identify exactly the same cells in different serial sections of the cell block on different slides ( figure 4 ) .
this is critical while interpreting and evaluating the coordinate properties such immunoprofile of particular cells by the scip approach to follow the same cells in different sections ( 6,7 ) . identifying and monitoring the level at which the cells are concentrated in the cell block .
the exposure of the dark colored signpost highlights the level at which most of the singly scattered cells in the cell block are expected to be located ( figure 4 ) .
this prevents the overcutting ( cutting past the level with most cells ) or undercutting ( not cutting deep enough into the level with highest concentration of sample cells ) in to the cell block and allow selection of the sections from the level in the cell block corresponding with highest concentration of cells .
the dark colored signpost in the sections also serves as a reference point to survey and identify exactly the same cells in different serial sections of the cell block on different slides ( figure 4 ) .
this is critical while interpreting and evaluating the coordinate properties such immunoprofile of particular cells by the scip approach to follow the same cells in different sections ( 6,7 ) .
although our protocol is standardized and reported for liquid based cervical cytology specimens , it can also be used to enhance diagnostic yield of many other non - gynecologic specimens such as effusion fluids , fna , brushings , cyst contents etc .
in addition the av marker would facilitate improved application of scip approach during immunohistochemical evaluation of cell block sections of these specimens .
the embedding medium may be replaced by other reagents with appropriate modifications at relevant steps .
hg may be replaced by plasma ( fibrinogen ) to be gelled by thrombin at room temperature ( 1 ) . | this video demonstrates shidham 's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups .
this technique uses histogel ( thermo scientific ) with conventional laboratory equipment.the use of cell block sections is a valuable ancillary tool for evaluation of non - gynecologic cytology .
they enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion .
most importantly , they allow for the evaluation of ancillary studies such as immunocytochemistry , in - situ hybridization tests ( fish / cish ) and in - situ polymerase chain reaction ( pcr ) .
traditional cell block preparation techniques have mostly been applied to non - gynecologic cytology specimens , typically for body fluid effusions and fine needle aspiration biopsies.liquid based cervicovaginal specimens are relatively less cellular than their non - gynecologic counterparts with many individual scattered cells . because of this , adequate cellularity within the cell block sections is difficult to achieve .
in addition , the histotechnologist sectioning the block can not visualize the level at which the cells are at the highest concentration . therefore , it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing . as a result
, the area of the cell block with the cells of interest may be missed , either by cutting past or not cutting deep enough .
current protocol for shidham 's method addresses these issues .
although this protocol is standardized and reported for gynecologic liquid based cytology specimens , it can also be applied to non - gynecologic specimens such as effusion fluids , fna , brushings , cyst contents etc for improved quality of diagnostic material in cell block sections . |
skin cancer is the most common form of cancer in the united states and causes significant morbidity and mortality .
inflammatory bowel disease ( ibd ) is a chronic autoimmune condition that is associated with increased risk of development of skin cancer .
proposed mechanisms predisposing ibd patients to skin cancer include chronic inflammation , cellular damage , and underlying immune dysfunction leading to altered tumor surveillance [ 24 ] .
use of immunosuppressants in ibd patients has been shown to lead to a 47-fold increased risk of skin cancer and approximately half of ibd patients are exposed to these medications within 5 years of diagnosis .
specifically , the use of biologic and immunomodulating agents increases skin cancer risk [ 26 ] .
similarly , immunosuppression has been shown to accelerate the development of skin cancer in transplant patients ; thus , routine skin exams are recommended [ 3 , 4 ] . according to the united states preventive service task force , there is insufficient evidence to assess the risk versus benefit for general skin cancer screening .
in addition , there is a lack of data to support that early detection of skin cancer reduces morbidity and mortality .
however , patients with ibd have different risk factors for skin cancer development compared to the general population . thus , these skin cancer screening recommendations may not be applicable to ibd patients . despite the lack of specific standardized guidelines for screening ,
there is general consensus among gastroenterologists that ibd patients should protect themselves from the sun and that annual skin cancer surveillance should be considered , especially for patients on biologic and immunomodulating agents [ 5 , 8 , 9 ] .
thus , it is important for ibd patients to be aware of their risk of skin cancer and to adopt preventive strategies for modifiable risk factors .
the aims of this study are to assess ibd patients ' risk factors for and knowledge of skin cancer .
we anticipate that this survey will help to identify gaps in patient education regarding skin cancer prevention in the ibd population .
patients 18 years of age or older with a diagnosis of inflammatory bowel disease were eligible for inclusion in the study . due to the study design ,
patients were recruited via email , by online sources , and by healthcare providers in gastroenterology offices at northwestern medicine .
eligible patients who agreed to participate were forwarded to an online , confidential , anonymous survey .
a cross - sectional research questionnaire queried patients about baseline demographics and ibd - related information , including medications , complications , and extent of their ibd .
in addition , study participants were questioned about their individual skin cancer risk factors , sun exposure and skin protection practices , behaviors in the event that they identified a mole , and general skin cancer knowledge .
topics addressed in this study were compiled after critical review of published literature on skin cancer and its risk factors and included a compilation of questions in formats derived from other validated skin cancer assessments [ 6 , 1016 ] .
answers were formatted with likert scales , true / false , and multiple - choice responses ( table 1 ) .
descriptive statistics ( percentages , mean ( sd ) ) analyzed the demographic and clinical characteristics of the study sample .
scores for each skin cancer category were summed to produce total scores for skin cancer prevention ( out of 28 ) , awareness ( out of 40 ) , knowledge ( out of 4 ) , protection ( out of 32 ) , and exposure ( out of 24 ) .
each score was compared by demographic and clinical variables via a series of independent samples t - tests and one - way analysis of variance ( anova ) .
relationships between continuous variables were evaluated via pearson 's correlations while categorical variables were analyzed via chi square .
statistical significance was set to p 0.01 for t - tests and anova to control for type i error due to multiple comparisons .
patient demographics are reported in table 2 . the mean age of participants was 43.5 years and 63% of patients were female .
the majority of study participants were caucasian ( 94% , n = 153 ) and non - hispanic ( 98% , n = 161 ) .
crohn 's disease was reported in 67% of patients , ulcerative colitis in 31% , and indeterminate colitis in 2% .
ninety - five percent of patients were currently receiving ibd treatment at the time of survey response .
approximately two - thirds ( n = 105 ) of patients reported either current or past treatment with immunomodulators ( which include imuran / azathioprine and 6-mercaptopurine ) and approximately two - thirds with biologics ( n = 103 ) . sunburn and skin cancer history
twelve percent of patients ( n = 19 ) reported a personal history of skin cancer , of which 7% were basal cell carcinoma , 2% squamous cell carcinoma , 1% melanoma , and 4% multiple types of skin cancer .
additionally , 34% ( n = 55 ) reported having a first - degree relative with skin cancer .
sixty - two percent ( n = 102 ) of patients had three or more episodes of bad sunburns .
the majority of patients ( 70% ) had seen a dermatologist , with 35% of patients receiving a full skin exam once per year , 30% less than once per year , and 24% having never received a skin exam .
half of patients ( 51% , n = 84 ) were self - referred to a dermatologist ; gastroenterologists referred 13% of patients for dermatology consultation and primary care referred 17% .
patient responses to standardized questions of skin cancer prevention , awareness , knowledge , protection , and sun exposure are reported in table 1 .
average scores for skin cancer prevention , awareness , knowledge , skin protection , and sun exposure were 16.24/28 , 34.34/40 , 2.93/4 , 16.1/32 , and 6.15/24 , respectively .
patients over 40 years of age scored higher on prevention ( 17.45/28 ) compared to patients 40 years old or younger ( 15.35/28 , p = 0.03 ) .
females scored higher on skin protection ( 16.94/32 compared to 14.53/32 for males , p = 0.02 ) and awareness ( 35.16/40 compared to 32.98/40 for males , p = 0.03 , table 4 ) .
there were no statistically significant differences in scores between participants with or without exposure to immunomodulators .
there was a difference in knowledge scores between those currently treated with biologic agents ( 3.01/4 ) compared to those with prior biologic exposure ( 2.91/4 ) or no exposure ( 2.85/4 , p 0.01 ) . additionally , those who had a personal history of skin cancer scored better on both skin protection ( 21.47/32 ) and prevention ( 20.56/28 ) questions compared to those without a personal history ( 15.33/32 , 15.75/28 , resp .
in addition , those with a family history scored higher on skin protection questions ( 18.46/32 ) compared to those without a first - degree relative with skin cancer ( 14.03/32 , p 0.001 ) .
when asked if ibd and its treatment increase the risk of skin cancer , 61% of patients agreed with the statement and 38% responded neutrally or disagreed ( table 5 ) ; patients currently or previously treated with immunomodulators scored higher than those who were not on these medications ( p = 0.000 ) .
patients with inflammatory bowel disease are at increased risk of development of skin cancer compared to the general population , independent of the use of immunosuppressants [ 2 , 3 ] .
thus , this questionnaire aimed to assess ibd patients ' actual and perceived risk factors for and knowledge of skin cancer as well as general skin protection strategies .
overall , we found that ibd patients are well informed about skin cancer and are cognizant of worrisome skin features . despite this insight , they often practice suboptimal skin protection and prevention . according to the social - cognitive theory of health behavior ,
knowledge of risk alone is insufficient for behavior change ; patients also require the perception of personal risk and skills training to change their habits .
this study highlights a potential opportunity for health professionals to teach and reinforce skin cancer prevention in ibd .
other than treating the underlying inflammatory state of ibd , many of the factors that place these patients at additional risk of skin cancer development , including effective ibd treatments and early age at diagnosis , are not easily modifiable
. however , similar to the general population , behavioral changes , such as avoidance of sun exposure and tanning beds and skin protection with use of sunscreen and protective clothing , can prevent or decrease the risk of skin cancer .
, a lack of awareness of skin cancer risk serves as a barrier to adoption of protective behaviors .
overall , we found that ibd patients do have a modest understanding of skin cancer and are aware of worrisome signs and risk factors for skin cancer occurrence .
however , despite their knowledge and awareness , our patient population does not practice optimal sun protection or prevention strategies if they were to find a new mole . furthermore , when asked if ibd and its treatment increase one 's skin cancer risk , 61% of patients agreed with this statement .
those with exposure to immunomodulators , which have been shown to increase the risk of skin cancer , did score higher for this variable .
a proportion of our ibd population still does not recognize that their ibd increases their personal risk of skin cancer .
individuals adopt protective behavior ( such as sun protection ) if they perceive a health threat ( such as skin cancer ) [ 18 , 19 ] . according to the health behavior model ,
if ibd patients do not perceive a personal risk of skin cancer development , they may not adopt effective skin protection behaviors .
thus , it is our role as clinicians to discuss this topic with our patients .
furthermore , patients must understand that the proposed behavioral changes can mitigate this threat and that they can overcome barriers to achieving this [ 17 , 18 ] .
accordingly , this highlights the importance of not only teaching patients about their own risk but also providing them with ways to minimize this risk with methods to protect oneself from the sun and its negative effects .
moreover , we found that patients with a personal and family history of skin cancer tend to address worrying skin lesions with more vigilance and practice better sun protection than those without such histories .
this further highlights that one 's perceived risk of skin cancer development motivates one to adopt appropriate skin protection strategies .
we found that females practice more appropriate sun protection and are better informed about skin cancer risk factors and worrisome skin features , which is consistent with prior studies .
previous reports have found that young age is associated with high - risk behavior for skin cancer development , less knowledge about skin cancer , and increased incidence of skin cancer , especially melanoma [ 3 , 1114 ] .
we found that patients over 40 years of age are more cognizant of when to seek medical attention for worrisome skin features .
ibd patients often present in early adulthood , thus increasing the cumulative risk of skin cancer development .
specifically , younger men with crohn 's disease have an increased risk of nonmelanoma skin cancer ; therefore , this is a particularly important group to target .
the questionnaire format may both over- and/or underestimate skin cancer risk factors and skin practices .
furthermore , given the anonymous nature of the survey , a diagnosis of ibd was self - reported and not confirmed via chart review .
in addition , this study is limited by a sample primarily recruited in gi clinics at one large academic medical center .
thus , the study population may not be representative of all patients with inflammatory bowel disease .
in addition , questionnaire items were compiled based on prior literature and skin cancer surveys ; however , this specific survey has not been validated .
furthermore , some findings were statistically significant using a p value of < 0.05 instead of 0.01 ; thus there is the possibility of a type i statistical error .
our study shows that the majority of survey participants did recognize the connection between skin cancer and ibd .
comparatively , quality improvement studies have found even lower vaccination rates in the ibd population .
encouragingly , several studies have demonstrated remarkable improvements in vaccination rates with implementation of educational programs and increased access to vaccines [ 2022 ] . as in vaccination studies , physician recommendations for
our study demonstrates that despite general knowledge about skin cancer and an overall understanding that they are at increased risk of skin cancer development , our ibd population continues to practice suboptimal skin protection .
educational modules may be necessary for ibd patients to further understand their personal risk of skin cancer development and to provide more information regarding appropriate preventive and protective behaviors .
according to the social - cognitive theory of health behavior , in addition to knowledge of risk , patients also require guidance in tools to minimize this risk .
given the young age at ibd diagnosis , there is ample opportunity to intervene , educate , and reduce the cumulative risk of skin cancer development in this population .
this study supports the hypothesis that primary prevention of skin cancer in the ibd population is suboptimal and emphasizes a potential opportunity for healthcare professionals to highlight this population 's risk and reinforce skin cancer prevention strategies .
implementation for improvement includes clinical reminders at each visit and educational modules on proper skin protection techniques . | objective . patients with inflammatory bowel disease ( ibd ) are at increased risk from skin cancer . aims include assessing ibd patients ' risk factors and knowledge of skin cancer and current skin protection practices to identify gaps in patient education regarding skin cancer prevention in ibd
. methods .
ibd patients 18 years were recruited to complete an online survey .
results .
164 patients ( mean age 43.5 years , 63% female ) with ibd ( 67% crohn 's disease , 31% ulcerative colitis , and 2% indeterminate colitis ) were included .
12% ( n = 19 ) of patients had a personal history and 34% ( n = 55 ) had a family history of skin cancer .
females scored better on skin protection ( 16.94/32 versus 14.53/32 , p 0.03 ) and awareness ( 35.16/40 versus 32.98/40 , p 0.03 ) .
patients over 40 years old scored better on prevention ( 17.45/28 versus 15.35/28 , p = 0.03 ) .
patients with skin cancer scored better on prevention ( 20.56/28 versus 15.75/28 , p 0.001 ) and skin protection ( 21.47/32 versus 15.33/32 , p 0.001 ) .
61% of patients recognized the link between skin cancer and ibd .
conclusions .
the majority of ibd patients are aware of the link between skin cancer and ibd ; however , skin protection practices are suboptimal .
this emphasizes the role of healthcare professionals in providing further education for skin cancer prevention in the ibd population . |
olfactory receptor neurons ( orns ) in fish and amphibia have been shown to respond to various groups of odorants , e.g. , prostaglandins ( sorensen et al . , 1988 ; kitamura et al . , 1994 ) , nucleotides ( kang and caprio , 1995 ) , bile acids ( kang and caprio , 1995 ; sato and suzuki , 2001 ) , and amino acids ( caprio and byrd , 1984 ; kang and caprio , 1995 ; vogler and schild , 1999 ; sato and suzuki , 2001 ; manzini et al . ,
the olfactory receptors ( ors ) to which these stimuli bind , their number , and their ontogenetic appearance are largely unknown . the way these stimuli are mapped onto the olfactory bulb depends primarily on the precise projections of the respective orns to the olfactory bulb .
these are unknown , too . in our animal model , the tadpole of xenopus laevis , four ors of class
cross adaptation of responses to amino acids showed that these stimuli fall into several classes ( caprio and byrd , 1984 ; caprio et al . , 1989 ) , and recent evidence suggests ( a ) that amino acids are mapped to a lateral area in the olfactory bulb ( friedrich and korsching , 1997 ; manzini et al . , 2002b ) and ( b ) that they are not transduced by the well - known camp - dependent olfactory transduction pathway ( manzini et al . , 2002b ; manzini and schild , 2003b ) .
according to the generally accepted scheme of olfactory coding every orn expresses one or ( nef et al . , 1992 ;
strotmann et al . , 1992 ; ressler et al . , 1993 ; vassar et al .
, 1993 ; chess et al . , 1994 ; malnic et al . , 1999 ) and
however , amino acids may also unspecifically bind to putative olfactory peptide receptors , or to any other receptors , in which cases the total number of receptors sensitive to amino acids could be much larger .
here we measured the responses of orns to amino acids in xenopus laevis tadpoles . as the amount of odorant profile data that could result from patch - clamp experiments
has proved limited in our hands we resorted to calcium imaging experiments of the olfactory mucosa ( manzini et al .
we imaged calcium responses upon application of amino acids in the main cavity of the olfactory epithelium of this species .
we set out to determine the characteristics of the tadpoles ' orn responses and , in particular , whether there were classes of identically responding orns and , if so , how many .
tadpoles of xenopus laevis ( stages 5156 ) ( nieuwkoop and faber , 1994 ) were chilled in a mixture of ice and water and decapitated , in accordance with the gttingen university committee for ethics in animal experimentation .
a block of tissue containing the olfactory mucosae , the olfactory nerves , and the anterior two thirds of the brain was cut out and kept in bath solution ( see below ) .
the tissue was glued onto the stage of a vibroslicer ( vt 1000s ; leica ) and cut horizontally into 120130-m thick slices .
1 , a and b , show a mucosa slice stained with biocytin / avidin by backfilling the receptor axons from the glomerular layer of the olfactory bulb .
the slice was counterstained with propidium iodide ( for staining procedures see manzini et al . , 2002a ) .
for the imaging experiments the tissue slices were incubated in bath solution ( see below ) containing fluo-4/am ( 50 m ; molecular probes ) for 1 h , and then transferred to a recording chamber .
fluo-4/am was dissolved in dmso ( sigma - aldrich ) and pluronic f-127 ( molecular probes ) .
orns of xenopus laevis tadpoles express multidrug resistance transporters ( manzini and schild , 2003a ) with a wide substrate spectrum , including calcium - indicator dyes . to avoid transporter - mediated destaining of the slices , mk571 ( 50 m ; alexis biochemicals ) , a specific inhibitor of the multidrug resistance - associated proteins ( mrp ; gekeler et al . , 1995 ; abrahamse and rechkemmer , 2001 ) ,
after incubation , the tissue slices were placed between two grids in a recording chamber to allow diffusion from both sides and placed on the microscope stage of an axiovert 100 m ( zeiss ) to which a laser scanning unit ( lsm 510 ; zeiss ) was attached . before starting the calcium imaging experiments ,
intracellular calcium was monitored using a laser - scanning confocal microscope ( zeiss lsm 510/axiovert 100 m ) .
the confocal pinhole was 80 m and excluded fluorescence detection from more than one cell layer .
fluorescence images ( excitation at 488 nm ; emission > 505 nm ) of the olfactory mucosa were acquired at 0.251.27 hz and 786.4 ms exposure time per image with 35 images taken as control images before the onset of odor delivery .
the fluorescence changes f / f were calculated for individual orns as f / f = ( f1 f2)/f2 , where f1 was the fluorescence averaged over the pixels of an orn , while f2 was the average fluorescence of that orn before stimulus application , averaged over three images .
the fluorescence changes f / f were exported from the zeiss 510 image acquisition program to ascii files containing the time courses of fluorescence changes for each responsive orn .
these data were used ( a ) for plotting ( figs . 2 and 3 ) and ( b ) for generating a binary response vector for each orn indicating to which out of the 19 stimuli applied the respective orn was sensitive to .
the responses of the 283 orns tested were thus represented as a 283 19 matrix ( see fig .
4 ) , the elements of which are 1 ( response ) or 0 ( no response ) . calculations on this matrix were done using a custom - written program in c++ .
the composition of the bath solution was ( in mm ) : 98 nacl , 2 kcl , 1 cacl2 , 2 mgcl2 , 5 glucose , 5 na - pyruvate , 10 hepes .
this is the physiological ph in this poikilothermal species ( howell et al . , 1970 ) .
as odorants , we used the amino acids ( sigma - aldrich ; listed in table i ) applied either as a mixture of 19 amino acids ( aa ) , as submixtures , or as single amino acids .
the amino acids were dissolved in bath solution ( 10-mm stock , each ) and used at a final concentration of 200 m in all of the experiments .
stimulus solutions were prepared immediately before use by dissolving the respective stock solution in bath solution .
the bath solution was applied by gravity feed from a storage syringe through a funnel drug applicator ( schild , 1985 ) to the recording chamber .
outflow was through a syringe needle placed close to the mucosa to ensure that odorant molecules were removed rapidly .
water - soluble mixtures of l - amino acids mixtures of l - amino acids following caprio and byrd ( 1984 ) .
the dilution of the stimulus in the mucosa was determined by first putting a confocal volume ( 1 fl = 10 l ) of a laser - scanning confocal microscope ( zeiss lsm 510/axiovert 100 ) in front of the funnel outlet and , second , in front of the epithelial surface and measuring the respective fluorescences .
for this control measurement we used the fluorescent probe tetramethylrhodamine ( tmr , 500 nm ; sigma - aldrich ) as a dummy stimulus .
the dilution factor was 0.91 0.02 ( mean sd , n = 7 ) .
the delay between tmr leaving the funnel outlet and reaching the steady - state concentration at the mucosal surface was < 1 s , and after the end of stimulation , tmr was completely rinsed from the mucosa within 15 s.
tadpoles of xenopus laevis ( stages 5156 ) ( nieuwkoop and faber , 1994 ) were chilled in a mixture of ice and water and decapitated , in accordance with the gttingen university committee for ethics in animal experimentation .
a block of tissue containing the olfactory mucosae , the olfactory nerves , and the anterior two thirds of the brain was cut out and kept in bath solution ( see below ) .
the tissue was glued onto the stage of a vibroslicer ( vt 1000s ; leica ) and cut horizontally into 120130-m thick slices .
1 , a and b , show a mucosa slice stained with biocytin / avidin by backfilling the receptor axons from the glomerular layer of the olfactory bulb .
the slice was counterstained with propidium iodide ( for staining procedures see manzini et al . , 2002a ) .
for the imaging experiments the tissue slices were incubated in bath solution ( see below ) containing fluo-4/am ( 50 m ; molecular probes ) for 1 h , and then transferred to a recording chamber .
fluo-4/am was dissolved in dmso ( sigma - aldrich ) and pluronic f-127 ( molecular probes ) .
orns of xenopus laevis tadpoles express multidrug resistance transporters ( manzini and schild , 2003a ) with a wide substrate spectrum , including calcium - indicator dyes . to avoid transporter - mediated destaining of the slices , mk571 ( 50 m ; alexis biochemicals ) , a specific inhibitor of the multidrug resistance - associated proteins ( mrp ; gekeler et al . , 1995 ; abrahamse and rechkemmer , 2001 ) ,
after incubation , the tissue slices were placed between two grids in a recording chamber to allow diffusion from both sides and placed on the microscope stage of an axiovert 100 m ( zeiss ) to which a laser scanning unit ( lsm 510 ; zeiss ) was attached . before starting the calcium imaging experiments ,
intracellular calcium was monitored using a laser - scanning confocal microscope ( zeiss lsm 510/axiovert 100 m ) .
the confocal pinhole was 80 m and excluded fluorescence detection from more than one cell layer .
fluorescence images ( excitation at 488 nm ; emission > 505 nm ) of the olfactory mucosa were acquired at 0.251.27 hz and 786.4 ms exposure time per image with 35 images taken as control images before the onset of odor delivery .
the fluorescence changes f / f were calculated for individual orns as f / f = ( f1 f2)/f2 , where f1 was the fluorescence averaged over the pixels of an orn , while f2 was the average fluorescence of that orn before stimulus application , averaged over three images .
the fluorescence changes f / f were exported from the zeiss 510 image acquisition program to ascii files containing the time courses of fluorescence changes for each responsive orn .
these data were used ( a ) for plotting ( figs . 2 and 3 ) and ( b ) for generating a binary response vector for each orn indicating to which out of the 19 stimuli applied the respective orn was sensitive to .
the responses of the 283 orns tested were thus represented as a 283 19 matrix ( see fig .
4 ) , the elements of which are 1 ( response ) or 0 ( no response ) .
the composition of the bath solution was ( in mm ) : 98 nacl , 2 kcl , 1 cacl2 , 2 mgcl2 , 5 glucose , 5 na - pyruvate , 10 hepes .
this is the physiological ph in this poikilothermal species ( howell et al . , 1970 ) .
as odorants , we used the amino acids ( sigma - aldrich ; listed in table i ) applied either as a mixture of 19 amino acids ( aa ) , as submixtures , or as single amino acids .
the amino acids were dissolved in bath solution ( 10-mm stock , each ) and used at a final concentration of 200 m in all of the experiments .
stimulus solutions were prepared immediately before use by dissolving the respective stock solution in bath solution .
the bath solution was applied by gravity feed from a storage syringe through a funnel drug applicator ( schild , 1985 ) to the recording chamber .
outflow was through a syringe needle placed close to the mucosa to ensure that odorant molecules were removed rapidly .
water - soluble mixtures of l - amino acids mixtures of l - amino acids following caprio and byrd ( 1984 ) .
the dilution of the stimulus in the mucosa was determined by first putting a confocal volume ( 1 fl = 10
l ) of a laser - scanning confocal microscope ( zeiss lsm 510/axiovert 100 ) in front of the funnel outlet and , second , in front of the epithelial surface and measuring the respective fluorescences . for this control measurement
we used the fluorescent probe tetramethylrhodamine ( tmr , 500 nm ; sigma - aldrich ) as a dummy stimulus .
the dilution factor was 0.91 0.02 ( mean sd , n = 7 ) .
the delay between tmr leaving the funnel outlet and reaching the steady - state concentration at the mucosal surface was < 1 s , and after the end of stimulation , tmr was completely rinsed from the mucosa within 15 s.
for this study we analyzed changes in the intracellular calcium concentration [ ca]i in 283 orns ( n = 49 slices ) of the olfactory epithelium of xenopus laevis tadpoles ( fig .
1 , a and b ) using fluo-4 as calcium - indicator dye and amino acids as stimuli . fig .
the encircled orns were responsive to a mixture of 19 amino acids ( aa ) as seen from the increase of the [ ca]i in fig . 1 , d f . at interstimulus intervals of 2 min
we applied the five submixtures of amino acids ( lcn , scn , bas , acid , and arom , see table i ) and subsequently the 19 single amino acids , one after another .
slice of the olfactory epithelium of a xenopus laevis tadpole and amino acid induced [ ca]i increases in individual orns in a mucosa slice .
( a ) overview of a horizontal slice of the olfactory epithelium of a xenopus laevis tadpole ( stage 54 , pc , principal cavity and oe , olfactory epithelium ) .
the neurons were backfilled through the nerve using biocytin / avidin staining ( green fluorescence ) , and then the slice was counterstained with propidium iodide ( red fluorescence ) .
( b ) higher magnification of a part of the olfactory epithelium shown in a. olfactory receptor neurons with dendrites , dendritic knobs , and cilia are easily recognizable .
( c ) fluorescence image of a mucosa slice ( stage 55 , image acquired at rest ) stained with fluo-4 .
the responses to amino acids of the orns indicated by arrows are shown in fig .
( d f ) sequence of three pseudocolored images of the slice showing that stimulation with a mixture of amino acids ( 200 m each ) transiently increases calcium - dependent fluorescence in the orns encircled in c. ( d ) image taken before the application of the amino acid mixture ( t = 0 s ) .
( e ) image taken at the peak of the response ( t = 15 s ) .
( f ) image taken after return to the base line fluorescence ( t = 52 s ) .
bars : ( a ) 50 m ; ( b f ) 20 m .
2 . orn # 1 was responsive to l - histidine and , accordingly , to the mixture of basic amino acids .
orn # 2 reacted upon application of l - leucine , l - methionine , l - cysteine , l - glutamine , l - asparagine , and l - arginine and to the respective submixtures , whereas orn # 3 was sensitive just to l - asparagine and the short chain neutral submixture .
the fourth orn shown in this figure responded to l - isoleucine , l - methionine , l - glutamine , and the submixtures of long chain neutral and short chain neutral amino acids .
the number of orns in a slice responding to amino acids varied from slice to slice , the average being 5.8 orns / slice ( n = 49 slices ) .
when orns were tested several times to the same stimulus , the responses exhibited a high degree of similarity ( fig .
3 ) . in some cases different stimuli induced different response time courses ( fig .
3 a , l - cysteine vs. l - methionine ; fig . 3 b , l - methionine vs. l - leucine ) , while in other cases the response to one stimulus appeared just down- or upscaled with respect to another one ( fig . 3 c , l - glutamine vs. l - isoleucine ) . amino acid induced changes in calcium - dependent fluorescence of four individual orns in a mucosa slice .
( a ) time course of [ ca]i transients of an orn ( orn # 1 in fig
the traces show the responses to the mixture of 19 amino acids ( aa ) , to the mixture of basic amino acids ( bas ) and to l - histidine .
no response to the mixtures of the long chain neutral ( lcn ) , the short chain neutral ( scn ) , the aromatic ( arom ) , and the acidic ( acid ) amino acids .
. 1 c ) responding to the mixture of aa , lcn , l - methionine , l - leucine ( though slightly weaker ) , scn , l - cysteine , l - glutamine , l - asparagine , bas , and to l - arginine .
no response to the mixtures arom or acid , nor to the remaining single amino acids of the responsive groups .
1 c ) responding to the mixtures of aa , scn , and to l - asparagine .
no response to the other mixtures , nor to the remaining single amino acids of the scn mixture .
1 c ) responding to the mixture of aa and lcn , to l - isoleucine and to l - methionine , the mixture of scn , and to l - glutamine .
no response to the mixtures bas , arom , or acid , nor to the remaining single amino acids of the responsive groups .
different amino acids elicit distinguishable and highly reproducible [ ca]i transients in individual orns in a mucosa slice .
time courses of amino acid induced [ ca]i transients of three individual orns ( a c ) .
the traces show responses to successive applications of the amino acids l - cysteine and l - methionine ( a ) ; l - methionine , l - cysteine , and l - leucine ( b ) ; and l - glutamine and l - isoleucine ( c ) .
shape and duration of the [ ca]i transients stayed approximately constant if the same amino acid was applied several times .
all 283 orns recorded were tested for their responses to 19 amino acids ( see table i ) , resulting in the 283 19 response matrix shown in fig .
we indicated a response of an orn to a particular stimulus by a 1 in the response matrix , while a 0 in the matrix means no response .
283 19 matrix representing the responses of the 283 orns each tested for 19 amino acids indicated by the common one - letter code for amino acids ( first line ) .
a 1 or a 0 in the matrix indicates whether or not a particular orn responded to a particular amino acid ( 1 = response ; 0 = no response ) .
a response was assumed if the following two criteria were met : ( a ) the first two intensity values after stimulus arrival at the mucosa , i ( t1 ) and i ( t2 ) , had to be larger than the maximum of the prestimulus intensities ; ( b ) i ( t2 ) > i ( t1 ) with t2 > t1 .
the frequency by which the 19 amino acids used elicited a response varied markedly from amino acid to amino acid ( fig .
l - methionine and l - proline were the most and the least effective stimuli , leading to responses in 64% and 3.5% of all responsive orns , respectively ( fig .
the histogram shows for each of the 19 amino acids used in how many orns ( out of 283 ) a response was observed .
results are plotted as relative numbers , i.e. , normalized to 283 . as most of the orns ( n = 283 ) responded to more than one amino acid ,
the sum of the response frequencies is much higher than 100% . as most orns ( 239 of 283 ) responded to more than one stimulus ( fig .
4 ) , the frequencies shown in fig . 5 sum up to a value > 100% . of the 283 responses recorded 204 patterns were unique in the sense that they differed from all other patterns .
while 168 of these occurred just once , 36 patterns occurred more than once . all orns that showed the same response pattern to the sequence of 19 amino acids formed a class of orns , respectively . among the 283 orns recorded
there were 36 classes , each of which contained between 2 and 13 identically responding orns ( table ii ) .
classes of identically responding orns are represented by their class index , the response pattern , and the member orns , respectively .
the orns of six classes ( class # 2 , 3 , 10 , 15 , 18 , and 32 ) responded to one stimulus each and these stimuli were l - histidine , l - arginine , l - lysine , l - asparagine , l - glutamine , and l - methionine . among the individual orns that responded differently from all others ,
there were four orns responding to one amino acid , i.e. , to l - glycine , l - cysteine , l - tryptophane , and l - leucine .
together there were 44 orns that responded to one stimulus , 40 of them being members of cell classes and 4 of them being individual orns .
as a next step of our evaluation we asked how many of the 283 orns responded to two stimuli , how many to three stimuli , and so forth .
the resulting frequency distribution ( including the 44 orns responding to one stimulus ) is shown in fig .
6 . it indicates a clear tendency of orns to respond to few rather than to many stimuli .
frequencies of orns ( n = 283 , 49 slices ) that responded to a certain number n of amino acids ( n out of 19 amino acids , table i ) .
the distribution was , however , rather skewed , with small numbers of effective stimuli occurring much more often than larger numbers .
the responses of an orn to multiple stimuli could be explained either by the expression of a relatively nonspecific receptor or by the expression of more than one receptor per orn . though the expression of more than one or in one orn contradicts all evidence in adult orns of higher vertebrates , it is a priori not excluded in larval stages of lower vertebrates . in this case , however , the sensitivity spectra of tadpole orns might be expected to change over time and stages .
we therefore split the dataset of fig . 4 in two subsets , i.e. , orns recorded at stage 51 , 52 , 53 , and orns recorded at stage 54 , 55 , 56 .
is arranged in a way that the first subset includes orns # 0143 ( first set of stages ) and the second subset includes orns # 144283 ( second set of stages ) .
frequency histograms of the number of effective stimuli in each subset of orns are plotted in fig .
7 , a and b. it is obvious that orns at later developmental stages respond to less stimuli , indicating that xenopus laevis tadpole orns indeed gain specificity over stages .
histograms of the number of effective amino acids per orn in two groups of orns of different stages .
( a ) frequency distribution of orns ( n = 143 , 27 slices ) responding to n stimuli , evaluated for earlier stages ( stages 51 , 52 , and 53 ) . ( b )
frequency distribution of orns of later stages ( n = 140 , 22 slices , stages 54 , 55 , and 56 ) . in the second subset ( later stages )
amino acids are chemically well - known and biologically relevant stimuli in xenopus laevis ( iida and kashiwayanagi , 1999 ; vogler and schild , 1999 ; manzini et al .
from an experimental point of view they are convenient stimuli because they form a finite and small group of stimuli and dissolve well in water . to obtain specificity profiles of orns to many odorants using the patch - clamp technique
first , it is rather troublesome to find individual orns that respond to a particular known group of odorants and , second , in the rare case where such an orn is found , the orn is notoriously lost before it has been tested for all of the odorants of that set ( see manzini et al . , 2002a , b ) .
a way out of this intrinsic problem is using calcium imaging in the slice of the olfactory epithelium . this way it is feasible to monitor responses of many orns in parallel , thereby increasing the detection probability of responses considerably .
the specificity profiles to amino acids of a number of orns can thus be determined simultaneously
. another important advantage of this method is that calcium responses can be observed over a long period of time , markedly longer than orns can usually be held on a patch pipette , thereby allowing to test all stimuli of a test set . to our knowledge
the data presented here is the largest olfactory response matrix ( 283 orns , each tested for 19 odorants ) reported so far .
in our study we set out to determine the specificity profiles of a large number of orns to the 19 amino acids listed in table i , using fluo-4 as calcium - indicator dye . using this approach we determined the specificity profiles to amino acids of 283 orns of xenopus laevis tadpoles ( stages 5156 ) .
as to the evaluation of the resulting data , we here concentrated on the three most prominent issues , i.e. , ( a ) reproducibility and variability , ( b ) response classes , and ( c ) changing selectivities over developmental stages .
the time course of the calcium responses of the orns showed a high degree of similarity ( fig .
interestingly , the time courses of the [ ca]i transients elicited by some amino acids on an individual orn differed from each other , i.e. , shape and duration clearly depended on the stimulus applied ( fig .
this could be explained by individual orns each expressing one unspecific receptor with different ligand affinities .
obviously , this interpretation would imply the existence of a large number of amino acid sensitive ors .
for instance in the case of l - histidine , there would be one or sensitive to l - histidine ( orns of class 2 , table ii ) , but in addition many more ors ( in the case of l - histidine 79 ors ) would be sensitive to a combination of amino acids that includes l - histidine ( fig .
, the differing [ ca]i transients in an individual orn elicited by different stimuli could be explained by the expression of more than one or per orn .
while this hypothesis is not consistent with other findings in adult higher vertebrates ( nef et al .
, 1992 ; strotmann et al . , 1992 ; ressler et al . , 1993 ; vassar et al .
a decision of whether the first interpretation , the second one , or a combination of both is true will presumably come from a more detailed characterization of xenopus laevis ors and single - cell pcr experiments in orns of this species .
the concentration of the amino acids used is important . throughout the experiments reported herein we used an amino acid concentration of 200 m .
though xenopus laevis orns respond to lower concentrations as well , the used concentration is sufficiently high to activate ors with high as well as with low affinity for amino acids , but not as high as in many other studies ( e.g. , michel et al .
, 1991 ; iida and kashiwayanagi , 1999 , 2000 ; sato and suzuki , 2000 , 2001 ) . of the 283 response profiles observed 36 patterns occurred more than once .
these were seen in a total of 115 orns ( see table ii ) , while 168 response profiles were recorded just once .
there were thus 36 classes of orns , each comprising a number of orns ( from 2 to 13 ) . among these 115
orns 40 responded to one amino acid , while the others responded to more than one amino acid .
what was surprising was the large number of different response patterns . at the beginning of our experiments we started out with the hypothesis that , assuming one or per orn , there would be a limited number between 10 and 30 of amino acid receptors and a correspondingly limited number of orn classes .
in contrast , our data showed 36 classes and , in addition , 168 individual response patterns that were different from all others .
assuming we had measured k more orns , what would have been the number of classes and the number of individual response patterns ?
36 + k classes and 168 individual patterns , or 36 classes and 168 + k individual patterns ?
this thought experiment shows that 36 is a minimum estimate for the number of amino acid sensitive orn classes .
explaining the diversity of response patterns delineated above by assuming one or per orn would again imply the existence of a large number of amino acid binding ors .
it has been shown that in higher vertebrates every orn expresses one or ( nef et al . , 1992 ;
strotmann et al . , 1992 ; ressler et al . , 1993 ; vassar et al .
, 1993 ; chess et al . , 1994 ; malnic et al . , 1999 )
admittedly , however , little is known about or expression in lower vertebrates , especially at larval stages .
therefore , it can not be excluded that orns of xenopus laevis tadpoles express a number of ors at lower stages , lose most of them during development , and finally express one or . in this case the sensitivity spectra of tadpole orns must be expected to change over stages , i.e. , under such a condition of narrowing selectivity over stages , orns would respond to less stimuli at higher stages than at lower stages . to verify this hypothesis we split the dataset of fig . 4 into two subsets , the first subset containing orns of earlier stages ( 51 , 52 , 53 ) , the second subset containing orns of later stages ( 54 , 55 , 56 ) .
tadpoles were staged after nieuwkoop and faber ( 1994 ) , using the size of the tadpole 's fore and hind limbs as a major classification criterion . however , as development is a continuous process , any categorization into discrete stages is necessarily error - prone .
in addition , distinguishing certain stages from each other ( e.g. , stage 52 from 53 ) is more difficult than distinguishing others ( e.g. , stage 53 from 54 ) . to minimize errors of stage classification
we then calculated histograms for either subset giving the frequencies by which the respective orns responded to a certain number of stimuli . the earlier stage subset ( fig . 7 a )
comprises orns responding to up to 19 amino acids , while the orns of the later stage subset ( fig . 7
b ) never responded to > 13 amino acids . while 58% of the early stage orns responded to seven or less amino acids , over 80% of the late stage orns responded to seven or less amino acids .
these data unambiguously show a trend demonstrating that orns of xenopus laevis gain specificity over stages .
thus , or expression patterns do not appear to be constant over stages . to substantiate this assertion
, the genes coding for amino acid ors need to be characterized , a task certainly beyond the scope of this study .
furthermore , it would be interesting to study whether or not the specificity of orns of adult xenopus laevis is consistently confined to one stimulus per orn , as seen in a portion of tadpoles orns .
we have as yet failed , however , to develop a slice preparation from adult xenopus ' water mucosa , which would allow us to answer this question . | in olfactory receptor neurons ( orns ) of aquatic animals amino acids have been shown to be potent stimuli . here
we report on calcium imaging experiments in slices of the olfactory mucosa of xenopus laevis tadpoles .
we were able to determine the response profiles of 283 orns to 19 amino acids , where one profile comprises the responses of one orn to 19 amino acids .
204 out of the 283 response profiles differed from each other .
36 response spectra occurred more than once , i.e. , there were 36 classes of orns identically responding to the 19 amino acids .
the number of orns that formed a class ranged from 2 to 13 .
shape and duration of amino acid - elicited [ ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid .
different amino acids , however , in some cases led to clearly distinguishable calcium responses in individual orns .
furthermore , orns clearly appeared to gain selectivity over time , i.e. , orns of later developmental stages responded to less amino acids than orns of earlier stages .
we discuss the narrowing of orn selectivity over stages in the context of expression of olfactory receptors . |
nephrotic syndrome ( ns ) in children is typically idiopathic , but may also occur secondary to other diseases including eosinophilic disorders1234 ) .
eosinophilic gastroenteritis ( eg ) is an eosinophilic disease characterized by eosinophilic inflammation of the gastrointestinal ( gi ) tract56 ) .
there have been few previous reports of these 2 distinct diseases coexisting in the same patient7 ) .
here we describe a rare case of eg presenting as abdominal pain with peripheral eosinophilia in prolonged ns .
an 18-year - old male was admitted with epigastric pain and nausea . during the previous 2 years he visited a local emergency department several times with a similar clinical presentation .
he had been treated for steroid - dependent ns since he was three years of age , and was taking 6 mg ( 0.1 mg / kg ) of oral deflazacort every other day when admitted .
there was no history of allergic disease such as food allergies , asthma or atopic dermatitis . on admission ,
the results of laboratory tests included leukocyte count , 18,010/l with 28.3% eosinophils ( absolute eosinophil count , 5,108/l ) ; hemoglobin , 15.5 mg / dl ; platelets , 58210/l ; blood urea nitrogen , 9.9 mg / dl ; creatinine , 0.42 mg / dl ; total protein , 4.2 g / dl ; albumin , 2.4 g / dl ; total cholesterol , 362 mg / dl ; aspartate aminotransferase ( ast ) , 18 iu / l ; alanine aminotransferase ( alt ) , 16
iu / l ; amylase , 234 iu / l ; and urine protein , ; and urinalysis showed no other abnormal findings .
abdomen and pelvis computed tomogarphic scans were performed due to elevated amylase , and revealed wall thickening from the distal esophagus to the proximal jejunum and distal jejunal loops without evidence of pancreatitis ( fig .
1 ) . when he was referred to our hospital after discharge from the local hospital , the patient 's physical examination demonstrated no specific findings .
additional laboratory values were as follows : leukocyte count , 14,810/l with 20.7% eosinophils of ( absolute eosinophil count , 3,063/l ) ; hemoglobin , 14.6 mg / dl ; platelets , 50110/l ; peripheral blood smear , mild leukocytosis with eosinophilia ; amylase , 68 iu / l ; total ige , 85.0 iu / ml ; anti - dsdna antibody , ( ) ; stool exam , ( ) ; anti - amoeba antibody ( ab ) igg , ( ) ; toxocariasis ab , ( ) ; urine protein , 4 + ; and spot urine protein / creatinine , 3.1 .
biopsy specimens of the duodenal mucosa showed eosinophilic infiltration in the lamina propria up to 35/high - power field ( fig .
renal biopsy was performed to evaluate the relationship between hypereosinophilic syndrome ( he s ) and renal disease .
the biopsy findings were ( 1 ) within the minimal change disease ( mcd ) spectrum with igm deposits ( igm nephropathy ) , and ( 2 ) mild tubular atrophy with interstitial fibrosis .
on the 6th hospital day , the patient complained of epigastric pain again and the quantity of oral deflazacort was increased to 54 mg ( 1.2 mg / kg ) per day , after which his symptoms improved . on the 11th hospital day , the patient was discharged with decreased proteinuria ( spot urine protein / creatinine , 2.0 ) and decreased eosinophilia ( absolute eosinophil count , 72/l ) . on follow - up ,
mild to moderate peripheral eosinophilia was a continuing issue , but the patient did not complained of further abdominal pain for 2 years .
there was severe peripheral eosinophilia ( absolute eosinophil count , 5,054/l ) , without proteinuria .
the results of both abdomen computed tomography and duodenal biopsies were similar with previous results .
biopsy specimens of the duodenal mucosa showed eosinophilic infiltration up to 30/high - power field .
his symptom was improved after treating with 50 mg of intravenous methylprednisolone iv for 3 days and he was discharged with decreased eosinophilia , on the 8th hospital day ( absolute eosinophil count , 318/l ) .
the patient reported here had peripheral eosinophilia with abdominal pain as a form of eg and mcd presenting as ns .
although the etiology of eg is unknown , a history of allergies is reported in one - third to one - half of all patients with eg , suggesting an allergic basis6 ) .
one interesting case report describes the occurrence of ns in a patient with eg and a food allergy . in this patient , a direct causal association between atopic eg and
ns was suggested by renal biopsy showing immune complex glomerulonephritis with bovine serum albumin7 ) .
to our knowledge , this is the first case report of child or adolescent with nonatopic eg and ns .
eg may also occur secondary to systemic diseases such as connective tissue disease or hes5 ) .
he s is characterized by marked ( > 1,50010/l ) and persistent ( > 6 months ) eosinophilia and multiorgan involvement without a clear etiology .
the most frequently involved organs are the cardiovascular , pulmonary , hematopoietic and nervous systems4 ) .
it may be difficult to differentiate between he s with intestinal involvement and eg5 ) .
eg is defined based on the following 3 criteria : the presence of gi symptoms , biopsy indicating eosinophilic infiltration of one or more areas of the gi tract from the esophagus to the colon , or characteristic radiological findings with peripheral eosinophilia , and no evidence of parasitic or extraintestinal disease8 ) .
mucosal eosinophilic gastroenteritis , the most common type , manifests as abdominal pain , diarrhea , protein - losing enteropathy and / orvomiting568 ) , while the peripheral eosinophilic count appears normal in 23% to 40% of eg patients6 ) . in our case ,
diagnosis was based on patient history ( recurrent abdominal pain with nausea ) , infiltration of eosinophils on duodenal biopsy and peripheral eosinophilia .
the patient had gi symptoms , but no extraintestinal signs such as skin nodules , lymphadenopathy or hepatosplenomegaly on physical examination , and no evidence of other organ involvement .
primary eg is a more appropriate diagnosis for our patient than gi involvement with he s .
however , patients with eg may , in rare cases , progress to he s , so long - term follow - up and careful exam for new signs of he s are necessary5 ) .
the mechanisms implicated in renal involvement are eosinophil cytotoxicity , mass effect due to eosinophilic infiltration and thromboembolic events secondary to cardiac damage10 ) . in our case ,
, renal disease may be less likely to develop secondary to he s in our patient .
corticosteroids are also the most effective anti - inflammatory drugs used in the treatment of eg568 )
. their beneficial effects result from ( 1 ) the suppression of the synthesis and effects of eosinophil survival factors , ( 2 ) the direct induction of eosinophil apoptosis and ( 3 ) the stimulation of engulfment by professional phagocytic cells12 ) . despite their different pathological mechanisms ,
the treatment of both eg and ns is the same , and thus there is a possibility that diagnosis of eg was delayed in our case . in summary , we report the first case of nonatopic eg with ns .
as many as 45% of children with ns will have a frequently relapsing course , and some will be steroid - dependent13 ) .
this case emphasizes that , in children , treatment with prolonged steroid therapy has the potential to mask other diseases , even those that may be fatal .
thus , pediatricians should be cautious when examining child or adolescent treated with steroids for long periods . | eosinophilic gastroenteritis is a rare disease characterized by prominent eosinophilic tissue infiltration of the gastrointestinal tract . here , we report a case of eosinophilic gastroenteritis in an 18-year - old patient with prolonged nephrotic syndrome who presented with abdominal pain and peripheral hypereosinophilia . during the previous 2 years
, he had visited local emergency department several times because of epigastric pain and nausea .
he had been treated with steroid - dependent nephrotic syndrome since 3 years of age .
tests ruled out allergic and parasitic disease etiologies .
gastroduodenoscopy with biopsy revealed marked eosinophilic infiltration in the duodenum .
renal biopsy findings indicated minimal change disease spectrum without eosinophilic infiltration .
the oral deflazacort dosage was increased , and the patient was discharged after abdominal pain resolved . to our knowledge , this is the first report of eosinophilic gastroenteritis in a patient with minimal change disease . |
the following case report highlights a potentially life threatening complication of undertaking an incision biopsy on a soft tissue tumour , namely surgically uncontrollable haemorrhage from an unsuspected vascular lake .
the case report demonstrates the value of interventional radiology for acute bleeding and radiotherapy for more chronic tumour bleeding .
a 75 year old male presented with a 3 month history of painful progressive left buttock swelling and non - productive cough .
past medical history included carcinoma of the prostate 5 years previously , which had been treated with surgery and radiotherapy .
examination revealed a fixed 10 by 10 cm mass in the left buttock extending over the sacro - iliac joint .
a pleural aspiration cytology confirmed a malignant effusion , although did not differentiate the cell type .
the pelvic mri scan demonstrated a large 10 x 10 x 5 cm heterogeneous lesion in the left buttock , occupying much of the posterior portion of the gluteus maximus and invading the iliac bone and sacrum .
the primary lesion was not operable and the presence of the malignant effusion denoted stage 4 disease , ( t2 , n0 , m1 ) .
percutaneous needle core biopsy of the lesion was inconclusive and incision biopsy was requested after drainage and talc pleurodesis of the pleural effusion . at the time of incision biopsy the capsule of the lesion
this resulted in profuse bleeding from a deep , fresh blood filled , cavity within the cystic tumour . a large vascular lake in the centre of the tumour was evident with a significant vascular flow rate .
the cavity was therefore tightly packed with haemostatic material and firm pressure applied to the wound , causing partial cessation of the haemorrhage .
full surgical control of the bleeding was not possible due to the fact that the tumour was in - operable and the feeding vessels originated from the internal iliac vessels running through the invaded bone of the ileum .
angiography revealed a tumour blush in the gluteal region supplied by the median sacral artery and the posterior division of the internal iliac artery ( iia ) .
the iia was catheterised revealing multiple feeding branches with extravasation from one ( figure 1 ) .
angiograms pre - embolisation with histo - acryl angiograms post - embolisation with histo - acryl over the ensuing 3 weeks , despite the initial successful embolisation the patient haemorrhaged on a further 2 occasions despite pressure dressings to the wound area . on both of these occasions
on each occasion , a different set of feeding vessels was noted to be bleeding and was embolised .
histology from the incision biopsy revealed a diagnosis of epithelioid angiosarcoma ( figure 2 ) which is known to be associated with the development of vascular lakes .
( cd31 immunohistochemistry : immunoperoxidase , original magnification x100 ) to prevent the occurrence of further haemorrhage , the patient was given a course of radiotherapy ( 30gy in 10 treatments over 14 days ) .
palliative chemotherapy to control respiratory symptoms from the pleural effusion was declined and the patient died 6 months after initial presentation .
the epithelioid phenotype of angiosarcoma was first described in 1976 by rosai and colleagues who documented cases of cutaneous angiosarcomas .
it was later documented by fletcher and colleagues in 1991 who described deeper lesions found in the soft tissues where they are now most commonly found .
( 1 ) epithelioid angiosarcomas are rare , high - grade , malignant , vascular tumours , which have an aggressive course and tend to recur locally , spread widely , and have a high rate of lymph node and systemic metastases.(2 ) it often presents in middle - aged and elderly males with a peak incidence in the seventh decade of life .
prognosis for these types of tumours is very poor with death usually occurring from 6 months to 2 years after presentation .
epithelioid angiosarcomas have been described arising in various soft tissues around the body and are most often metastatic in origin .
histologically these tumours consist of solid and infiltrative sheets of epithelioid cells characterised by large , oval to round cells with abundant eosinophilic cytoplasm , vesicular nucleus and eosinophilic nucleolus.(3 ) in most cases there are vascular channels or cystically dilated spaces .
vascular lakes are therefore a common finding associated with these types of sarcoma . a distinctive starry - sky histological pattern has also been reported in epithelioid angiosarcomas .
immunohistochemically the neoplastic cells show positivity for endothelial cell markers such as cd31 , cd34 , factor viii - related antigen , and vimentin.(4 - 5 ) due to cytokeratin expression and epithelioid growth pattern these tumours can occasionally be misdiagnosed as carcinomas .
endothelial differentiation can be confirmed ultrastructually by the presence of weibel - palade bodies , intermediate filaments and basal lamina .
intracytoplasmic vacuoles containing red blood cells are often present in some cells.(4 ) irradiation of soft tissues for the treatment of various primary cancers is well documented as a cause for several types of sarcoma . as in this case
, the patient had undergone a course of radiotherapy for prostate cancer several years prior to presenting with the pelvic sarcoma and this therefore may have been the causative factor .
other risk factors for the development of angiosarcomas include carcinogens , chronic lymphoedema and hormonal status .
however one such case documented a lesion of the mons pubis 4 years following radiotherapy for vulval carcinoma.(5 ) angiography and tumour embolisation is a well established treatment of tumours both pre- and post - operativley .
tumour embolisation may be carried out for acute bleeding from tumour vessels , and to reduce the blood supply to tumours prior to surgery .
treatment usually consists of delivering one or two fractions of 5 - 8 gy each on successive days . | epithelioid angiosarcoma is a rare , highly malignant tumour with a poor prognosis .
we present the case of a 75 year old man who underwent an incision biopsy to diagnose the soft tissue tumour and suffered from surgically uncontrollable haemorrhage .
the case report demonstrates the value of interventional radiology for acute bleeding and radiotherapy for more chronic tumour bleeding . |
depression and anxiety are common mental health disorders ; however , the risks for depression and anxiety are not distributed equally .
women are twice as likely as men to experience depression and generalized anxiety disorder due to a variety of biological and social factors [ 1 , 2 ]
. women 's hormonal fluctuations associated with menstruation , pregnancy , and childbirth may increase their vulnerability to mental distress .
social factors such as poverty , race , and neighborhood also increase women 's risk for depression and anxiety .
low - income women 's risk of depression is almost double that of their nonpoor counterparts [ 2 , 3 ] .
poverty contributes to daily worries about basic needs and limits the ability to engage in recreational activities .
poor women are more likely to experience financial troubles , relationship problems , poor health , and unemployment which can contribute to the onset of depression [ 35 ] .
poverty may combine with a lack of social support to make low - income women vulnerable to anxiety and depressive disorders . for women , racial differences in risk for depression and anxiety vary widely . in some studies , african american women have higher rates of depression and anxiety compared to white women , while other data demonstrate that african american women have lower rates [ 2 , 711 ] . lower rates of anxiety and depression among african american women may be attributed to psychosocial resources , emotional resilience , social support , and ethnic identity .
higher rates in african american women are generally attributed to greater levels of poverty , poor health , and stigma about mental health care [ 1214 ] .
anxiety disorders among african american women are particularly understudied , which contributes to uncertainty about its risk factors and rates in this population .
neighborhood disadvantage can obstruct the creation of bonds among residents , reducing social support and social control that serve as a protective factor against psychological and economic stressors . by increasing daily hassles and reducing protective factors , poor neighborhoods increase an individuals ' vulnerability to depression when negative events occur .
women who live in poor neighborhoods have fewer opportunities for employment , education , recreational activities , and positive social interactions which can cause or exacerbate depression or anxiety .
african american women are more likely to live in poor neighborhoods than their white counterparts . to date , few studies have been able to compare rates of depression and anxiety in african american and white women while examining both individual and neighborhood characteristics .
we used a unique cohort of african american and white women from the pathways to adulthood , a follow - up of a longitudinal cohort of inner city women and their children in baltimore , md , usa . our aims were to ( 1 ) examine what individual and neighborhood socioeconomic factors are associated with depression and anxiety and ( 2 ) determine whether these relationships varied between african american and white women .
the johns hopkins perinatal collaborative study in conjunction with the pathways to adulthood follow - up was a retrospective study of three generations of families initially living in the inner city baltimore .
the perinatal collaborative study enrolled 2307 inner - city women ( referred to throughout as first - generation mothers ( g-1s ) ) who were selected at random at the time of their first prenatal visit to a public obstetric clinic at johns hopkins hospital between 1959 and 1965 .
the study later collected data on the 2694 children of these first - generation mothers who were born between 1960 and 1965 ( children referred to as second generation ( g-2s ) throughout ) .
these children were initially studied prospectively with data gathered between birth and 8 years of age regarding their neurologic and cognitive development , health , behavior , and family , and neighborhood socioeconomic characteristics . from 1992 to 1994 , the pathways to adulthood study collected additional information from the 1758 g-2s ( then aged 27 to 33 ) about their lives from age 9 to present .
follow - up data included information on education , employment , family composition , health , health care usage , and income .
in addition to these data that were collected directly from the g-2 individuals , data regarding the neighborhood characteristics of each g-2 at birth , at 11 - 12 years of age , at 16 - 17 years of age , and at age of follow - up ( ages 2733 ) were obtained through census data . for a full description of sample population and methods ,
see . our final sample ( n = 989 g2 females ) included the 75% of female respondents who provided information ( in - person or via telephone ) for the pathways to adulthood study and had information on the depression or anxiety measures .
the general health questionnaire ( ghq-28 ) is a self - administered screening questionnaire aimed to detect probable psychiatric disorders in primary care settings .
the threshold for a case ( individuals who probably need further evaluation ) is 4 - 5 depending on the population . for greater specificity , we defined a case as a ghq score greater than or equal to 5 .
the ghq-28 has four subscales : somatic , anxiety , social dysfunction , and depression and that each contains seven questions .
the subscales provide individual diagnostic or profile information ; however , they have no diagnostic thresholds . for our study , a ghq depression subscale score greater than or equal to 4
was defined as severe depression and a ghq anxiety subscale score greater than or equal to 4 was defined as anxiety . we examined individual characteristics ( including demographic age , race , number of children , and marital status ) and health - related characteristics ( current drinking status and self - reported health rating ) .
although we had two measures for income , self - reported total household income and personal income , the high rate of missing values in both measures precluded their use in the analysis .
we treated assets as a continuous variable ranging from 0 to 6 , with one point given for a yes
response when asked about each of six assets ( current personal checking account ; current ira or pension , own house or condo , car , truck , or motorcycle ownership , credit or charge account , and current savings account ) . after factor analysis showed home ownership to be the most robust measure , we included homeownership in our model .
each respondent 's address at the time of the interview was linked to data from the appropriate census tract .
we used cross - tabulations to compare all categorical variables by race , neighborhood , and weight status .
we used chi - square statistics as the corresponding measure of heterogeneity . for continuous variables , we determined summary measures ( mean and standard error ) for each sub - group .
next , we used linear regression to examine the multivariate associations between our outcomes depression and anxiety subscale scores and the covariates of interest . during this process
, we included those in which we had substantive a priori interest based on prior literature and included those that were of at least borderline statistical significance during forward stepwise selection ( p < 0.10 ) . for multivariate analysis
, we used linear regression clustered on census tract while controlling for marital status , race , age at first child , number of children , years of education , homeownership , neighborhood poverty , and percent african american in the neighborhood .
the point estimate is the same as in nonclustered linear regression ; only the standard error is corrected .
all analyses were done using intercooled stata ( version 11.0 ; stata corporation , college station , tex , usa ) .
a summary of baseline sociodemographic , health , and neighborhood characteristics of the cohort is presented in table 1 .
white women scored significantly higher than african american women on total ghq score ( 4.42 versus 3.12 ) , mean depression score ( 0.57 versus 0.25 ) , and mean anxiety score ( 1.80 versus 1.22 ) , all p < 0.05 .
although household income was not significantly different between white and african american women ( $ 35,638 versus $ 31,807 , p = 0.18 ) , they differed on many other socioeconomic indicators . compared to their white counterparts ,
african american women had more years of education ( 12.87 versus 10.89 for whites ) , were more likely to have completed college ( 14% versus 3% ) , and had higher personal income ( p < 0.05 ) .
white women had a higher mean number of assets than their african american counterparts ( 3.17 versus 2.48 , p < 0.05 ) , and african american and white women did not differ on the number of children or age of first child ( p > 0.05 ) .
however , white women were significantly more likely to be married ( 59% versus 27% , p < 0.05 ) , which may , in part , account for the differences in both assets and household income .
african american and white women also substantially differed by current neighborhood characteristics ( table 1 ) .
white women lived in neighborhoods with an average household income that was significantly higher than african americans ( $ 37,578 versus $ 28,655 ) .
white women were less likely to live in poor neighborhoods ( 25% versus 58% ) or to report a concern with crime ( 25% versus 36% ) , all p < 0.05 .
the neighborhoods in which the african american study participants lived had a significantly higher proportion of african americans than their white counterparts . on average , white women lived in neighborhoods that were 8% african american , while african women lived in neighborhoods that were predominantly african american ( 72% ) .
approximately 30% of the sample had a ghq score greater than or equal to 5 ( table 2 ) .
they had fewer years of education ( 12 versus 12.7 ) , fewer assets ( 2.2 versus 2.8 ) , lower family income ( $ 29,790 versus $ 33,505 ) , and personal income ( $ 12,520 versus $ 16,487 ) , all p values < 0.05 .
women with a greater ghq score had a higher bmi ( 27.3 versus 26.1 ) , were more likely to be smokers ( 52% versus 42% ) , and were less likely to report good or excellent health ( 32.1 versus 65.2 ) , all p values < 0.05 .
overall , over half the population in both groups lived in poor neighborhoods that were predominately african american , and a significant proportion had concerns about crime within their neighborhoods . in multivariate logistic models , only white race and poor self - rated health were associated with a higher ghq score .
women with good or excellent health were 75% less likely to have a ghq score greater than 4 ( or 0.25 , 95% ci 0.170.36 ) .
african american women were almost 50% less likely than their white counterparts to have a positive ghq score ( or 0.56 , 95% ci 0.320.99 ) .
women with higher depression scale ratings ( 4 ) differed significantly from their less depressed counterparts ( table 3 ) .
more depressed women had fewer years of education ( 10.9 versus 12.6 ) and were less likely to have a college degree ( 0% versus 12% ) , all p < 0.05 .
depressed women had lower assets ( 0.8 versus 2.7 ) , lower household income ( $ 21,300 versus $ 32 , 633 ) , and lower personal income ( $ 8,591 versus $ 15,482 ) , all p < 0.05 .
depressed women were less likely to be married ( 8% versus 34% ) and had a lower age at first child 's birth ( 18.7 versus 21.2 ) , all p < 0.05 .
they had a higher bmi ( 29 versus 26 ) , were more likely to be drinkers ( 64% versus 44% ) , and less likely to report good health ( 24% versus 57% ) , all p < 0.05 .
depressed women lived in neighborhoods with lower median household incomes ( $ 24,594 versus $ 30,045 , p < 0.05 ) .
however , they did not differ significantly from their counterparts on other neighborhood characteristics such as percent african american , neighborhood poverty , and neighborhood crime .
women with higher anxiety ratings ( 4 ) differed significantly from their less anxious counterparts ( table 4 ) .
women with a high anxiety score were less likely to be african american ( 76% versus 83.6% ) , had fewer years of education ( 11.9 versus 12.6 ) , and had more children ( 1.72 versus 1.47 ) , all p < 0.05 .
in addition , more anxious women had fewer assets ( 1.9 versus 2.76 ) , lower family income ( $ 26,325 versus 33,654 ) , and lower personal income ( $ 12,336 versus 15,917 ) , all p < 0.05 .
more anxious women were more likely to be smokers ( 53% versus 43% ) and less likely to report very good or excellent health ( 34% versus 60% ) , all p < 0.05 .
table 5 shows the factors associated with score in ghq depression domain . decreased self - rated health , lower age at first child
, being unmarried , and white race were significantly associated with a higher depression score . in multivariable analysis , women who were african american ( b = 0.44 ) , were married ( b = 0.21 ) , had older age at first child ( b = 0.02 ) , or reported good or excellent health had lower ghq depression scores , all p < 0.05 . a 30-year - old white unmarried woman who had
her first child at age 16 would have an average depression score of 0.95 ( 95% ci 0.711.20 ) , while an african american woman under the same circumstances would have an average score of 0.48 ( 95% ci 0.330.62 ) .
a 30-year - old woman who was married and had her first child at 21 would have a depression score of 0.62 ( 95% ci 0.420.82 ) if she were white and 0.13 ( 95% ci 0.020.29 ) if she were african american
african american women ( b = 0.77 ) , those with older age at first child ( b = 0.06 ) , and those who reported good or excellent health ( b = 0.99 ) had lower anxiety scores , all p < 0.05 . the average white woman in our sample had an anxiety score of 1.72 ( 95% ci 1.292.14 ) if she reported good or excellent self - rated health and 2.7 ( 95% ci 2.273.14 ) if she did not .
the average african american woman in our sample had an anxiety score of 0.81 ( 95% ci 0.491.13 ) if she reported good or excellent health and 1.8 ( 95% ci 1.452.15 ) if she did not .
in our study african american women had significantly lower mean depression scores compared to white women .
our work is consistent with a number of studies that have shown that african americans have lower rates of depression than their white counterparts [ 2 , 711 ] .
although african american women live with greater stress than white women , their coping strategies may make them less vulnerable to the impact of chronic strains [ 25 , 25 ] . additionally , our study indicated african american women had significantly lower anxiety scores than white women .
very little research has focused on the rates of generalized anxiety disorder among african american women and white women .
a large portion of anxiety research focuses on specific population subgroups ( e.g. , postmenopausal women , mothers of preterm infants ) and subcategories of anxiety ( e.g. , women with hiv , social phobia , etc . ) .
consensus has not yet been reached on african american prevalence and presentation of anxiety disorders [ 15 , 26 ] .
our study is one of the few to report a decreased risk of both depression and anxiety for african american women compared to white women . for african american and white women in similar circumstances , african american women consistently had lower rates of both depression and anxiety .
however due to historic and ongoing socioeconomic disparities , african american women experience significantly more challenging life experiences than their white counterparts ( i.e. , discrimination , neighborhood poverty , and low income ) [ 27 , 28 ] . unmarried women and women who had children at a younger age had higher depression scores .
this is consistent with the literature that shows that adolescent mothers have an increased rate of depression and anxiety due to stressful life events [ 29 , 30 ] .
prior work also shows that marriage provides economic , social , and psychological support , which contributes to a person 's well - being .
these social supports and structured routine can encourage healthy behaviors and discourage harmful ones [ 8 , 31 , 32 ] . health status was also associated with depression and anxiety scores .
poor health can increase daily hassles and reduce pleasure , increasing one 's risk for mental distress [ 5 , 13 ] .
conversely , depression and anxiety can increase one 's perception of poor health and can worsen outcomes for preexisting conditions . determining causality is beyond the scope of this study .
however , simply knowing what factors are associated with greater psychological distress can still lead to better efforts to screen for depression and to target mental health services .
the ghq-28 was developed to determine people in a general population who need further evaluation for mental health disorders .
the threshold for a case , those who probably need further evaluation , is 4 - 5 , depending on the population .
we used similar cut - offs for the depression and anxiety scale of greater than or equal to 4 to determine diagnosis .
the pathways to adulthood data had a limited geographic focus , children born in the johns hopkins hospital catchment area .
this group had a higher proportion of african americans and higher poverty rates than the usa as a whole .
our findings suggest racial differences in anxiety and depression may be due , in part , to differences in socioeconomic status or neighborhood environment .
one strength of this study is the use of census data for aggregate neighborhood measures .
using a different data source to determine individual and group measures avoids one source of error because the study population may not be a representative sample of the population .
an additional limitation , also faced by other researchers studying neighborhood , was characterizing the neighborhood environment solely through some proxy measures based on census data . despite the wealth of census information , other variables that may be more directly associated with depression or anxiety
asset mapping may allow a fuller characterization of neighborhood and a deeper understanding of what factors confer health risks . in this cross - sectional study of african american and white women with similar rates of poverty
, we sought to quantify the association between measures of anxiety and depression and individual and neighborhood socioeconomic status and to determine whether these relationships varied by race . at similar levels of disadvantage , white women had higher scores for depression and anxiety .
we found an increased risk for depression in unmarried women and those with younger motherhood .
women with lower reported self - rated health had higher scores for both depression and anxiety .
neither neighborhood poverty nor racial composition was in and of itself a predictor of greater risk for anxiety or depression ; however , the significant risk factors cluster in disadvantaged neighborhoods .
our work highlights the importance of universal screening for depression or anxiety with more in - depth surveillance based on risk factors rather than on racial classification . | background .
we examined factors associated with depression and anxiety in a cohort of low - income baltimore women .
methods .
we used pathways to adulthood data , a cohort of adults aged 27 to 33 who were born in baltimore between 1960 and 1965 .
our outcomes were a score of > 4 on the general health questionnaire ( ghq-28 ) across the depression or anxiety domains .
linear regression clustered on census tract was used for multivariate analysis .
results . in multivariable analyses , unmarried women , white women , those with lower self - rated health , and younger mothers had higher depression scores .
only lower self - rated health and white race were associated with a higher anxiety score .
neither neighborhood poverty nor racial composition was a predictor for anxiety or depression ; however , the significant risk factors cluster in disadvantaged neighborhoods . conclusion .
our work highlights the importance of universal screening for depression or anxiety with more in - depth surveillance based on risk factors rather than on race . |
ogilvie 's syndrome is rare type of acute intestinal pseudoobstruction associated with spontaneous massive dilation of cecum and proximal colon in the absence of mechanical obstruction .
colonic pseudoobstruction is most often seen in postsurgical patients and in medical patients with multiple comorbidities .
exact etiology of this syndrome is still unknown but it is widely believed that an imbalance in the autonomic innervations , either an increase in sympathetic activity or decrease in parasymapathetic activity of distal colon is a main contributor of colonic motility impairment .
this article describes the case of a 46yearold white british man with no significant previous medical and surgical history , who developed ogilvie syndrome after rectal prolapse repair and total hemorrhoidectomy .
the clinical characteristics , diagnostic methods , and management of ogilvie 's syndrome are reviewed .
a 46yearold male without any previous significant medical history was initially admitted for elective surgical repair of recta prolapse and total hemorrhoidectomy as a day case .
he was doing well postsurgery and was safely discharged from the hospital later that evening with regular analgesics , that is , paracetamol and ibuprofen with lactulose .
he reported back to the hospital next morning with complaints of severe spasmodic pain in the lower abdomen and urinary retention for few hours , he opened his bowels twice after he was discharged from the hospital . on examination
lower abdomen was extremely tender on palpation and vital signs were normal except sinus tachycardia .
initial impression was bladder spasms , therefore he was catheterized and was given tramadol 100 mg iv , buscopan 40 mg iv , and cyclizine 50 mg iv .
his symptoms were slightly relieved but still complaining of severe abdominal pain , on reassessment there was a tender palpable mass in right iliac fossa , which was thought to be distended cecum due to air or fecal impaction . to rule out acute intestinal obstruction , urgent erect
plain radiograph revealed massive dilatation of large intestine with few air fluid levels but there were no signs of intestinal perforation ( fig
. 1 ) and a provisional diagnosis of acute colonic pseudoobstruction ( ogilvie 's syndrome ) was made .
treatment was started with nasogastric tube suction , intravenous fluids , regular enemas , and periodic change in position .
over postoperative day 2 , the patient 's condition did not improve and few doses of intravenous neostigmine were given prior to attempted colonic decompression .
a rectal tube was inserted after endoscopic decompression of the colon . despite these interventions ,
his condition deteriorated and his lower abdominal pain worsened . computed tomography ( ct ) imaging showed stigmata of recent hemorrhoid surgery and gas filled distension of cecum and proximal large bowel with localized pneumatosis coli and small volume of adjacent free fluid
the patient proceeded to emergency laparotomy where a significantly distended colon with evidence of cecal ischemia was found .
gross examination of specimen showed hemorrhages and ulceration , histopathology revealed early necrotizing phlebitis and thrombi formation , ulceration , and granulation tissue involving lamina propria and submucosa .
though uncommon , os is still a potentially dangerous condition with life threatening complications like colonic ischemia , perforation , and peritonitis .
the incidence of cecal perforation is 340% and associated mortality rate is as high as 40% 1 , 2 , 3 .
these propositions focus on the increased sympathetic activity or decreased parasympathetic activity or combination of both can cause the phenomenon of os .
this condition is most commonly associated with recent surgical intervention of any kind , trauma to spinal cord or brain , old age , respiratory failure , metabolic imbalance , electrolytes disorders , malignancy , myocardial infarction , congestive heart failure , pancreatitis , recent neurological event like stroke or subarachnoid hemorrhage , renal insufficiency , hypothyroidism , and medications , for example , narcotics , opioids , anticholinergics , and antidepressants .
the studies have documented that 95% of the cases of os are associated with medical or surgical conditions , rest are idiopathic 1 , 4 , 5 .
the main clinical features are severe abdominal pain and distension , nausea and vomiting , constipation , and fever . abdominal tenderness with some degree of guarding and rebound tenderness may be detected .
hypoactive bowel sounds , mass in right iliac fossa , and empty rectum are some other signs that are frequently picked up on physical examination .
the maximum upper limit for normal cecum is 9 cm ; a diameter of more than 1012 cm increases the risk of perforation .
other differentials would include acute or chronic megacolon , diverticulitis , hirschprung 's disease , and carcinoma .
patients should be mobilized or periodically rolled from side to side and to the kneechest position in an effort to promote expulsion of colonic gas .
intervention should be considered if there is no improvement or cecal dilation of more than 12 cm .
neostigmine is main pharmacological agent used in treatment of os , it results in rapid decompression in 7590% of the patients .
cardiac monitoring during neostigmine infusion is indicated for possible bradycardia that may require atropine administration .
contraindications of neostigmine include bradycardia of less than 60/min , systolic blood pressure lower than 90 mmhg and bronchospasm 6 .
several new cytokinetic agents have also been reported to use in os like 5hydroxytryptamin4 receptor antagonist ( e.g. , cisapride ) and motilin receptor agonists 7 .
methylnaltrexone(opioid receptor antagonist ) and erythromycin(prokinetic agent ) have also been documented as therapeutic options .
decompression by colonoscopy is indicated in patients who fail to respond to neostigmine , the success rate is up to 70% with morbidity only 0.22% 8 , 9 , 10 , 11 .
recurrence can be reduced by placing long indwelling compression tubes ; still some patients require serial colonscopic decompressions .
surgical interventions are the last resort which can be either cecostomy or hemicolectomy with end to end anastomosis . in cases of acute dilatation without ischemia or perforation
, tube cecostomy is the preferred option 1 , 2 , 4 , 8 , 12 , 13 .
all efforts should be made postoperatively to improve splanchnic circulation and prevent complications with fluid resuscitation and prophylactic antibiotics .
complications of surgery include intestinal fistula , abdominal compartment syndrome , wound infection , dehiscence , abdominal sepsis , and incisional hernia .
prognosis is related to the underlying condition but with aggressive therapy development of perforation is unusual .
mortality rate has been reported to be 14% in medically treated patients and 30% in surgically treated patients 8 .
acute colonic pseudoobstruction ( ogilvie 's syndrome ) remains uncommon condition with serious complications . the underlying defect in motility of the colon
the key to diagnosis is early recognition of clinical features and requesting radiological images like xrays and ct scan to aid the diagnosis .
conservative management with ng tube insertion , rectal tubes and intravenous neostigmine ( 22.5 mg ) is the mainstay of treatment .
if conservative therapy fails or cecal distension on radiology is > 12 cm then surgical intervention is required to prevent perforation .
it is extremely important to anticipate os in postsurgical patients with acute abdomen . in private hospital where immediate facility of advanced radiology scan and senior input may not be readily available
, it is crucial for junior doctors to recognize the condition in initial assessment and involve surgical consultant early .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
| key clinical messagethis case highlights the rare diagnosis of ogilvie 's syndrome after minor surgery in a private hospital where facilities and expertise are generally sparse . it shows the importance of knowledge of the subject , proper assessment , accurate diagnosis , and early input from seniors is crucial to prevent ischemia and perforation of colon that carries high mortality
. |
the nutrition care process ( ncp ) , developed by the american dietetic association ( now the academy of nutrition and dietetics ) , was designed to improve the consistency and quality of individualized care for patients / clients or groups and the predictability of the patients / client outcome .
it consists of four distinct , interrelated steps - nutrition assessment , nutrition diagnosis , nutrition intervention , and nutrition monitoring and evaluation .
the ncp provides dietetic professionals with a framework for critical thinking and decision - making .
it is thought that use of the ncp can lead to more efficient and effective care and greater recognition of the role of dietetic professionals in all care settings . also the academy of nutrition and dietetics developed international dietetics and nutritional terminology ( idnt ) to describe the unique functions of dietetic professionals in each step of ncp .
now the dietetic professionals have shown significant interest in ncp and are trying to implement ncp in many countries [ 4 - 6 ] . also in korea , the attention to ncp has been increasing among the dietetic professionals .
several korean hospitals attempted to apply ncp into their clinical practices and made many efforts to improve ncp implementation consistently for years .
the korean dietetic association ( kda ) recognized the needs to gather information on ncp and the necessity of ncp implementation in korea .
accordingly kda undertook a few projects , and the ' international dietetics and nutritional terminology ( idnt ) , second edition ' was translated into korean in march 2012 .
the korean society of hospital dietitians ( kshd , the organization under kda ) held the workshops and education programs on ncp for hospital dietitians , and in 2012 , organized a taskforce team to develop ncp education programs for dietetic professionals . not only kshd but some local branches of kda also constructed education programs . besides kda
, several academic societies have made an effort to enhance the abilities to apply and to teach ncp .
moreover ncp is included in the standard education curriculum for clinical dietitian training . as clinical dietitian
is now certified by the ministry of health and welfare ( legislated in 2010 ) , it is recommended that clinical dietitian candidates fully experience ncp at clinical dietitian training institutes .
these various situations provoke the dietetic professionals ' attention to ncp in korea . in this circumstance
, we conducted the survey to investigate the status of ncp implementation in korean hospitals .
also we surveyed the perception and the opinions on ncp of clinical nutrition managers of general hospitals in korea .
the questionnaires were delivered to 82 general hospitals located nationwide via e - mail in may 2012 .
these 82 hospitals had experiences of attending the second hospital evaluation program conducted by the ministry of health and welfare ( 2007 - 2009 ) and the clinical nutrition managers of those hospitals are recognized to possess relatively higher awareness of the professional performance of clinical dietitians at their own fields in korea . of the 82 questionnaires delivered , 37 responses were collected , for a response rate of 45.1% .
the questions included in the first part were on the information on structural characteristics of hospitals related to clinical nutrition care performance .
a pretest was conducted and the initial questionnaire was corrected and revised according to results of the pretest .
descriptive statistics analysis , chi - square test , and student t - test were conducted .
the questionnaires were delivered to 82 general hospitals located nationwide via e - mail in may 2012 .
these 82 hospitals had experiences of attending the second hospital evaluation program conducted by the ministry of health and welfare ( 2007 - 2009 ) and the clinical nutrition managers of those hospitals are recognized to possess relatively higher awareness of the professional performance of clinical dietitians at their own fields in korea . of the 82 questionnaires delivered , 37 responses were collected , for a response rate of 45.1% .
the questions included in the first part were on the information on structural characteristics of hospitals related to clinical nutrition care performance .
a pretest was conducted and the initial questionnaire was corrected and revised according to results of the pretest .
descriptive statistics analysis , chi - square test , and student t - test were conducted .
sixty - eight percent of hospitals were tertiary care hospitals ( designated by ministry of health and welfare among general hospitals , and should be satisfied the assigned levels of disease severity of treated , staffs , facilities , and equipment etc ) and 45.7% of hospitals ( 16 hospitals ) were located in seoul .
the responses to questions regarding perception and knowledge on ncp and idnt are shown in table 2 .
more than 90% of respondents answered that they had known ncp or idnt , and major routes to know them were academic programs and the education programs of the kda .
most respondents had taken education on ncp and more than 75% of respondents had taken education on idnt . seminars or workshops held by the kshd played important roles in providing hospital dietitians with the opportunities to know ncp and idnt .
respondents answered that the known levels of dietitians working in their hospitals were not relatively high , especially idnt . although there were shown no significant differences ,
the percentages of respondents perceived ncp and idnt were higher in those who worked in the hospitals implementing ncp ( table 3 ) .
however there were significant differences in dietitians ' knowledge levels on ncp and idnt between the hospitals implementing ncp and the hospitals not implementing ncp .
the percentages of dietitians responded " very well " and " well " were higher in the hospitals implementing ncp .
ncp was implemented in 9 hospitals among surveyed hospitals and table 4 shows the status of ncp implementation in these hospitals .
mean duration of ncp implementation was 21.9 months , but the duration varied greatly depending on the hospital ( 2 to 52 months ) . in a - third of those hospitals , ncp was applied to all nutrition therapy cases .
computerized programs were using in ncp implementation in two - thirds of hospitals implementing ncp .
idnt had been used in ncp records in 7 hospitals and the steps of ncp using idnt were different depending on the hospital ( figure 1 ) .
the clinical nutrition managers of 21 hospitals among 26 hospitals not implementing ncp responded that they planned to introduce ncp in the near future .
the percentages of ncp steps responded as the most difficult to implement were similar among 3 steps except nutrition assessment step .
the major benefits of ncp implementation thought by the clinical nutrition managers from ncp implementing hospitals were the easy decision - making and the quality improvement of nutrition care .
lack of knowledge and increased time spent were pointed out as the major difficulties in settlement of ncp implementation .
these were also the major reasons that made the clinical nutrition managers hesitate to introduce ncp to their hospitals .
the results of this survey showed that ncp implementation in korea is in an early stage .
most clinical nutrition managers perceived ncp and idnt and had the education experiences on ncp and idnt .
this low ncp implementation rate is going to be a problem not only in the improvement of nutrition care , but in the training of clinical dietitian candidates .
many clinical nutrition managers were concerned about the lack of knowledge and skills of their dietitians and the increase of time spent with adopting ncp .
it was consistent with the responses on difficulties in implementing ncp from the clinical nutrition managers in hospitals that had adopted ncp .
however many clinical nutrition mangers responded ' easy decision - making ' and ' efficient communication ' as the benefits of ncp implementation .
this suggests that there is no need to delay ncp implementation because of the concern in the extra time spent . in the initial stage
, it may become an issue because dietitians have to change their way of work , but the working time may decrease after the adapting to the new method .
in fact , the periods of ncp implementation were less than a year in about a half of hospitals that had adopted ncp . the barriers to adopt ncp and difficulties in implementing ncp pointed out from our survey results were similar to the responses reported in the u.s .
idnt was used in many hospitals implemented ncp , but the extent of idnt use varied depending on the hospital .
idnt was used at nutrition diagnosis step in all hospitals using idnt , but used at other steps of ncp in less than a half of those hospitals .
nutrition diagnosis is the new step that distinguishes ncp from the existing process and requires using idnt . as a result
, nutrition diagnosis terminology will be familiar and widely used in practice compared to other terminologies ( nutrition assessment terminology , nutrition intervention terminology , and nutrition monitoring & evaluation terminology ) .
it was found that nutrition diagnosis terminology was the most familiar and was most widely used in the u.s . .
our survey was carried out on a small scale and administered to limited subjects , clinical nutrition managers .
therefore it is possible that there were differences between our results and the thought of overall working dietitians in clinical fields .
the role of clinical manager is important to adopt ncp , however dietitians who practice nutrition therapy in the fields play an important role also .
the extensive comprehensive survey will be needed and it will provide us with valuable information needed to implement ncp and to improve ncp implementation .
implementation of ncp is required to enhance efficiency , effectiveness , and quality of nutrition therapy . to successfully implement ncp in korean hospital
, ongoing education and training programs should be developed to provide adequate knowledge and help dietitians to cope with the barriers to ncp implementation . | the nutrition care process ( ncp ) , developed by the american dietetic association , is a significant issue to dietetic professionals in many countries and there are rising needs for ncp implementation in korea .
we surveyed clinical nutrition managers of korean general hospitals regarding the perception of ncp , the status of ncp implementation , and the opinions on ncp .
the questionnaire was collected from 35 hospitals .
most clinical nutrition managers perceived ncp , but ncp implementation in hospital was at early stage .
ncp was implemented in a fourth of the surveyed hospitals and many clinical nutrition managers responded that the lack of knowledge and the concern on increasing working time were major barriers to implementing ncp . to successfully implement ncp in korean hospital , ongoing education and training programs should be developed to provide adequate knowledge and help dietitians to cope with the barriers . |
flowers of n. indicum were collected from the local area of vidharbha region and authenticated from the department of botany , amravati university , amravati .
flowers were dried in shade and coarsely powdered weighing 1 kg was extracted with ethyl acetate by maceration process for 7 days .
the extract of n. indicum flower ( nie ) was found to be 10.7 g with percentage practical yield of 1.070% w / w .
healthy male sprague dawley rats ( 200250 g ) were used for the pharmacological screening .
the animals were housed in polypropylene cages with wire mesh top and husk bedding and maintained under standard environmental conditions ( 25c 2c , relative humidity 60% 5% , light - dark cycle of 12 h each ) and fed with standard pellet diet ( trimurti feeds , nagpur ) and water ad libitum for the entire animal study .
animals were housed and treated according to the rules and regulations of the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) and institutional animal ethics committee ( iaec ) .
the protocol for all the animal studies was approved by the iaec with research project number 650/02/c / cpcsea/04/2009 .
animals were grouped into six groups of six animals each , as follows :
control group : animals were not subjected for social isolation and not treated with any drugnegative control group : animals were subjected for 21 days isolation in dark room without any treatmentlow dose group ( nie 200 mg / kg p.o . ) : animals were subjected to social isolation and treated with 200 mg / kg of n. indicum extract once daily p.o.high dose group ( nie 400 mg / kg p.o . ) : animals were subjected for social isolation and treated with 400 mg / kg of n. indicum extract once daily p.o.dzm group : animals were subjected for social isolation and treated with dzm ( 2 mg / kg ) alone i.p . before 1 h prior to the experimentve group : animals were subjected for social isolation and treated with vitamin e ( 100 mg / kg ) alone p.o .
control group : animals were not subjected for social isolation and not treated with any drug negative control group : animals were subjected for 21 days isolation in dark room without any treatment low dose group ( nie 200 mg / kg p.o . ) : animals were subjected to social isolation and treated with 200 mg / kg of n. indicum extract once daily p.o .
high dose group ( nie 400 mg / kg p.o . ) : animals were subjected for social isolation and treated with 400 mg / kg of n. indicum extract once daily p.o .
dzm group : animals were subjected for social isolation and treated with dzm ( 2 mg / kg ) alone i.p . before 1 h prior to the experiment ve group : animals were subjected for social isolation and treated with vitamin e ( 100 mg / kg ) alone p.o .
once daily for 21 days . behavioral and oxidative stress parameters ( in blood and tissue )
were studied in socially isolated rats and normal rats ( social isolation for 21 days ) .
after 21 days of social isolation , behavioral study was carried out in animals using various behavioral models such as elevated plus maze ( epm ) and light and dark model .
three main parameters were studied , namely , percent number of open arm entries , time spent in open arms , and percent number of closed arm entries . increase in percent number of open arm entries and time spent in open arms indicated a reduction of anxious behavior .
one area is small , black , nonilluminated and other is larger , white , and brightly light .
number of entries and time spent in the light compartment were used for the estimation of anxiety .
suspension of red blood cells ( rbcs , 5% ) was prepared by adding phosphate buffer saline ( pbs ) ( 8 ml ) to packed cells .
about 0.5 ml of 5% rbc was mixed with 5 ml of distilled water , shaken for 5 min , and then kept at 4c for 5 min .
ethanol mixtures was added , shaken vigorously to precipitate hemoglobin , and then 0.15 ml of distilled water was added .
the mixture was centrifuged to get a clear erythrocyte lysate . after receiving the treatments for 21 days ,
brain was removed and thoroughly washed with ice - cooled 0.1 m pbs containing 0.1 mmol / l phenyl methanesulfonyl fluoride .
this tissue was blotted dry and homogenized in 0.1 m pbs in an ice bath to prepare a 10% suspension .
this suspension was then centrifuged at 16,000 rpm for 1 h in a cooling centrifuge at 0c .
the supernatant was employed to assess the parameters of oxidative stress after estimating the protein content .
lipid peroxidation ( lpo ) was determined by the molar extinction coefficient of malondialdehyde ( mda ) ( 1.56/105 ) and expressed in terms of nanomoles of mda / ghb .
the activity of catalase ( cat ) enzyme was determined in the erythrocyte lysate , as the decrease in absorbance was measured spectrophotometrically at 240 nm for 1 min .
the activity of superoxide dismutase ( sod ) was determined spectrophotometrically in the erythrocyte lysate ; increase in the absorbance was measured at 420 nm for 3 min .
one unit of enzyme activity represents 50% inhibition of the rate of auto - oxidation of pyrogallol , as determined by change in absorbance / minute at 420 nm .
blood - reduced glutathione ( gsh ) was measured by addition of 0.2 ml of whole blood to 1.8 ml of distilled water followed by 3.0 ml of precipitating mixture .
it was centrifuged at 2000 rpm for 5 min , and 1 ml of supernatant was added to 1.5 ml acid ; 5 ml of phosphate solution , followed by the addition of 0.5 ml of dithionitrobenzoic , 5-dithiobis ( 2-nitrobenzoic acid ) reagent .
flowers of n. indicum were collected from the local area of vidharbha region and authenticated from the department of botany , amravati university , amravati .
flowers were dried in shade and coarsely powdered weighing 1 kg was extracted with ethyl acetate by maceration process for 7 days .
the extract of n. indicum flower ( nie ) was found to be 10.7 g with percentage practical yield of 1.070% w / w .
healthy male sprague dawley rats ( 200250 g ) were used for the pharmacological screening .
the animals were housed in polypropylene cages with wire mesh top and husk bedding and maintained under standard environmental conditions ( 25c 2c , relative humidity 60% 5% , light - dark cycle of 12 h each ) and fed with standard pellet diet ( trimurti feeds , nagpur ) and water ad libitum for the entire animal study .
animals were housed and treated according to the rules and regulations of the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) and institutional animal ethics committee ( iaec ) .
the protocol for all the animal studies was approved by the iaec with research project number 650/02/c / cpcsea/04/2009 .
animals were grouped into six groups of six animals each , as follows :
control group : animals were not subjected for social isolation and not treated with any drugnegative control group : animals were subjected for 21 days isolation in dark room without any treatmentlow dose group ( nie 200 mg / kg p.o . ) : animals were subjected to social isolation and treated with 200 mg / kg of n. indicum extract once daily p.o.high dose group ( nie 400 mg / kg p.o . ) : animals were subjected for social isolation and treated with 400 mg / kg of n. indicum extract once daily p.o.dzm group : animals were subjected for social isolation and treated with dzm ( 2 mg / kg ) alone i.p . before 1 h prior to the experimentve group :
animals were subjected for social isolation and treated with vitamin e ( 100 mg / kg ) alone p.o .
control group : animals were not subjected for social isolation and not treated with any drug negative control group : animals were subjected for 21 days isolation in dark room without any treatment low dose group ( nie 200 mg / kg p.o . ) : animals were subjected to social isolation and treated with 200 mg / kg of n. indicum extract once daily p.o .
high dose group ( nie 400 mg / kg p.o . ) : animals were subjected for social isolation and treated with 400 mg / kg of n. indicum extract once daily p.o .
dzm group : animals were subjected for social isolation and treated with dzm ( 2 mg / kg ) alone i.p . before 1 h prior to the experiment ve group : animals were subjected for social isolation and treated with vitamin e ( 100 mg / kg ) alone p.o . once daily for 21 days .
behavioral and oxidative stress parameters ( in blood and tissue ) were studied in socially isolated rats and normal rats ( social isolation for 21 days ) .
after 21 days of social isolation , behavioral study was carried out in animals using various behavioral models such as elevated plus maze ( epm ) and light and dark model .
three main parameters were studied , namely , percent number of open arm entries , time spent in open arms , and percent number of closed arm entries .
increase in percent number of open arm entries and time spent in open arms indicated a reduction of anxious behavior .
one area is small , black , nonilluminated and other is larger , white , and brightly light .
number of entries and time spent in the light compartment were used for the estimation of anxiety .
three main parameters were studied , namely , percent number of open arm entries , time spent in open arms , and percent number of closed arm entries .
increase in percent number of open arm entries and time spent in open arms indicated a reduction of anxious behavior .
one area is small , black , nonilluminated and other is larger , white , and brightly light .
number of entries and time spent in the light compartment were used for the estimation of anxiety .
suspension of red blood cells ( rbcs , 5% ) was prepared by adding phosphate buffer saline ( pbs ) ( 8 ml ) to packed cells .
about 0.5 ml of 5% rbc was mixed with 5 ml of distilled water , shaken for 5 min , and then kept at 4c for 5 min .
subsequently , 0.4 ml of 3:5 chloroform ethanol mixtures was added , shaken vigorously to precipitate hemoglobin , and then 0.15 ml of distilled water was added .
the mixture was centrifuged to get a clear erythrocyte lysate . after receiving the treatments for 21 days ,
brain was removed and thoroughly washed with ice - cooled 0.1 m pbs containing 0.1 mmol / l phenyl methanesulfonyl fluoride .
this tissue was blotted dry and homogenized in 0.1 m pbs in an ice bath to prepare a 10% suspension .
this suspension was then centrifuged at 16,000 rpm for 1 h in a cooling centrifuge at 0c .
the supernatant was employed to assess the parameters of oxidative stress after estimating the protein content .
lipid peroxidation ( lpo ) was determined by the molar extinction coefficient of malondialdehyde ( mda ) ( 1.56/105 ) and expressed in terms of nanomoles of mda / ghb .
the activity of catalase ( cat ) enzyme was determined in the erythrocyte lysate , as the decrease in absorbance was measured spectrophotometrically at 240 nm for 1 min .
the activity of superoxide dismutase ( sod ) was determined spectrophotometrically in the erythrocyte lysate ; increase in the absorbance was measured at 420 nm for 3 min .
one unit of enzyme activity represents 50% inhibition of the rate of auto - oxidation of pyrogallol , as determined by change in absorbance / minute at 420 nm .
blood - reduced glutathione ( gsh ) was measured by addition of 0.2 ml of whole blood to 1.8 ml of distilled water followed by 3.0 ml of precipitating mixture .
it was centrifuged at 2000 rpm for 5 min , and 1 ml of supernatant was added to 1.5 ml acid ; 5 ml of phosphate solution , followed by the addition of 0.5 ml of dithionitrobenzoic , 5-dithiobis ( 2-nitrobenzoic acid ) reagent .
suspension of red blood cells ( rbcs , 5% ) was prepared by adding phosphate buffer saline ( pbs ) ( 8 ml ) to packed cells .
about 0.5 ml of 5% rbc was mixed with 5 ml of distilled water , shaken for 5 min , and then kept at 4c for 5 min .
subsequently , 0.4 ml of 3:5 chloroform ethanol mixtures was added , shaken vigorously to precipitate hemoglobin , and then 0.15 ml of distilled water was added .
after receiving the treatments for 21 days , animals were sacrificed using deep ether anesthesia .
brain was removed and thoroughly washed with ice - cooled 0.1 m pbs containing 0.1 mmol / l phenyl methanesulfonyl fluoride .
this tissue was blotted dry and homogenized in 0.1 m pbs in an ice bath to prepare a 10% suspension .
this suspension was then centrifuged at 16,000 rpm for 1 h in a cooling centrifuge at 0c .
the supernatant was employed to assess the parameters of oxidative stress after estimating the protein content .
lipid peroxidation ( lpo ) was determined by the molar extinction coefficient of malondialdehyde ( mda ) ( 1.56/105 ) and expressed in terms of nanomoles of mda / ghb .
the activity of catalase ( cat ) enzyme was determined in the erythrocyte lysate , as the decrease in absorbance was measured spectrophotometrically at 240 nm for 1 min .
the activity of superoxide dismutase ( sod ) was determined spectrophotometrically in the erythrocyte lysate ; increase in the absorbance was measured at 420 nm for 3 min .
one unit of enzyme activity represents 50% inhibition of the rate of auto - oxidation of pyrogallol , as determined by change in absorbance / minute at 420 nm .
blood - reduced glutathione ( gsh ) was measured by addition of 0.2 ml of whole blood to 1.8 ml of distilled water followed by 3.0 ml of precipitating mixture .
it was centrifuged at 2000 rpm for 5 min , and 1 ml of supernatant was added to 1.5 ml acid ; 5 ml of phosphate solution , followed by the addition of 0.5 ml of dithionitrobenzoic , 5-dithiobis ( 2-nitrobenzoic acid ) reagent .
table 1 shows that there was a significant ( p < 0.001 ) increase in the closed arm entries and decrease in the open arm entries of negative control as compared to the control group .
nie 200 mg / kg p.o . , nie 400 mg / kg p.o . ,
0.001 ) decrease in closed arm entries and a significant ( p < 0.001 ) increase in the open arm entries as compared to negative control group .
there was a significant ( p < 0.001 ) increase in the time spent in the closed arm and decrease in time spent in the open arm of negative control group as compared to control group .
nie 200 mg / kg p.o . , nie 400 mg / kg p.o . , and
dzm group show a significant ( p < 0.001 ) decrease in time spent in the closed arm and a significant ( p < 0.001 ) increase in the time spent in the open arm as compared to the negative control group .
effect of nerium indicum flower extract on anxiety by elevated plus maze test after 21 days of social isolation table 2 shows that there was a significant ( p < 0.001 ) increase in the dark box entries of negative control as compared to control group .
nie 200 mg / kg p.o . , nie 400 mg / kg p.o . , and
dzm group show a significant ( p < 0.001 ) decrease in dark box entries and a significant ( p < 0.001 ) increase in light box entries as compared to the negative control group .
there was a significant ( p < 0.001 ) increase in the time spent in dark box and decrease in the time spent in the light box of negative control as compared to control group .
dzm group show a significant ( p < 0.001 ) decrease in time spent in dark box and a significant ( p < 0.001 ) increase in the time spent in light box as compared to the negative control group .
effect of nerium indicum flower extract on anxiety by light and dark model after 21 days of social isolation table 3 shows that there was a significant ( p < 0.001 ) increase in the lpo and decrease in sod , gsh , and cat levels of negative control as compared to control group , and in nie 200 mg / kg p.o . , nie 400 mg / kg p.o . , and ve group ,
there was a significant ( p < 0.001 ) decrease in lpo and increase in sod , gsh , and cat levels in blood .
effect of nerium indicum flower extract on oxidative stress parameters in blood after 21 days of social isolation table 4 shows that there was a significant ( p < 0.001 ) increase in the lpo and decrease in sod , gsh , and cat levels of negative control as compared to control group in brain tissue . in nie 200 mg /
treated group , there was a significant ( p < 0.05 ) decrease in the lpo and increase in cat level in brain tissue . in
there was a significant ( p < 0.001 ) decrease in the lpo and increase in sod , gsh , and cat levels in brain tissue .
effect of nerium indicum flower extract on oxidative stress in brain after 21 days of social isolation
the present study evaluates the anxiolytic activity of nie by the virtue of its antioxidant potential . in this study , anxiety was induced by isolating the animals socially for 21 days .
there are different models used for the induction of anxiety such as novelty - induced hyponeophagia , social interaction , and open field exploration .
social isolation enhances the oxidative stress and thereby alters and causes anxiety - like behavior . here , in the present investigation
, it was observed that there was a significant increase in the oxidative stress and anxiety level in socially isolated animals .
the epm and dark and light model are most commonly used models for the study of anxiogenic or anxiolytic effect of drug in rodents .
the results of the present study showed that nie exhibited a significant ( p < 0.001 ) anxiolytic activity by an increase in the percent of entries and time spent in open arms and decrease in the percent of entries and time spent in closed arms in socially isolated animals , as compared to negative control . whereas in dark and light test , treatment with nieshowed a significant ( p < 0.001 ) increase in the number of entries and time spent at light box when given at 200 mg / kg p.o . and 400 mg / kg p.o
literature also suggest that social isolation alters glucocorticoid production and neurotransmitter system , which induce stress .
it was also observed that production of reactive oxygen species is increased in anxiety , and the drug possessing antioxidant property effectively manages the anxiety - like behavior .
this has enforced the research in finding antioxidants in foods and medicinal plants ; as a result , approximately , 4000 antioxidants have been identified .
this study shows that social isolation significantly ( p < 0.001 ) increases the lpo level , and there is a decrease in the levels of sod , cat , and gsh found in the blood and brain of anxious rats compared to normal rats . whereas the treatment with nie to anxious rats significantly ( p < 0.001 ) decreases the lpo level and increases the levels of sod , cat , and gsh
the results suggest that nie upregulates the activities of some antioxidant enzymes intracellular activities in anxiety .
the beneficial antioxidant effects of n. indicum could be as a result of inhibition of specific pathways that are activated as a consequence of increased oxidative stress in the progression of anxiety .
| aim : the aim of this study was to analyze the ethyl acetate extract of nerium indicum ( nie ) flower for its antioxidant effect in anxious sprague
dawley rats.materials and methods : animals were divided into six groups ( n = 6 ) and treated with 200 mg / kg and 400 mg / kg p.o .
of nie for 21 days to assess its preventive and curative effects .
anxiety was induced by isolating animals socially for 21 days . elevated plus
maze ( epm ) and light and dark model were used for measuring anxiety in animals .
oxidative stress parameters such as lipid peroxidation ( lpo ) , superoxide dismutase ( sod ) , catalase ( cat ) , and reduced glutathione ( gsh ) in blood and brain tissue homogenate were monitored after 21 days of social isolation in animals.results:rats were treated with nie 200 mg / kg and 400 mg / kg p.o .
both the treatments showed a significant ( p < 0.001 ) increase in the number of open arm entries and time spent in open arm in epm when compared with the negative control .
results also demonstrated that there was a significant ( p < 0.001 ) increase in the number of lightbox entries and time spent in light box in light and dark model when compared with negative control .
there was a significant ( p < 0.001 ) improvement in endogenous anti - oxidants such as sod , cat , reduced gsh , and decreased levels of lpo in blood and brain tissue when compared with the negative control.conclusion:the present study suggests the role of nie in the treatment of anxiety , possibly by modulating the oxidative stress . |
celem pracy jest ocena moliwoci diagnostyki nowotworw neuroendokrynnych jelita cienkiego przy uyciu ultrasonografii poprzez identyfikacj zmian w krezce .
w okresie od 1996 do 2013 roku zgromadzono 17 chorych ( 9 kobiet i 8 mczyzn w rednim wieku 57 lat ) z nowotworem neuroendokrynnym w jelicie cienkim .
wszystkie objte retrospektywn analiz osoby ( n = 17 ) miay wykonane badanie usg jamy brzusznej wedug protokou opisanego wczeniej .
w badaniu ultrasonograficznym udao si uwidoczni pierwotny nowotwr neuroendokrynny u 13 z 17 chorych ( 76,5% ) .
u pozostaych 4 osb guz zlokalizowany w jelicie cienkim zosta rozpoznany na podstawie enteroklizy tk ( n = 3 ) i enteroklizy klasycznej ( n = 1 ) .
somatostatynowa scyntygrafia receptorowa i enterokliza tk dostarczyy dodatkowych informacji co do stopnia zaawansowania procesu chorobowego w stosunku do wynikw badania usg w 7 przypadkach .
wrd 17 chorych z rozpoznanym ultrasonograficznie nowotworem neuroendokrynnym jelita cienkiego u wikszoci ( n = 11 , 64,7% ) zmiany w krezce jelita byy dominujcym objawem patologicznym , ktry pozwoli podejrzewa , a nastpnie zlokalizowa zmian pierwotn .
hipoechogeniczne wzy chonne krezkowe miay najwiksze wymiary ( 918 mm ) , byy dobrze odgraniczone , a wiksze z nich wykazyway unaczynienie .
wszystkie byy pojedyncze , przewanie nieostro odgraniczone , a w dwch zmianach obecne byy niewielkie zwapnienia .
w badaniu usg przezbrzusznym u 2/3 chorych z guzem neuroendokrynnym w jelicie cienkim wystpuj wtrne zmiany w krezce , ktre s atwiejsze do zobrazowania ni samo ognisko pierwotne .
from 1996 to 2013 , 17 patients ( 9 women and 8 men , aged 2362 , mean age 57 ) with a neuroendocrine tumor in the small intestine were subject to a retrospective analysis .
most of them reported non - specific gastrointestinal symptoms ( 13 of 17 ) . in each case
all patients ( n = 17 ) had an abdominal us examination conducted in accordance with the previously mentioned protocol .
ultrasound examinations were conducted with the use of various systems using convex probes with the frequency of 2.55 mhz and linear probes with the frequency of 512 mhz .
all abdominal regions were scanned thoroughly , including the available loops of the small intestine , the mesentery and the colon .
all scans were performed by one person , a physician with 25-year experience in ultrasound imaging .
moreover , the examined group was diagnosed with the use of multiphasic computed tomography or ct enteroclysis ( n = 16 ) , somatostatin receptor scintigraphy ( n = 11 ) and conventional enteroclysis ( n = 2 ) .
also , biochemical tests , including non - specific and specific markers helpful in net diagnosis , were performed in each patient .
the ultrasound examinations visualized foci of neuroendocrine tumors in 13 of 17 patients ( 76.5% ) . in the remaining 4 patients ,
tumors in the small bowel were localized in ct enteroclysis ( n = 3 ) and conventional enteroclysis ( n = 1 ) .
the localization was also confirmed by somatostatin receptor scintigraphy in 3 of 4 such patients .
somatostatin receptor scintigraphy and ct enteroclysis supplemented the ultrasound result by providing information about the stage of the disease in 7 cases .
this concerned the number of involved mesenteric lymph nodes ( n = 4 ) , metastases to the lungs ( n = 2 ) and to the ovary in one woman .
the examinations revealed single intramural nodules with the size of 1535 mm in the small intestine of 17 patients .
secondary mesenteric changes , which were striking ultrasound signs of an existing pathology in 11 patients ( 64.7% ) , were analyzed in greater detail ( tab .
it should also be mentioned that the hypoechoic mesenteric lymph nodes were the largest ( 918 mm ) and well - circumscribed ( fig . 1 and 2 ) .
in most cases , only single nodes were visible ( n = 9 ) , but two patients presented 23 pairs of lymph nodes they were all localized in the vicinity of the primary lesion .
all lesions were single , poorly circumscribed , and slight calcifications were detected in two cases ( fig .
3 , 4 , 5 , 6 a and 6 b ) . figures 7a and 7b illustrate a similar case : a net focus ( 20 mm ) in the terminal ileum and a hypoechoic mass ( 35 mm ) in the mesentery .
ct enteroclysis confirmed the presence of these lesions and additionally revealed slight enlargement of four mesenteric lymph nodes ( fig .
7 c and 7 d ) and two slight metastatic foci in the lower field of the right lung ( not shown in figures ) .
metastatic lymph nodes ( n ) in the mesentery reflect the presence of a small intestinal carcinoid tumor ( not shown in the figure ) one view shows irregular infiltration of a carcinoid tumor of the terminal ileum ( c ) and a sentinel lymph node ( n ) carcinoid tumor ( 12 mm ) of the small bowel ( not shown in the figure ) . in this case , an irregular hypoechoic and vascularized infiltration is visible in the mesentery an irregular hypoechoic mass in the mesentery is the only manifestation of a carcinoid tumor in the ileum ( not shown in the figure ) two views of a hypoechoic mass ( 4 cm ) in the mesentery of a 32-year - old patient with the body mass of 122 kg as the only ultrasound sign of a carcinoid tumor in the ileum a sizeable , partially calcified hypoechoic mass ( m ) in the mesentery is a prominent manifestation of a carcinoid tumor in the ileum ( not shown in the figure ) the same case .
the vascularization of the lesion shown in a different view one view captures a carcinoid tumor in the ileum ( c ) and a hypoechoic mass in the mesentery , which is greater than the primary lesion the same patient .
this view shows only the bean - shaped carcinoid tumor in the intestine , visualized with the use of a linear probe the same patient , but the lesions are visualized in ct enteroclysis . the tumor of the terminal ileum ( c ) is of an identical shape to that visualized by ultrasonography .
moreover , two affected mesenteric lymph nodes are visible the same patient . a different ct layer .
it presents the mass ( m ) in the small intestine and two different affected mesenteric lymph nodes ( n ) .
moreover , two metastatic foci were detected in the right lung ( not shown here ) secondary mesenteric changes in 17 patients with small bowel nets detected by ultrasonography the wrinkled and thickened mesentery was hyperechoic ( fig .
8 a ) , and in one patient mesenteric retraction had caused small bowel obstruction ( fig .
a net was manifested by the heterogeneously thickened mesentery , in which a focal lesion was not visualized with certainty ( fig .
9 ) . in five cases , the affected lymph nodes accompanied a hypoechoic mesenteric mass .
carcinoid tumor ( 2 cm ) of the ileum with features of hyperemia and with the markedly hyperechoic mesentery the same patient .
dilated loops of the small intestine surrounding the thickened mesentery ( m ) indicate ileus thickened mesentery shows lesions that are difficult to visualize .
they are an ultrasound representation of a carcinoid tumor in the small intestine ( not shown in the figure )
out of all nets in the gastrointestinal tract , those in the small bowel are characterized by the greatest ability to metastasize , both locally and distantly .
list the following 5-year survival rates depending on the localization of the primary focus : 8590 in the appendix , colon and rectum , 73 in the stomach , 68 in the cecum and 67 in the small bowel .
the aforementioned authors have not demonstrated a strong relationship in their multivariate analysis . despite small sizes , nets in the small bowel rapidly metastasize to the lymph nodes : 1 cm lesions metastasize in 1525% of cases , tumors between 1 and 2 cm in 5880% of cases , and larger masses ( greater than 2 cm ) in over 70% of cases .
the role of abdominal ultrasound ( apart from its endoscopic version ) in diagnosing nets is rather limited .
the literature usually mentions that us can detect metastatic foci in the liver and lymph nodes , or the method is not included at all in the diagnostic algorithms of small bowel nets .
there are only case reports that present single cases of nets in the small intestine diagnosed by ultrasonography .
the attempts to introduce ultrasound contrast agents have not changed the attitude to abdominal ultrasonography either .
however , it seems that , in experienced hands , this modality can be useful in the initial diagnosis of patients with suspicions of nets in the small intestine .
these changes are sometimes more visible than the primary focus concealed in the intestinal wall .
this issue was the main subject of the analysis conducted in our 17 patients with small bowel nets .
all hypoechoic changes are well visible against the mesentery of intermediate or slightly increased echogenicity .
mesenteric thickening and centrally folding structure are usually manifested by a pattern of slightly increased reflection intensity compared with the normal mesentery , and that is why such a reaction can be more difficult to detect . to date , pathological masses is the mesentery , which can signal the presence of a net in the small intestine , have been the subject of only few papers .
of 29 patients with a tumorous carcinoid mass , 30 foci were detected in the mesentery .
their sizes ranged from 0.5 cm to 6.0 cm ( mean 3.3 cm ) and most of them ( 24/30 , 70% ) contained calcifications of various types .
21 specimens were tested histologically , but only 7 ( 33% ) showed fragments of the lymph node architecture .
however , the authors of this paper do not specify the percentage of patients with nets in whom mesenteric changes were found . in our patients ,
it must be therefore concluded that a hypoechoic mass in the mesentery is not a rare phenomenon in patients with nets in the small bowel and can be an important manifestation of such a pathology in an abdominal us scan .
this finding should prompt the ultrasonographer to assess the small intestine carefully , particularly when such a pathological process is suspected clinically .
it is obvious that a change detected in the mesentery can also be a different neoplasm , either primary ( e.g. lymphoma , liposarcoma , mesothelioma , etc . ) or secondary ( e.g. metastases of colorectal cancer or carcinoma of the small intestine or ovary , etc . )
the differential diagnosis should also include inflammatory lesions ( tuberculosis , sarcoidosis , etc . ) and other processes ( amyloidosis , fibromatosis , etc . ) .
however , the detection of the affected mesenteric lymph nodes as the only finding requires even more pathologies to be ruled out , mainly numerous infectious lesions and crohn 's disease .
fragmentary thickening of the mesentery in nets is difficult to differentiate from mesenteritis ( particularly its fibrotic form ) which can also lead to small bowel obstruction .
sonography should be treated as an initial examination in the diagnostic algorithm of nets because of its limited ability to assess the generalization of the pathological process , the possibility of multiple nets in the small bowel ( which is relatively frequent ) and increased risk of the development of other neoplastic lesions , either synchronous or metachronous .
in an abdominal us examination , 2/3 of patients with neuroendocrine tumors in the small bowel manifest secondary lesions in the mesentery which are easier to visualize than the primary focus .
thirty percent of them are manifested as hypoechoic masses.the detection of such lesions should prompt the search for the primary focus in the small intestine .
in an abdominal us examination ,
2/3 of patients with neuroendocrine tumors in the small bowel manifest secondary lesions in the mesentery which are easier to visualize than the primary focus .
the detection of such lesions should prompt the search for the primary focus in the small intestine .
authors do not report any financial or personal links with other persons and organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication . | neuroendocrine tumors make up an interesting pathology of a variable clinical picture , prognosis , localization , endocrine activity and degree of malignancy.aimthe aim of this paper is to assess whether ultrasonography can be helpful in diagnosing neuroendocrine tumors in the small intestine by analyzing changes in the mesentery.material and methodsfrom 1996 to 2013 , we encountered 17 patients ( 9 women and 8 men at the mean age of 57 ) with a neuroendocrine tumor in the small intestine .
the diagnosis was confirmed in all patients by pathomorphological examinations .
all retrospectively analyzed patients ( n = 17 ) had an abdominal us examination conducted in accordance with the previously mentioned protocol.resultsprimary neuroendocrine tumors were visualized ultrasonography in 13 of 17 patients ( 76.5% ) . in the remaining 4 patients , tumors in the small bowel
were diagnosed in ct enteroclysis ( n = 3 ) and conventional enteroclysis ( n = 1 ) .
somatostatin receptor scintigraphy and ct enteroclysis supplemented the ultrasound result by providing information about the stage of the disease in 7 cases . in most of the 17 patients with a neuroendocrine tumor diagnosed by ultrasound ( n = 11 , 64.7% ) , changes in the mesentery were prevailing pathological signs that raised suspicion and , consequently , helped localize the primary lesion .
the hypoechoic mesenteric lymph nodes were the greatest ( 918 mm ) , well - circumscribed , and the largest of them showed signs of vascularization .
the size of hypoechoic lesions in the mesentery ranged from 25 to 53 mm .
these lesions showed moderate blood flow .
all of them were single , usually poorly circumscribed ; two lesions showed slight calcifications.conclusionsin an abdominal us examination , 2/3 of patients with neuroendocrine tumors in the small bowel manifest secondary lesions in the mesentery which are easier to visualize than the primary focus .
30% of them are manifested as hypoechoic masses .
the detection of such lesions should prompt the search for the primary focus in the small intestine . |
since the advent of icodextrin in 1994 , its use is continuously increasing in peritoneal dialysis ( pd ) patients .
it has proven advantage in ultrafiltration failure , high transport peritoneal membranes , and diabetics as compared to standard glucose based dialysate . with successful result of a case of single nocturnal icodextrin exchange in a patient with residual renal function and incremental dialysis in literature
, a protocol was made to offer incremental dialysis to all end - stage renal failure patients with significant residual renal function and opting pd as renal replacement therapy .
we retrospectively analyzed the 5-year data for outcome of ico - alone patients and compared it with that of a cohort of contemporary patients on conventional pd .
this was a single - center , retrospective analysis of patients between october 2006 and october 2011 .
all adult patients opting for pd and significant urine output were asked for urinary clearance ( kt / v ) .
those having a urinary kt / v of about 1 were offered incremental dialysis and initiation with single nocturnal icodextrin exchangeico - alone group .
all others were initiated with conventional pd ( 3 exchanges of 2 l standard glucose - based dialysate ) .
adequacy was done at 1 , 3 , and 6 months and then 6 monthly .
patients in ico - alone group , falling short of adequacy or if clinically indicated with oliguria and/or fluid overload , were shifted to conventional pd .
those falling short of adequacy in conventional pd were offered 3 exchanges of 2.5 l or 4 exchanges per day .
outcomes ( peritonitis , exit site infection rates , and mortality ) were compared between the two groups .
continuous variables were compared between groups by independent sample t - test and categorical variables by chi - square test .
patient survival was calculated using kaplan meier survival curves and both the groups were compared by log rank test ( mantel cox ) . in view of intention to treat
continuous variables were compared between groups by independent sample t - test and categorical variables by chi - square test .
patient survival was calculated using kaplan meier survival curves and both the groups were compared by log rank test ( mantel cox ) . in view of intention to treat
forty - six adult patients were initiated on pd between october 2006 and october 2011 .
baseline characteristics of study patients are shown in table 1 and laboratory parameters in table 2 .
table 1 shows creatinine before these patients were initiated on dialysis and most of them were covered with hemodialysis during their break in period , whereas table 2 creatinine values are at initiation of pd .
there was no difference in demographics and laboratory parameters at initiation or at the end of the study , except for the urine output , which was 1265 316 in the ico - alone group and 551 504 in the conventional group ( p = 0.000 ) .
mean period on therapy for ico - alone was 18.8 14.7 months , whereas , for conventional dialysis , it was 13.5 8.3 months ( p = 0.15 ) .
distribution of patients in the study baseline characteristics of patients in the study laboratory parameters of patient at initiation of pd and at the end of the study calculated median period on ico - alone was 9.6 months .
peritonitis rate was 1 episode in 56.22 vs 25.29 patient - months and exit site infection was 1 episode in 56.2 vs 189.71 patient - months in ico - alone and conventional pd group , respectively . over the study period ,
two patients were shifted following oliguria : one patient at 3.2 months following coronary bypass surgery and another at 15 months following peritonitis .
remaining 3 patients were shifted at 6.4 , 7.2 , and 9.6 months for inadequate dialysis .
there was only 1 death in ico - alone group while on treatment .
there were 3 more deaths of ico alone group patients afterwards while on conventional pd .
patient survival was 42.84 months ( se 7 ) in ico - alone vs 25.29 months ( se 9.2 ) in conventional dialysis ( p = 0.01 ) [ figure 2 ] .
median patient survival in ico - alone and in conventional pd groups ( 42.84 [ se 7 ] vs 25.29 [ se 9.2 ] months ,
p=0.01 )
incremental dialysis has been studied and reported earlier for patients of end - stage renal failure with residual renal function .
we are the first one to recommend icodextrin single exchange in selected group of patients .
earlier , foggensteiner et al . , initiated such patients on icodextrin single exchange , but had very high rates of sterile peritonitis ( 46% ) and had to switch to glucose - based dialysate .
however , later , review of literature showed several cases of such peritonitis reported from across the world and the possible reason was high dose of peptidoglycan in the pd fluid .
the incidence of sterile peritonitis subsequently dropped significantly with regulation of peptidoglycan concentration in the pd fluid by the manufacturer . in our study , none of our patients had sterile peritonitis during ico - alone exchange . in our study , the median period sustainable on single icodextrin exchange was 9.6 months , which is similar to that in other studies in literature .
foggensteiner et al . , reported a median actuarial survival on single exchange of 297 days and viglino et al .
, initiated pd with 2 exchanges per day and their median survival before incremental dialysis was 9.7 months .
none of the studies in the literature compared the pd outcomes between those on incremental dialysis vs conventional dialysis .
our study for the first time compared outcome and complications on ico - alone to that of conventional pd .
there was only 1 death in ico - alone group and that too was not related to dialysis .
survival in ico - alone group was better than in the conventional group ; this may reflect and reinforce the common understanding that residual renal function is the most important factor determining the outcome of pd patients .
it also confirms the fact that renal clearance and not the total clearance determines the survival outcome in pd patients .
ico - alone patients had lesser peritonitis rates , which could be because of lesser number of connections - disconnections per day . however , exit site infection was higher in them , as 1 patient had recurrent infections .
however , the protocol did offer advantages and possible better quality of life , which needs to be studied and documented in larger number of patients . to conclude , ico - alone single nocturnal exchange per day is a viable option in incremental pd to initiate dialysis in patients of end - stage renal failure with residual renal function without any compromise in outcomes . | we analyzed the outcome of incremental dialysis with single nocturnal icodextrin exchange peritoneal dialysis ( pd ) as the initial treatment for end - stage kidney failure in patients who have significant residual renal function .
all adult patients opting for pd as renal replacement therapy , having residual renal function , and urinary kt / v of 1.0 were offered incremental dialysis with single nocturnal icodextrin exchange as initial treatment .
adequacy of dialysis was calculated at 1 , 3 , and 6 months and then 6 monthly .
patients were shifted to conventional pd if short of adequacy or if clinically indicated .
median period on ico - alone ,
peritonitis , exit site infection rates , and patient survival , while on this protocol , were calculated .
these outcomes were compared with the cohort of contemporary patients on conventional pd .
thirteen patients were initiated on ico - alone dialysis between october 2006 and october 2011 .
the baseline characteristics were similar when compared with cohort of conventional pd patients , except urine volume , which was more in ico - alone group ( 1265 316 vs. 551 504 , p = 0.000 ) . total kt / v at 3 months ( 1.63 0.6 vs. 1.7 0.2 , p = 0.6 ) and at 1 year ( 1.64 0.5 vs. 1.53 0.3 ,
p = 0.6 ) was similar to the cohort of conventional pd patients .
median period on ico - alone was 9.6 months .
peritonitis rate was 1 episode in 56.22 vs 25.29 patient - months and exit site infection was 1 episode in 56.2 vs 189.71 patient - months in ico - alone and conventional group , respectively .
patient survival was 42.84 months in ico - alone vs 25.29 months in conventional dialysis ( p = 0.01 ) . in conclusion ,
single icodextrin exchange offers adequate dialysis in patients with residual renal function ( kt / v = 1 ) for a median period of 9 months . |
1.26 mol of lipids in chloroform ( typical lipid composition : dopc(1,2-dioleoyl - sn - glycero-3-phosphocholine):dopg(1,2-di-(9z - octadecenoyl)-sn - glycero-3-phospho-(1'-rac - glycerol ) ) : mpb ( 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-[4-(p - maleimidophenyl ) butyramide ) = 4:1:5 molar ratio , all lipids from avanti polar lipids , alabaster , al ) were dispensed to glass vials , and the organic solvents were evaporated under vacuum overnight to prepare dried thin lipid films .
the lipid films were rehydrated in 10 mm bis - tris propane at ph 7.0 with cargo proteins for 1 hr with rigorous vortexing every 10 min , and then sonicated in alternating power cycles of 6 watts and 3 watts in 30s intervals for 5 min on ice ( misonix microson xl probe tip sonicator , farmingdale , ny ) .
the liposomes formed in this first step were induced to undergo fusion by addition of divalent cations such as mg and ca at a final concentration of 10 mm .
the resulting mlvs were incubated with 1.5 mm dtt ( maleimide : dtt molar ratio of 2:1 ) for 1 hr at 37c to conjugate opposing bilayers of maleimide - functionalized lipids and form crosslinked icmvs ; the resulting vesicles were recovered by centrifugation at 14,000 g for 4 min , and washed twice with deionized water .
for pegylation , the particles were incubated with 2 kda peg - thiol ( laysan bio , arab , al ) in a 1.5-fold molar excess of peg - sh to maleimide groups for 1 hr at 37c .
the final products were either stored in pbs at 4c or lyophilized in the presence of 3% sucrose as a cryoprotectant and stored at 20c .
for some assays , simple liposomes or mg - fused mlvs were harvested prior to crosslinking with ultracentrifugation at 115 k g using an optima ultracentrifuge for 6 hrs ( beckman coulter ) . for encapsulation studies , ovalbumin ( ova , worthington , lakewood , nj ) , siv - gag
( advanced bioscience laboratories , kensington , md ) , and flt-3l ( peprotech , rocky hill , nj ) were labeled with alexa - fluor 555 ( invitrogen , carlsbad , ca ) for direct fluorometric quantification of the amount of protein entrapped .
ova was also encapsulated in drvs and plga nanoparticles as described previously43,44 . in some experiments ,
icmvs were loaded with a recombinant vivax malaria protein ( vmp ) as an irrelevant antigen control49 ( provided by dr .
capped - thiol ova was prepared by incubating 1 mg of ova with 1.5 mm tcep for 1 hr at rt , followed by incubation with 1.5 mm ethyl - maleimide ( pierce , rockford , il ) at 37 c for 1hr .
release of ova labeled with alexa - fluor 555 from lipid vesicles was quantified in rpmi media supplemented with 10% fetal calf serum at 37c using dialysis membranes with mw cutoff of 100 kda . at regular intervals ,
the releasing media were removed for quantification of fluorescence , and an equal volume of fresh media were replaced for continued monitoring of drug release .
residual ova remaining at the end of the time - course was determined by lipid extraction of vesicles with 1% triton x-100 treatment and measuring released protein by fluorescence spectrophotometry .
ova release assays were also performed in hank s buffered saline solution supplemented with 500 ng / ml of phospholipase a ( sigma , st .
louis , mo ) . to examine stability of encapsulated cargo molecules , monoclonal rat igg encapsulated in icmvs
was retrieved with 1% triton x-100 treatment and analyzed with sds - page under non - reducing conditions with silver staining ( pierce ) .
groups of c57bl/6 mice ( jackson laboratories ) were immunized s.c . in the tail base with indicated doses of ova ( with or without tlr agonists , mpla ) .
frequencies of ova - specific cd8 t - cells and their phenotypes elicited by immunization were determined by flow cytometry analysis of pbmcs at selected time points following staining with dapi ( to discriminate live / dead cells ) , anti - cd8 , anti - cd44 , anti - cd62l , and siinfekl / h-2k peptide - mhc tetramers ( becton dickinson ) . to assess functionality of primed cd8 t - cells , pbmcs were stimulated ex vivo with 1 m ova - peptide siinfekl for 6 hrs with golgiplug ( becton dickinson ) , fixed , permeabilized , stained with anti- ifn- and cd8 , and analyzed by flow cytometry .
anti - ova igg titers , defined as the dilution of sera at which 450 nm od reading is 0.5 , were determined by elisa analysis of sera from immunized mice .
statistical analysis was performed with jmp 5.1 ( sas institute inc , cary , nc ) .
data sets were analyzed using one- or two - way analysis of variance ( anova ) , followed by tukey s hsd test for multiple comparisons .
1.26 mol of lipids in chloroform ( typical lipid composition : dopc(1,2-dioleoyl - sn - glycero-3-phosphocholine):dopg(1,2-di-(9z - octadecenoyl)-sn - glycero-3-phospho-(1'-rac - glycerol ) ) : mpb ( 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-[4-(p - maleimidophenyl ) butyramide ) = 4:1:5 molar ratio , all lipids from avanti polar lipids , alabaster , al ) were dispensed to glass vials , and the organic solvents were evaporated under vacuum overnight to prepare dried thin lipid films .
the lipid films were rehydrated in 10 mm bis - tris propane at ph 7.0 with cargo proteins for 1 hr with rigorous vortexing every 10 min , and then sonicated in alternating power cycles of 6 watts and 3 watts in 30s intervals for 5 min on ice ( misonix microson xl probe tip sonicator , farmingdale , ny ) .
the liposomes formed in this first step were induced to undergo fusion by addition of divalent cations such as mg and ca at a final concentration of 10 mm .
the resulting mlvs were incubated with 1.5 mm dtt ( maleimide : dtt molar ratio of 2:1 ) for 1 hr at 37c to conjugate opposing bilayers of maleimide - functionalized lipids and form crosslinked icmvs ; the resulting vesicles were recovered by centrifugation at 14,000 g for 4 min , and washed twice with deionized water .
for pegylation , the particles were incubated with 2 kda peg - thiol ( laysan bio , arab , al ) in a 1.5-fold molar excess of peg - sh to maleimide groups for 1 hr at 37c .
the final products were either stored in pbs at 4c or lyophilized in the presence of 3% sucrose as a cryoprotectant and stored at 20c .
for some assays , simple liposomes or mg - fused mlvs were harvested prior to crosslinking with ultracentrifugation at 115 k g using an optima ultracentrifuge for 6 hrs ( beckman coulter ) .
for encapsulation studies , ovalbumin ( ova , worthington , lakewood , nj ) , siv - gag ( advanced bioscience laboratories , kensington , md ) , and flt-3l ( peprotech , rocky hill , nj ) were labeled with alexa - fluor 555 ( invitrogen , carlsbad , ca ) for direct fluorometric quantification of the amount of protein entrapped .
ova was also encapsulated in drvs and plga nanoparticles as described previously43,44 . in some experiments ,
icmvs were loaded with a recombinant vivax malaria protein ( vmp ) as an irrelevant antigen control49 ( provided by dr .
capped - thiol ova was prepared by incubating 1 mg of ova with 1.5 mm tcep for 1 hr at rt , followed by incubation with 1.5 mm ethyl - maleimide ( pierce , rockford , il ) at 37 c for 1hr .
release of ova labeled with alexa - fluor 555 from lipid vesicles was quantified in rpmi media supplemented with 10% fetal calf serum at 37c using dialysis membranes with mw cutoff of 100 kda . at regular intervals ,
the releasing media were removed for quantification of fluorescence , and an equal volume of fresh media were replaced for continued monitoring of drug release .
residual ova remaining at the end of the time - course was determined by lipid extraction of vesicles with 1% triton x-100 treatment and measuring released protein by fluorescence spectrophotometry .
ova release assays were also performed in hank s buffered saline solution supplemented with 500 ng / ml of phospholipase a ( sigma , st .
louis , mo ) . to examine stability of encapsulated cargo molecules , monoclonal rat igg encapsulated in icmvs was retrieved with 1% triton x-100 treatment and analyzed with sds - page under non - reducing conditions with silver staining ( pierce ) .
groups of c57bl/6 mice ( jackson laboratories ) were immunized s.c . in the tail base with indicated doses of ova ( with or without tlr agonists , mpla ) .
frequencies of ova - specific cd8 t - cells and their phenotypes elicited by immunization were determined by flow cytometry analysis of pbmcs at selected time points following staining with dapi ( to discriminate live / dead cells ) , anti - cd8 , anti - cd44 , anti - cd62l , and siinfekl / h-2k peptide - mhc tetramers ( becton dickinson ) . to assess functionality of primed cd8 t - cells , pbmcs were stimulated ex vivo with 1 m ova - peptide siinfekl for 6 hrs with golgiplug ( becton dickinson ) , fixed , permeabilized , stained with anti- ifn- and cd8 , and analyzed by flow cytometry .
anti - ova igg titers , defined as the dilution of sera at which 450 nm od reading is 0.5 , were determined by elisa analysis of sera from immunized mice .
statistical analysis was performed with jmp 5.1 ( sas institute inc , cary , nc ) .
data sets were analyzed using one- or two - way analysis of variance ( anova ) , followed by tukey s hsd test for multiple comparisons .
| vaccines based on recombinant proteins avoid toxicity and anti - vector immunity associated with live vaccine ( e.g. , viral ) vectors , but their immunogenicity is poor , particularly for cd8 + t - cell ( cd8 t ) responses .
synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines , but in general these materials have failed to elicit cd8 t responses comparable to live vectors in preclinical animal models . here , we describe interbilayer - crosslinked multilamellar vesicles ( icmvs ) formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles .
icmvs stably entrapped protein antigens in the vesicle core and lipid - based immunostimulatory molecules in the vesicle walls under extracellular conditions , but exhibited rapid release in the presence of endolysosomal lipases .
we found that these antigen / adjuvant - carrying icmvs form an extremely potent whole - protein vaccine , eliciting endogenous t - cell and antibody responses comparable to the strongest vaccine vectors .
these materials should enable a range of subunit vaccines and provide new possibilities for protein therapeutic delivery . |
an 8-year - old girl presented with a progressively enlarging mass in the right conjunctival fornix composed of normal appearing eyelashes . the patient had a history of aberrant conjunctival eyelash growth that had caused recurrent conjunctivitis in her right eye over the past few years .
the mass was surgically removed and the pathology report revealed it to be a conjunctival dermoid .
mature hair follicle growth from the conjunctiva is another possible presentation of a conjunctival dermoid that can be cured by simple surgical excision .
dermoids are the most common orbital tumor of childhood.1 this mass can have numerous ocular presentations and can occur in a variety of locations ; the classic example is lesions occurring on the superotemporal orbital wall .
dermoids have also been reported on the inferotemporal cornea , the caruncle and the conjunctiva.2,3 while these heterogeneous presentations are classified under the general heading of dermoid , depending on the location , each can have a different underlying histology .
proper identification of dermoid type can be important both for preoperative surgical planning and postoperative prognosis .
an 8-year - old hispanic girl was referred to our institution for a growth in her right conjunctival fornix .
her mother had noticed the mass over the past few months and felt that it was enlarging .
our patient did not have any other medical conditions and denied undergoing prior trauma or surgery .
her only ocular history involved multiple , recurrent episodes of redness in the right eye that were diagnosed and treated as either bacterial or viral conjunctivitis by the referring physician .
the mother also reported that she had discovered pieces of hair in her daughter s right eye tear film over the past couple of years . on presentation , the patient s eye looked relatively normal , and she had uncorrected visual acuity of 20/20 in both eyes , with an otherwise unremarkable gross examination
. slit lamp examination of her right eye revealed a mildly injected palpebral conjunctiva with a moderate - sized , fibrous , tan - colored mass that had arborizing , centrally coursing blood vessels . upon manipulation of the mass ,
a large tuft of normal appearing eyelashes emanated from the inferior conjunctival fornix ( figure 1 ) .
the mass was easily mobile , firm and non - fluctuant , so a decision was made to surgically remove the mass .
the patient was taken to the operating room , and the base of the mass was carefully resected with sharp westcott scissors . the tumor had a wide based pedicle and was easily removed .
the remaining conjunctival edges were easily reapproximated without tension , so no sutures were placed .
on post - operative day one , the patient was comfortable and the conjunctival fornix was completely healed .
she returned for her post - operative month one visit and had a pristine inferior conjunctival fornix , without any evidence of tumor recurrence .
the pathology specimen revealed nonkeratinized stratified squamous epithelium with multiple goblet cells and an underlying chronic mononuclear inflammatory cell reaction .
the bulk of the lesion was made of relatively loose , irregular connective tissue with multiple small hair shafts in longitudinal and transverse cross - section and a small amount of adipose tissue .
the pathology report was consistent with dermoid choristoma of the conjunctiva ( figure 2 ) .
the differential diagnosis for a pediatric conjunctival tumor includes nevi ( 64% ) , choristomas ( 10% ) , vascular tumors ( 7% ) , juvenile xanthogranulomas ( jxg ) and lymphoid masses.4 the presence of normal conjunctival epithelium , hair shafts , and adipose tissue in our patient was consistent with a choristoma .
the clinical picture of the tan , fibrous mass with central vessels could have been mistaken for a pyogenic granuloma , but the patient had no history of surgery or trauma , and histopathology did not report any granulation tissue .
our patient s dermoid was composed of sequestered conjunctival tissue and not typical epidermal tissue . a literature search for dermoid choristoma , conjunctival dermoid , and
the main distinction between these two dermoid types depends on the presence or absence of two elements : keratin and goblet cells .
conjunctival dermoids have goblet cells interspersed amongst non - keratinized stratified squamous cells . in contrast ,
epidermal dermoids are composed of keratin producing stratified squamous cells without any goblet cells . while initially , this seems like a rather small distinction , there are a few salient features worth noting .
conjunctival dermoids are rare tumors with martinez et al finding only 14 cases in the literature in 1998.5 we found no other cases in the literature since then and this case is the first described one presenting with externalized hair follicles in the conjunctival fornix . the largest case series published in the literature involved 7 cases over a 50-year period .
jekobiec et al reviewed all dermoid cysts at their institution from 1929 to 1977 and reanalyzed the corresponding pathology slides to confirm or refute the original diagnosis.1 twelve of the 128 cases were given a new diagnosis because they had a non - keratinized cyst lining with conjunctival elements .
of these 12 , five were determined as cysts and the other seven were classified as dermoids ; the distinction between the two depended on the presence or absence of adnexal structures .
these seven conjunctival dermoids were noted to be unique in that they presented in a different location ( superonasal vs. superotemporal ) compared to epidermal dermoids , and did not cause adjacent bony defects on radiography .
the authors also mentioned how easily these tumors were removed because they had no surrounding attachments .
similarly , our patient s surgery was relatively simple and she enjoyed an excellent result .
orbital and corneal limbal dermoids are made of keratin producing epithelial cells , with the former causing bony erosion , and both are much more difficult to remove as compared to our experience and that of jekobiec et al.6 understanding the distinction between the two types of dermoids can be important for both pre - operative surgical planning and patient counseling . in conclusion ,
mature hair follicle growth from the conjunctiva is another possible presentation of a conjunctival dermoid that can easily be cured by simple excision . | purposeto describe a previously unreported presentation of a conjunctival dermoid.case reportan 8-year - old girl presented with a progressively enlarging mass in the right conjunctival fornix composed of normal appearing eyelashes .
the patient had a history of aberrant conjunctival eyelash growth that had caused recurrent conjunctivitis in her right eye over the past few years .
the mass was surgically removed and the pathology report revealed it to be a conjunctival dermoid .
the patient had an excellent surgical result with normal cosmetic appearance.conclusionmature hair follicle growth from the conjunctiva is another possible presentation of a conjunctival dermoid that can be cured by simple surgical excision . |
overproduced free radicals or a weakened natural antioxidant system can often lead to the oxidative stress that may eventually result in oxidative injury and diseases .
modern medical studies have shown that a variety of free radicals caused by oxidation in various tissues is one of the important factors of organic damage and pathological changes .
some serious diseases such as cardiovascular disease , diabetes , acquired immune deficiency syndrome , neurodegenerative diseases , inflammation , cancer , and aging process are related to oxidative damage caused by free radicals .
it has been widely recognized nowadays that lowering oxidative stress can provide clinical benefits to a variety of pathological conditions , and antioxidant treatment as research focus has thus been regarded as a viable therapy to alleviate the oxidative injury in these disorders . under physiological conditions ,
a number of free radical scavengers in human body can effectively scavenge free radicals , which keeps the organisms healthy under the normal concentrations of free radicals .
natural non - enzymatic scavengers contain vitamin c , vitamin e , tea polyphenols , and phenolic acid compounds , which exist widely in natural chinese herbal medicines and foods .
et franch . ) ling [ figure 1 ] , an herb of genus pyrethrum in composite family , is mainly distributed in qinghai , sichuan , yunnan and tibet eastern part in china .
p. tatsienense spends cold in nature and bitter taste , which has the functions of clearing away the heat - evil , expelling superficial evils , dispelling wind , eliminating dampness , anti - inflammatory and analgesia .
thus p. tatsienense has been widely used in folk and clinic in tibet for the notable therapeutic effects of treating hepatitis , headaches , head injuries , ulcers , wounds , etc . .
the early phytochemical studies by our group showed that flavonoids mainly revealing luteolin as mother nucleus were present in considerable amounts in p. tatsienense , and most of flavonoids exist in the forms of glucoside .
as we all know , therapeutic properties of flavonoids are diverse as antioxidant [ 6 - 9 ] , anti - inflammatory , anti - neoplastic , hepatoprotective , neuroprotective activities .
recently , ethanol extracts of p. tatsienense were found to have the hepatoprotective activity in d - galactosamine liver injury models , which reveals that flavonoids in p. tatsienense may be the active ingredients of hepatoprotective combined with the outcomes of phytochemical studies .
the important clinical impacts of oxidative stress on liver disease mainly include changes in gene expression , inflammation trigger , liver fibrosis , cancer , and apoptosis .
reactive oxygen free radicals as the starting and regulating factors of liver cell damage may cause necrosis or apoptosis through direct or immune mechanism and hence the antioxidant activity is of great significance to protect liver cell damage .
ling antioxidant activities of the 11 flavonoids isolated from p. tatsienense [ table 1 ] , named as tricin ( 1 ) , 4-methoxy - tricin ( 2 ) , apigenin ( 3 ) , luteolin ( 4 ) , luteolin-7-o--d - glucoside ( 5 ) , apiolin-7-o--d - glucoside ( 6 ) , apiolin-7-o--d - glucuronic acid methyl ester ( 7 ) , quercetin-7-o--d - glucoside ( 8) , 6-hydroxy - luteolin-7-o - b - d - glucoside ( 9 ) , luteolin-7-o--d - glucoside aid ( 10 ) , and apigenin-7-o--d - glucoside acid ( 11 ) , were studied in some detail by the classic antioxidant method of 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay in vitro in this experiment , and meaningful conclusions are drawn from the point of structure - activity relationships .
et franch . ) ling were collected in tibet province in china , and authenticated by prof .
chen - chen zhu ( institute of clinical pharmacology , guangzhou university of chinese medicine ) .
a voucher specimen ( pt-201102 ) was deposited in herbarium of institute of clinical pharmacology , guangzhou university of chinese medicine .
dpph radical and a - tocopherol were obtained from sigma chemical company ( usa ) .
the 11 flavonoids were isolated from ethanol extracts of p. tatsienense by the measures of normal phase , reversed phase , sephadex lh20 and molecular sieve column chromatograph .
their purities were over 98 % by normalization of the peak areas detected by high - performance liquid chromatography - ultra violet ( uv ) ( elite , china ) .
absorbance measurements were performed with a uv1000 uv - vis spectrophoto meter ( techcomp , china ) .
a total of 8.3 mg dpphwas resolved in 200 ml of methanol to obtain 0.043 mg / ml stock solution .
the 11 flavonoids and a - tocopherol were resolved in methanol to obtain 1.0 mg / ml stock solution , and then diluted by methanol to get 0.1 , 0.3 , 0.5 , 0.8 , and 1.0 mg / ml solutions , respectively .
each mixture contained 1.95 ml of dpph solution and 50 l of sample solutions , which reacted at room temperature for 20 min .
the absorbance value of methanol as blank control is a0 , and the absorbance value of reaction solution is a. lower absorbance of the reaction mixture indicates higher radical scavenging activity .
a - tocopherol was taken as positive control and the percentage dpph inhibition of the test samples was calculated as : inhibition % = ( 1 a / a0) 100 % where a is the absorbance of the tested sample or positive control , a0 is the absorbance of the reaction mixture without sample .
the measurements were performed in triplicate and the data were recorded as mean standard deviation . the ic50 value was defined as the final concentration of 50% free radical inhibition and was calculated by linear regression analysis .
statistical calculations were carried out using the spss ( spss inc . , chicago , il , usa ) .
et franch . ) ling were collected in tibet province in china , and authenticated by prof .
chen - chen zhu ( institute of clinical pharmacology , guangzhou university of chinese medicine ) .
a voucher specimen ( pt-201102 ) was deposited in herbarium of institute of clinical pharmacology , guangzhou university of chinese medicine .
dpph radical and a - tocopherol were obtained from sigma chemical company ( usa ) .
the 11 flavonoids were isolated from ethanol extracts of p. tatsienense by the measures of normal phase , reversed phase , sephadex lh20 and molecular sieve column chromatograph .
their purities were over 98 % by normalization of the peak areas detected by high - performance liquid chromatography - ultra violet ( uv ) ( elite , china ) .
absorbance measurements were performed with a uv1000 uv - vis spectrophoto meter ( techcomp , china ) .
a total of 8.3 mg dpphwas resolved in 200 ml of methanol to obtain 0.043 mg / ml stock solution .
the 11 flavonoids and a - tocopherol were resolved in methanol to obtain 1.0 mg / ml stock solution , and then diluted by methanol to get 0.1 , 0.3 , 0.5 , 0.8 , and 1.0 mg / ml solutions , respectively .
each mixture contained 1.95 ml of dpph solution and 50 l of sample solutions , which reacted at room temperature for 20 min .
the absorbance value of methanol as blank control is a0 , and the absorbance value of reaction solution is a. lower absorbance of the reaction mixture indicates higher radical scavenging activity .
a - tocopherol was taken as positive control and the percentage dpph inhibition of the test samples was calculated as : inhibition % = ( 1 a / a0) 100 % where a is the absorbance of the tested sample or positive control , a0 is the absorbance of the reaction mixture without sample .
the measurements were performed in triplicate and the data were recorded as mean standard deviation .
the ic50 value was defined as the final concentration of 50% free radical inhibition and was calculated by linear regression analysis .
all values are shown with confidence interval and the p < 0.05 . statistical calculations were carried out using the spss ( spss inc . , chicago , il , usa ) .
dpph assay is the classics method to evaluate free radical scavenging activity of natural products .
dpph are the stable purple free radicals with maximum absorption wavelength at 517 nm , which can accept an electron or hydrogen radical to become a stable diamagnetic molecule .
the percentages of scavenging activity of 11 flavonoids at different concentrations were analyzed by taking a - tocopherol as the positive drug .
the percentages of scavenging activity were plotted against the different sample concentration to obtain the ic50 values .
the smaller the clearance rate ic50 value is , the stronger the antioxidant activity of flavonoid is .
experimental results showed that dpph free radical scavenging ability of flavonoids increased with the adding concentration of sample , which indicated that flavonoids could effectively scavenge free radicals with certain dose - effect relationships .
as shown in table 1 , comparing the ic50 values of flavonoid 1 with 3 , antioxidant activity of the former was obviously larger than the latter although the numbers and substitutional positions of phenolic hydroxyls of these two compounds are the same .
it is speculated that the ortho - positions of 4 hydroxyl are substituted by the two methoxyls as electron donors , which are favorable for the increase of conjugative effect of semi - quinonoid skeletons after hydrogen abstraction . in other word ,
methoxyl groups remaining at the ortho - positions of phenolic hydroxyl in aromatic ring play a synergistic effect on the antioxidant activity without considering its steric effect
. the ic50 value of flavonoid 2 could not be detected , which showed this compound had no dpph scavenging activity owing to not containing phenolic hydroxyls in its structure . when c-7 position of flavonoid 4 is replaced by glucose , flavonoid 5 is obtained .
the ic50 value of the latter was larger than that of the former , which indicated that antioxidant activity of flavonoid 5 decreased due to the reduced phenolic hydroxyls after glycosidation .
although glucose ring has several hydroxyls , it does not have the ability to delocalize electrons owing to not having conjugated system in its structure .
thus hydroxyls of glucose ring can not be viewed as active sites to scavenge dpph free radicals .
the antioxidant activity will decrease after phenolic hydroxyls glycosidation , the absorbance and distribution of flavonoids in vivo will change because of the increase of volume and polarity of the compounds .
the same antioxidant effect will be obtained if flavonoid glycosides can form aglycone under the action of hydrolases in vivo . on the other hand , free radicals clearance rate ic50 value of flavonoid 4 , 5 , 8 , 9 , and 10 are comparable with a - tocopherol , and the antioxidant activity of these five flavonoids are significantly stronger than the other six flavonoids .
the reasons should be that the positions of c-3 , 4 in b ring of these five flavonoids were all replaced by two hydroxyls , which lead to the significant increase of antioxidant activity .
in other word , the catechol hydroxyls at c-3 , 4 positions in flavonoids are the most important active sites .
it is presumably that intramolecular hydrogen bond is formed by semi - quinonoid free radical with 4 hydroxyl after the first hydrogen abstraction , and further quinone is formed after the second hydrogen abstraction occurring at 4 hydroxyl [ figure 2 ] .
thus the o - dihydroxyls at c-3 , 4 positions in flavonoids are more advantageous to the high delocalization effect of electrons .
although flavonoid 10 has two sets of o - dihydroxyls at c-3,4 positions in b ring and at c-5,6 positions in a ring respectively , the ic50 value of flavonoid 10 was a little lower than that of flavonoid 9 .
this result indicated that the catechol hydroxyls at c-5 , 6 positions in a ring of flavonoid had an insignificant effect on the antioxidant activity .
the reason probably is that the intramolecule hydrogen bond are formed by the hydroxyl at c-5 position in a ring with the carbonyl oxygen at c-4 position in c ring , which decrease the ability of hydrogen abstraction of o - dihydroxyls at c-5,6 positions in flavonoid 9 .
in addition , antioxidant activity of phenolic hydroxyls in a ring of flavonoid decreased significantly because of electrophilic effect of c ring .
the possible scavenging 1,1-diphenyl-2-picrylhydrazyl mechanism occurring at c-3 , 4 positions of fl avonoids 4 , 5 , 8 , 9 , and 10
the degree of antioxidant activity depends mainly on the ability of compounds providing protons to neutralize free radicals at the initial stage of oxidation or terminating the radical chain reaction .
the main process of flavonoids scavenging free radicals is that phenolic hydroxyls of flavonoids can react with free radicals to terminate chain reaction .
the ability of flavonoids scavenging free radicals depends mainly on the ease or complexity of the breakdown of phenolic hydroxyls and the stability of semi - quinonoid free radicals after hydrogen abstraction reaction .
the bond dissociation energy of phenolic hydroxyl in the aromatic ring is a key mark to measure the strength of phenolic hydroxyl .
the relationships between the structures of flavonoid and antioxidant activity are as follows : ( 1 ) the increase of phenolic hydroxyls among flavonoids is favorable for the antioxidant activity , ( 2 ) antioxidant activity will increase remarkably when the c-3 , 4 positions in b ring of flavonoids are replaced by hydroxyls , ( 3 ) the ortho position of phenolic hydroxyl containing methoxyl ( electron donor group ) will be favorable for the improvement of antioxidant capacity , ( 4 ) antioxidant activity decreases when flavonoids are glycosylated , which reflects that antioxidant capacity mainly depends on the number of phenolic hydroxyls and nearly has nothing to do with the hydroxyl in sugar ring , ( 5 ) the substitutional positions of phenolic hydroxyls have more effects on antioxidant activity than the numbers of phenolic hydroxyls have .
this result is agreement with theoretical calculation results which combined phenolic bond dissociation enthalpy with radical scavenging ability .
the above studies indicate that flavonoids mainly revealing luteolin as mother nucleus isolated from p. tatsienense can effectively eliminate free radicals , which provide valuable clues for further clarifying the mechanism of preventing or treating hepatic injury induced by free radicals , especially for deeply analyzing the pharmacological functions of p. tatsienense in vivo from the point of antioxidation . | aim : antioxidant activity is one of the important indexes for estimating medicinal value for the traditional chinese medicine . the aim of this study is to investigate the antioxidant activity of 11 flavonoids mainly revealing luteolin as mother nucleus isolated from pyrethrum tatsienense.materials and methods : the antioxidant activity of 11 flavonoids was measured in vitro using the classical 1,1-diphenyl-2-picrylhydrazyl removal method .
the percentages of scavenging activity of 11 flavonoids were analyzed by taking the choice of a - tocopherol as positive drugs , and the scavenging activity was plotted against the sample concentration to obtain the ic50 values.results:ten flavonoids containing phenolic hydroxyl groups have different levels of antioxidant activity .
antioxidant activity mainly depends on the numbers and the substitutional positions of phenolic hydroxyls in b ring .
when c-3 ' , 4 ' positions in b ring of flavonoids are replaced by hydroxyl groups , the antioxidant activity improved remarkably .
phenolic hydroxyl groups in a ring contribute some to antioxidant activity because of the electrophilic effect of c ring , and the numbers and substitutional positions of methoxyl and glycosyl have a little effect on the antioxidant activity.conclusion:structure-activity relationships of antioxidant activity about flavonoids isolated from p. tatsienense are concluded , which will be beneficial to deep understanding the pharmacological functions of this tibetan medicine in vivo from the point of antioxidation . |
in the previous issue of critical care , rodriguez - nunez and coworkers report their experience with terlipressin in 16 children with refractory septic shock . over the past few years
there has been much interest in the use of terlipressin in such settings , both in adults [ 2 - 6 ] and children [ 7 - 10 ] .
the objectives of treatment are twofold : to maintain oxygen delivery above a critical threshold and to increase mean arterial pressure ( map ) to a level that allows distribution of cardiac index ( ci ) sufficient for adequate organ perfusion . among the catecholamines ,
noradrenaline ( norepinephrine ) and dopamine are often favoured . however , vascular responsiveness to catecholamines diminishes over time , and patients may die in states of intractable shock .
the vascular hyporeactivity to catecholamines is caused , among other mechanisms , by excessive nitric oxide formation associated with an activation of atp - sensitive potassium channels and reduction in calcium entry through voltage - gated calcium channels .
this action is not impaired during sepsis , and vasopressin has been shown to be effective in reversing catecholamine - resistant hypotension in patients with septic shock .
vasopressin is not available in all countries , and some hospital pharmacies dispense lysine vasopressin , or terlipressin ( glypressine ; ferring company , berlin , germany ) , which is the form of vasopressin that is present in pig .
the first clinical trial evaluating the efficacy of terlipressin in septic shock was performed in a small case series of eight patients .
terlipressin was administered as a single bolus of 1 mg ( the dosage used in gastroenterological indications ) in patients with septic shock refractory to catecholamine / hydrocortisone / methylene blue .
a significant improvement in blood pressure was achieved in these patients during the first 5 hours .
cardiac output was reduced , which might have impaired oxygen delivery ; no other adverse effect was observed .
another study was conducted in 15 patients with catecholamine - dependant septic shock ( noradrenaline 0.6 g / kg per min ) .
an intravenous bolus of 1 mg terlipressin was followed by an increase in map and a significant decrease in ci
gastric mucusal perfusion was evaluated by laser doppler flowmetry and was increased after administration of terlipressin . in the latter study , rather low doses of noradrenaline were used ( 0.75 g / kg per min at baseline ) and the study patients could not really be considered ' catecholamine resistant ' .
terlipressin was used in patients with intractable hypotension despite use of > 2.0 g / kg per min noradrenaline and 25 g / kg per min dopamine . in these ' catecholamine - resistant ' patients ,
terlipressin ( 1 or 2 mg intravenously ) was able to reverse the intractable hypotension , with a concomitant decrease in heart rate and ci . in this study oxygen delivery and consumption
the terlipressin - induced fall in oxygen delivery and consumption emphasizes the need to monitor ci closely when this drug is used in patients with sepsis .
the additional use of a potent positive inotropic drug such as dobutamine is of interest . despite a decrease in oxygen delivery and consumption
such a dependence on oxygen supply is usually associated with some degree of tissue ischaemia and a subsequent increase in lactate concentration .
we speculated that terlipressin could have modulated the hyperdynamic metabolic response during endotoxaemia and exerted anti - inflammatory effects , thereby decreasing the oxygen needs of tissues .
four studies were published prior to the start of 2006 [ 7 - 10 ] .
like the ones conducted in adults , these studies have serious limitations , including administration of the drug in desperate cases and evaluation of small numbers of patients .
one serious concern is raised by the high incidence of ischaemia during terlipressin administration . in nine patients without signs of ischaemia ,
interestingly , in seven other patients with signs of ischaemia before use of terlipressin , signs of ischaemia improved in four of them .
such a heterogeneous response is intriguing and emphasizes the needed ( at least in adults ) for close monitoring of ci and systemic vascular resistance .
another important consideration with use of terlipressin is its effects on regional haemodynamics and organ function . at present
renal function and gastric mucosal perfusion are improved [ 4 - 6 ] , but no control groups were evaluated in two of these studies . therefore , further studies are need to determine the safety of terlipressin when used in patients with septic shock . in conclusion ,
use of terlipressin may be considered in patients with ( truly ) refractory septic shock despite adequate fluid resuscitation and high - dose conventional vasopressors .
if terlipressin is a last resort therapy , then the advantages ( increased map , and improved renal function and perfusion of gastric mucosa ) should be weighed against unresolved issues , namely effects on other organs and risk for severe and irreversible ischaemia , not to mention the ( unknown ) effects on the microcirculation .
| use of terlipressin , an analogue of vasopressin , can be considered in septic shock patients with intractable hypotension and high cardiac output in whom fluid resuscitation and high - dose conventional catecholamines have failed .
the effects of this agent on organ function are poorly evaluated in humans .
the limited number of patients evaluated precludes any analysis of adverse outcomes and prognosis . |
infection is acquired either by contact with infected animals or consumption of contaminated milk or dairy products .
the disease has major economic impact not only due to time lost by patients from normal daily activities but also due to the loss in animal husbandry [ 1 , 2 ] .
indeed , brucellosis is characterised by abortion and loss of fertility in farm animals [ 24 ] . in humans ,
patients may present with an intermittent fever , joint pain ( including arthritis ) , neurologic manifestations , and so forth .
this could be due to the absence of erythritol ( a 4-carbon sugar alcohol which is the preferred carbon source for brucella ) in human placentas as opposed to ruminant placentas [ 7 , 8 ] .
however , a recent study has demonstrated that brucella replicates in several human trophoblast subpopulations and can interfere with the invasive capacity of extravillous trophoblast - like cells in vitro .
studies investigating an association between human brucellosis and abortion in vivo are scarce . in rwanda ,
the incidence of human brucellosis is not documented despite a number of reports on cattle brucellosis . because of the potential risk of brucella - associated abortion in infected women , the aim of this study was to establish the seroprevalence of brucellosis in women presenting with abortion / stillbirth .
this study also aims to document the risk factors for human brucellosis and the potential reservoir of pathogens .
the study was conducted in huye district , located in the southern province of rwanda .
blood samples were collected from women presenting with abortion / stillbirth of unknown cause at the district hospital ( kabutare ) and the referral hospital ( butare university teaching hospital ) .
permission to conduct the study was obtained from the medical director of each hospital as well as the head of department of the gynaecology and obstetrics .
institutional approval was obtained from the national university of rwanda ( currently part of the university of rwanda ) .
all the prospective study participants had the aims and procedures explained to them and only those who consented were enrolled into the study .
blood samples were collected into silicon - coated tubes without anticoagulant for isolation of sera .
the blood was stored at 4c overnight and centrifuged the following day and the serum was extracted .
briefly , a drop of the rose bengal coloured brucella antigen ( transak , belgium ) was placed near a drop of serum on a clean slide placed on a white paper .
if an agglutination reaction occurred , the sample was recorded as a positive case ( and negative otherwise ) .
the samples were collected from june to october 2006 and were processed all along . a total of 60 samples were collected and processed .
a questionnaire was used to record demographic data ( age , residency ) , clinical presentation ( abortion , stillbirth ) , profession , and milk consumption habits . in order to determine the local prevalence of cattle brucellosis ,
the first was the kabutare farm , located within a veterinary school near the district hospital ( kabutare hospital ) .
the second was isar - songa farm , part of a veterinary research institution in the southern province of rwanda ( isar - songa is currently part of the rwanda agricultural board ) .
randomly selected cows ( from the local population ) presenting at the kabutare farm 's abattoir were also tested for brucellosis during the study period .
blood samples ( 27 in total ) were collected from the jugular vein and serum was extracted and processed as described in the previous paragraph ( rbpt ) .
a total of sixty women were tested for brucellosis and 15 ( 25% ) were positive as shown in table 1 .
eleven ( 11 ) of them ( 73.3% ) had aborted while 4 ( 26.7% ) presented with a stillbirth .
the majority ( 76.7% ) of tested women were between 18 and 33 years old .
table 2 shows that 13 out of the 15 seropositive women ( 86.7% ) consumed cow 's milk , with 7 of them consuming it raw .
six of the 15 women ( 40% ) were in regular contact with farm animals by profession ( livestock - farming ) .
records from the kabutare veterinary school 's farm showed that the prevalence of brucellosis in cattle declined ( 9.6% to 4.6% ) from the year 2002 to 2006 ( see table 3 ) .
these records were of tests done on cattle from the local population coming for insemination ( natural or artificial ) .
twenty - seven ( 27 ) cows randomly selected from the kabutare farm 's abattoir were tested during the study period , and 2 cases ( 7.4% ) were positive .
records from isar - songa farm revealed that a single test was done on their livestock in 2004 and 50 cows out of 603 ( 8.3% ) tested positive . according to the staff in charge of the livestock
the current study , conducted in rwanda , revealed a high prevalence ( 25% ) of human brucellosis among women presenting with either abortion or stillbirth . to our knowledge , this is the first study to document the presence of human brucellosis in rwanda .
moreover , the study has shown that some cases of abortion / stillbirth may be due to this pathology in humans .
a recent study has demonstrated that pathogenic brucellae are able not only to proliferate in human trophoblasts but also to interfere with the invasive capacity of extravillous trophoblasts - like cells in vitro .
findings from the current study , together with findings by salcedo and colleagues , give insights into the otherwise controversial brucella - associated abortion in humans .
the next step will be to isolate brucella spp . from placental trophoblasts of aborting women .
results from the current study indicate that consumption of raw ( cow 's ) milk , followed by contact with farm animals , is the main risk factor associated with brucella seropositivity .
this is consistent with findings from a study conducted in iran , where 79% of infected patients contracted the disease via consumption of unpasteurized dairy products while 21% had a history of animal contact .
however , a lower prevalence of brucellosis in cattle ( 7.4% ) , with a declining trend in the local main farms , was observed in the current study .
these findings suggest that milk from a single contaminated cow is consumed by more than one family .
moreover , while the tested cows were randomly selected , our study population was selected based on a suspicious symptomatology . the declining trend in cattle brucellosis from the consulted farms
may be the result of control efforts in these institutions following documentations on cattle brucellosis .
there was no direct association between infected cattle and affected women in the current study as it was impossible to trace these cows specifically .
however , the incidence in local farms is an indication on the local reservoir given the herds ' movements and the trading of milk and dairy products . in conclusion
, this study has shown that human brucellosis exists in rwanda and that some cases of abortion / stillbirth may be associated with this pathology .
consumption of raw milk is the principal risk factor for contamination , followed by contact with farm animals .
education of the population regarding milk consumption habits could therefore extensively reduce the prevalence of infection .
this study provides a basis for a larger study to document the national prevalence of human brucellosis ( for control purposes ) and calls for more mechanistic studies to link brucellosis and reproductive problems in humans . | the incidence of human brucellosis is not documented in rwanda despite several reports on the disease in cattle . because brucellosis has been associated with abortion , the aim of this study was to investigate the prevalence of positive serology in women presenting with abortion and/or stillbirth .
the study was done in huye district , in the southern province of rwanda , and the patients were recruited from both the university teaching hospital of butare ( chub ) and kabutare district hospital .
serum samples were collected and the rose bengal plate test ( rbpt ) was performed on each sample .
a questionnaire was also used to investigate potential contacts with animals and/or consumption of raw milk .
a total of 60 women were recruited and 15 ( i.e. , 25% ) were brucella seropositive .
the questionnaire showed that those with seropositivity either were in contact with domestic animals ( cattle , goat , or sheep ) or were consuming raw cow 's milk .
human brucellosis appears to be of public health importance in rwanda and more attention should be drawn on the disease .
the current study provides a basis for larger studies to establish the incidence of human brucellosis in rwanda .
more mechanistic studies will also demonstrate the pathogenicity of brucella in human placentas . |
the neuroimaging intermediate phenotype is conceptually analogous to an intermediate phenotype for common complex medical disorders .
it is logical to assume that genes would show stronger associations with the biological substrates contributing to risk of a disorder , with measurable quantitative traits along a pathophysiologic causal pathway , intermediate to the end complex syndrome .
intermediate phenotypes in other realms of medicine include lipid level as an intermediate phenotype for heart disease , sodium homeostasis as an intermediate phenotype for hypertension , and body mass index as an intermediate phenotype for diabetes .
we favor the term intermediate phenotype over the more popular term endophenotype , though the two terms are essentially interchangeable .
the term endophenotype ( which was introduced into psychiatric genetics in the 1970s ) initially referred to a trait that is internal that may be discoverable by a
biochemical test or microscopic examination , but is not external or overtlymanifest . also , the term endophenotype does not emphasize the concept of intermediacy in pathogenicity .
criteria for the establishment of a neuroimaging - based intermediate phenotype for schizophrenia , as in other fields of medicine , are that the intermediate phenotype : ( i ) is heritable ; ( ii ) is found with increased frequency in healthy relatives of ill probands ; ( iii ) exists temporally before the onset of the clinical illness in the pathophysiological pathway to the emergence of the clinical syndrome .
as expounded in a review by tan et al , evidence for each of these criteria has accumulated for the syndrome of schizophrenia , with cognitive dysfunction often integral and assayable at the brain level by task - based neuroimaging intermediate phenotypes .
each of these criteria are consistent with the assumption that the intermediate phenotype is genetically and biologically less complex than the clinical syndrome , and that genes showing association at the syndromal level will show greater effect sizes ( penetrance ) on variation in intermediate phenotypes . according to the neuroimaging genetics paradigm , to simply demonstrate that a susceptibility gene for schizophrenia impacts brain function is a necessary but not sufficient biological proof of a mechanism of susceptibility .
this is because many , if not most , genes expressed in the brain , are apt to have a brain effect of some sort .
a sine qua non of this proof is to show that the physiological intermediate phenotype associated with a susceptibilitygene for schizophrenia is itself linked to illness risk . to make this link
, it is necessary to demonstrate that the physiological intermediate phenotype is a characteristic of individuals who are at increased genetic risk but do not manifest the clinical syndrome .
the ideal samples in which to demonstrate this are unaffected relatives , eg , cotwins , siblings . this has been done for a number of brain - associated intermediate phenotypes related to increased risk for schizophrenia , including cognitive dysfunctions and neuroimaging phenotypes .
thus , the study of healthy relatives as a target population is critical for establishing the link between genetic association with clinical risk , and genetic association with biological risk .
having identified a neuroimaging phenotype related to increased genetic risk for illness , investigators can ask the question of whether genetic variation in a gene of interest maps onto the specific phenotype , as an indication of its putative neural mechanism of risk .
neuroimaging studies of only affected subjects is confounded by illness - associated epiphenomena that are difficult to control , including smoking history , medical comorbidities , chronic illness burden , or prolonged neuroleptic exposure .
this makes results in patient samples difficult to interpret , as the associations may reflect an interaction of the gene with any of these epiphenomena . instead , the imaging genetics paradigm to test a specific gene - association hypothesis , le , the association of variation in a putative susceptibility gene and brain function linked to increased genetic risk , is best performed in healthy subjects .
healthy individuals possess common at - risk genotypes , but are not themselves symptomatic or clinically ill , thereby reducing the effect of confounding variables .
this approach isolates the simple biologic effect of the genetic variation on brain function(figure 1 ) .
in contrast to functional neuroimaging , structural imaging has not yielded robust intermediate phenotypes for schizophrenia , although a myriad of structural mri studies have revealed whole brain and region - specific volume and other differences between patients with schizophrenia and controls . as discussed elsewhere ,
variation in structural mri measurements mav be attributable to artifacts other than the disease itself , physiological alterations in brain tissue ( eg , tissue perfusion , body fat , or water content ) , or differences in image acquisition and analysis techniques .
alterations in body weight , alcohol intake , steroid administration and hormonal status , can also change brain volume . of critical importance in evaluating patients with chronic schizophrenia , medication can alter brain volume , sometimes rapidly ; lithium carbonate reportedlyincreased cortical gray matter volume by 3% in patients with bipolar affective disorder after 4 weeks of treatment , and multiple studies have reported basal ganglia volume change with neuroleptics over a duration of time intervals , including longitudinal studies of first - episode patients .
furthermore , there has been a lack of reliability in reports of the heritability of brain structures , with volume reductions in unaffected siblings inconsistent across brain regions and different studies , and not consistently overlapping with genetic liability to schizophrenia , decreasing the utility of structural volumetric indices as intermediate phenotypes . increasingly , a growing number of research groups have used resting - state fmri to map brain networks as well as diffusion tensor imaging ( dti ) to investigate white matter abnormalities in patients with schizophrenia compared with controls . these modalities and investigations ,
however , are beyond the current focus of imaging genetics in the present review , but have been reviewed in depth elsewhere .
we would emphasize two caveats with respect to these approaches : ( i ) the analysis of resting fmri patterns is very sensitive to variation in head motion and to the mental state of the subject , making problematic comparisons between ill and well samples ; and ( ii ) dti is based on highly derived data which are highly susceptible to many artifacts and biological events unrelated to white matter structure , rendering interpretation of the results also highly problematic . in considering activation - based fmri as applied to schizophrenia research there are methodological assumptions and limitations to recognize the same limitations of fmri in general in its application to elucidating other cns disorders or any cognitive function .
firstly , brain mapping is predicated on the assumption of a modular organization of the brain , that there is a functional segregation with specialized and spatially separated modules , with interconnection of the entities and functional integration by distributed systems .
only according to this assumption can fmri be then employed to reveal hierarchical decompositions of brain functional units .
another assumption is that a brain structure can be conceptualized as an information processing entity , with an input , a local processing capacity , and an output .
yet , the traditional cortical input - elaboration - output scheme , a correlate of the perception - cognition - action tripartite model , may be an oversimplification . in contributing to neuronal output
, a change in balance between excitation and inhibition may predominate over a more straightforward hierarchical connectivity feedforward and feedback model .
further , as for all hemodynamic - based modalities , fmri measures a surrogate signal of brain function , but then justifies the assignment of a functional role to an active area , presuming the change in signal results from the change of activity of a neuronal population . also , the blood oxygen level - dependent ( bold ) signal reflects neuronal mass activity that may limit its neurobiological inferences , even while being advantageous in some global instances . according to one estimate
there are about 90 to 100 000 neurons under 1 mm of cortical surface , or as another metric , an unfiltered fmri voxel contains 5.5 million neurons , and 2.2 - 5 .
lastly , for task - based fmri neuroimaging studies , using a block design , a subtraction method is required to compare a task state with an investigator - designed control state , requiring a detailed task analysis to determine subtraction components and their interactions . despite the above select critiques
, fmri remains the best tool at present for gaining insight into brain function , as many of these considerations are rendered relatively nonsys tematic because of the principle of every subject serving as his / her own control and the comparison of signals from one state to another .
thus , fmri activation approaches allow for the ability to test discrete hypotheses about definable brain functions , especially the impact of genetic variation on these functions .
early studies mapped single genes selected based on a measure of candidacy , to an area of brain activation .
examples in addition to comt mentioned above , include the association of a grm3 variant with inefficient prefrontal function during a working memory task , or the association of the short ( s ) variant in a variable repeat sequence in the promoter of the serotonin transporter gene , 5-httlpr , with altered amygdala activity during an emotionally evocative task .
illustrative of genetic vulnerability maps of brain function based on imaging genetics approaches , rasetti et al reviewed neuroimaging intermediate phenotypes of schizophrenia and the gene variants associated with them , catalogued by cognitive task . for example , working memory tasks have revealed abnormal engagement of : dorsolateral prefrontal cortex associated with cacna1c , comt , gad1 , grm3 , drd2 , kcnh2 , mthfr , and rgs4 ; ventrolateral prefrontal cortex associated with comt , prodh , and rgs4 ; thalamus and hippocampus associated with nrg1 , daoa / g72 , and disci ; and parietal lobe associated with prodh .
cognitive control tasks ( designed to challenge executive function of goal - directed behavior in the presence of conflict ) have identified abnormal engagement of the anterior cingulate cortex associated with comt , drd2 , and maoa ; of the dorsolateral prefrontal cortex ( dlpfc ) associated with dtnbpl , drd2,maoa , comt : and of the parietal cortex associated with drd2 , and maoa .
memory encoding tasks recently identified abnormal engagement of hippocampus parahlppocampus region and association of the hippocampus with bdnf , comt , disci , grm3 , and kcnh2 .
it is worthwhile to note that most association studies of brain function have used single gene variants and risk haplotypes emerging from linkage studies and more recently genome wide association studies , with differing levels of genetic evidence for each candidate gene , though there has been no systematic approach to date to selecting genes for imaging genetics studies .
imaging genetics approaches have progressed to associating gene variants with multiple regions of activation , with disease - relevant risk circuits and putative distributed functional networks , rather than isolated , single regions . after all
thus , circuit - based phenotypes would be expected to have greater fidelity in showing genetic association at the level of brain function , since in principle , the more realistic the phenotype , the stronger the genetic association . as schizophrenia is an emergent property of neural system function , not isolatable to a singular brain region or localized regional defect , but likely attributable to network - based neurointegrative deficits , neuroimaging and intermediate phenotyplng strategies
have progressed to better understand distributed networks associated with increased genetic risk . to identify a functional network or interregional coupling , functional connectivity between spatially remote regions
statistical analyses used for functional connectivity include mapping based on seed voxel correlations , principal component analysis , independent component analysis , and partial least squares methods .
the functional connectivity literature within schizophrenia research has largely focused on pfc connectivity , especially the dlpfc and anterior cingulate , and dlpfc interaction with the medial temporal lobe , specifically the hippocampal formation ( hf ) , and interaction with the dlpfc - thalamus . for the dlpfc
, abnormal connectivity has been identified in multiple studies in patients with schizophrenia and in high - risk subjects and various genetic associations have been established with this putative circuit , during working memory tasks .
for example during the n - back working memory task , comt - associated activation changes mapped onto a prefrontal - parietal - striatal circuit , involving the dlpfc , ventrolateral prefrontal cortex ( vlpfc ) , the posterior parietal regions , and the striatum .
a prodh schizophrenia risk haplotype was associated with increased striatal - frontal functional connectivity , while the protective haplotype was associated with decreased striatal - frontal functional connectivity .
a 7 single - nucleotide polymorphism ( snp ) haplotype of pppirib ( encoding darpp-32 ) was associated with functional coupling and increased activation of the striatum and prefrontal cortex .
while some functional connectivity studies have employed a classic intermediate phenotype strategy , testing for regions of correlation in affected subjects as well as unaffected relatives , many studies appear to query the association of risk susceptibility gene variants with correlated regions in healthy subjects or affected patients alone , with the putative neural circuit then pending validation as an intermediate phenotype .
psycho - physiological interaction ( ppi ) analysis is an alternative approach to estimating connectivity , and measures a regionally specific response in terms of an interaction between a cognitive ( or sensorimotor ) process and activity in another part of the brain .
the supposition is that the remote region is either the source of afferents that confer functional specificity on the target region or is activated by efferents that are specifically active during the task .
ppi , therefore , allows for the exploration of the effects of an independent variable ( eg , genotype ) on taskrelated differences in interregional connectivity . as a specific example , combining information about activity in the parietal region , mediating attention to a particular stimulus , and information about the stimulus , ppi aims to identify regions that respond to that stimulus when , and only when , activity in the parietal region is high .
if such an interaction exists , then one might infer that the parietal area is modulating responses to the stimulus for which the area is selective .
while this approach offers a deeper functional probe of network activity than simple time series correlation analyses , it is still based on a correlation of activity and not on a directional model of activity in one region influencing activity in another . in one study , during the n - back task , dlpfc - hf coupling was identified in both patients with schizophrenia and their unaffected relatives , and associated with a znf804a risk allele , using seeded connectivity as well as ppi approaches .
ppi analysis showed a reduction in task load - related modulation of coupling between the right dlpfc and bilateral hf , in patients and siblings , compared with controls .
further , subjects homozygous for the risk - associated allele of znf804a showed a disruption in task - related modulation of right dlpfc - left hf coupling in the ppi analysis .
the seeded connectivity analysis showed similar results to the ppi analysis in dlpfc - hf coupling .
overall then , functional connectivity analysis offers some insight into correlation between different brain regions , but is limited in that it does not account for directionality , influence , or causality between putatively interacting regions ; it makes no assumptions about the nature of underlying pathways , their structure , nor anatomical connectivity .
so while correlative methods provide a way to characterize neural functional networks by temporal coherence of inter - regional activation patterns , it yields neither an understanding of driving neural origins nor of the directionality of the observed network .
in contrast to functional connectivity approaches , effective connectivity analyses promise extended insight , referring explicitly to the influence that one neuronal system exerts over another , and may be used to better explain integration within a distributed neural system .
models employed in analyzing imaging data to uncover effective connectivity are based on regression models , or structural equation models , and these models may be linear or nonlinear .
dynamic causal modeling ( dcm ) is a type of effective connectivity analysis that yields directional , pathway information and allows for a quantification of the influence of a given neural region over another .
dcm analysis , introduced in 2003 for fmri data , is a bayesian framework for inferring hidden neuronal states from measurements of brain activity ; it is a hypothesisdriven approach , requiring an a priori definition of a set of interconnected neural areas that mediate a given function of interest .
dcms are generative models of brain responses , which provide estimates of neurobiologically interpretable quantities including strength of synaptic connections among neuronal populations and their context - dependent modulation .
causality in dcm is based on control theory , ie , causal interactions among hidden state variables that are expressed by differential equations that describe how the present state of one neuronal population causes dynamics in another via synaptic connection , and how these interactions change under the influence of external perturbations ( eg , experimental manipulations ) or brain activity .
dcm tests hypotheses about neuronal mechanisms , allowing one to specify a generative model of measured brain data , which is a probabilistic mapping from experimentally controlled manipulations to observed data , via neuronal dynamics .
dcm has begun to be applied to imaging genetics . using a dcm approach , distributed circuits that putatively underlie working memory
prefrontal - parietal and prefrontal - striatal circuits were identified in healthy , normal subjects , and comt , drd2 , and akt1 functional variants were associated with the circuits .
the goal of the study was to engage a hypothesis - driven strategy to study component dopamine signaling processes , both d2-mediated and non - d2-mediated aspects of dopamine signaling .
the investigators developed a working memory task that allowed dissociation of working memory into sub - processes , specifically maintenance of information and manipulation of information . in accordance with the dcm approach ,
models of prefrontal - subcortical - parietal networks were generated ( each model 's nodes , connections , and inputs were generated ) during working memory maintenance and manipulation events , and the optimal model with the highest group bayes factor was determined .
the best dcms for maintenance were primarily prefrontal - parietal connections , while for manipulation , the circuit that best fit the data was a prefrontalstriatal network .
these results fit remarkably well with data from nonhuman primates about subprocesses in working memory and the principal networks engaged .
the cortical network engaged during maintenance is presumed to be a non - d2 dominated network , and indeed , only comt showed association with activity in this network .
in contrast , the cortical - striatal network is expected to be d2-dominated , and all three genes showed effects on this network .
this study illustrates the greater fidelity of genetic association based on more realistic models of brain information processing . in a study using nonlinear dcm
, subjects at high familial risk of schizophrenia performed a sentence completion task , and the connection strength of the mediodorsal ( md ) thalamus and inferior frontal gyrus ( ifg ) was investigated , revealing lower connection strength in the at - risk subjects .
bayesian model selection was used to compare the optimal bilinear and nonlinear models , and bayesian model averaging was used to assess the connection strengths with the gating from the md thalamus and the ifg , with nonlinear models providing better explanation of the data . in another study , dynamic causal models were applied to fmri data to investigate how brain connectivity during an associative emotional learning task is affected by different pppirib variants ( darpp32-encoding ) , in healthy subjects . a pppirib variant was associated with increased connectivity between the inferior frontal gyrus ( ifg ) , amygdala and parahippocampal gyrus ( phg ) , with directionality of the connectivity determined to be from the ifg to the phg .
in addition to emerging effective connectivity analyses by dcm , connectivity is being explored from a more systems - level , hierarchical perspective , using graph theory metrics to describe the structural and functional composition of neural circuits . in graph theory , the correlated activity across multiple , distributed preselected brain regions can be expressed in terms of a graph , having various quantitative parameters , such as nodes , hubs , edges , pathway length , and connectivity strength .
the hubs of these networks correspond to the most highly interconnected neural regions , which typically map to the association cortices of the human brain .
the strength of each node is defined as its average connectivity with all other nodes , and the graph 's size is defined by the number of nodes in the largest connected component ; a larger graph size indicates fewer disconnected nodes .
accumulating evidence suggests that the small - world topological properties of brain functional networks are altered in patients with schizophrenia . in one study , in 31 patients with schizophrenia
compared with 31 healthy controls , functional connectivity between 90 cortical and subcortical regions was estimated by partial correlation analysis and thresholded to construct a set of unidirected graphs .
the healthy subjects demonstrated efficient small - world properties , whereas topological parameters of brain networks strength and degree of connectivity were decreased in patients with schizophrenia , especially in the prefrontal , parietal , and temporal lobes , consistent with a hypothesis of dysfunctional integration . in another study , in a sample of 203 patients with schizophrenia ,
compared with 259 healthy controls , multimodal network organization was noted to be abnormal , as measured by topological and distance metrics of anatomical network organization , abstracted from fmri data .
patients with schizophrenia , compared with controls , demonstrated reduced hierarchy throughout the small - world regime , and increased connection distance in the multimodal cortical network .
the loss of frontal hubs and the emergence of nonfrontal hubs was also noted , supporting the hypothesis of schizophrenia as a dysconnectivity syndrome , impacting the efficiency of a frontally dominated hierarchical network of multimodal cortical connections .
though the impact of genetic variation on network topology based on graph analyses has not yet been reported , moderate levels of heritability have been found for brain graph topology measured in a twin study using eeg , suggesting that genetic variation may impact small - world organization and brain graph metrics .
just as imaging genetics will continue to incorporate increasingly sophisticated analytic methodologies , so too will imaging genetics evolve to incorporate increasingly sophisticated models of genetic risk , reflective of the increasingly apparent polygenic complexity of psychiatric syndromes .
genome - wide association studies ( gwas ) have indicated a highly significant polygenic component of schizophrenia risk , possibly involving up to thousands of common alleles of very small effect , at the population level . while early imaging genetics used intermediate phenotypes to assess the impact of single gene variants , recent studies have increasingly tended towards epistatic models of gene interaction . in 2007 , tan et al reported a two - way risk variant epistasis : the comt - val risk allele , associated with reduced prefrontal dopamine , and the grm3 risk allele , related to suboptimal glutamatergic function , interacted to give disproportionately inefficient dlpfc activation in a working memory task .
nicodemus et al reported the first 3-way interaction using neuroimaging genetics to assess the risk susceptibility of the nrgi molecular pathway , finding epistasis between nrgi , and its tyrosine kinase receptor erbb4 , in a 3-way interaction with a variant of akt1 .
the statistical interaction was biologically validated by fmri , in which healthy individuals carrying all three at - risk genotypes for nrgi , erbb4 , and akt1 were disproportionately less efficient in dlpfc processing than any other combinations of one or two at - risk genotypes . of note , lower - level interactions were not observed between nrgi , erbb4 , and akti , suggesting that the interaction , and the nrgi pathway , was necessary for the observed fmri effect of inefficiency .
other reports of epistasis in neuroimaging genetics include association of variants of with altered dlpfc activation , during working memory tasks including disci - cit - ndeli , mthfr - comt , and comtrgs4 .
imaging genetics is further evolving towards modeling increasing genetic complexity , by utilizing a polygenic risk score or propensity score of genetic risk for schizophrenia in fmri studies .
a range of options for constructing a polygenic score may be considered , selection of markers according to their p - values in association studies , and different methods for weighting markers in the score .
only a handful of studies utilizing a polygenic risk score have been reported to date , using both functional and structural neuroimaging , and for multiple psychiatric syndromes .
walton et al calculated a genetic risk score for schizophrenia , the additive effect of 41 snps from 34 putative risk genes , and found a positive relationship between the genetic risk score and left dlpfc inefficiency during a working memory task .
holmes et al reported a structural anatomic association with polygenic risk for major depressive disorder ( mdd ) in a sample of 1050 healthy young adults with no history of psychiatric illness . using risk scores derived from large mdd gwas analyses ,
an mdd polygenic score was found to be associated with reduced cortical thickness in the left medial prefrontal cortex , a structural variation that is believed to influence vulnerability to mdd . in a third study , increasing polygenic risk allele load for bipolar affective disorder ( bpad ) was associated with increased activation in limbic regions previously implicated in bpad , including the anterior cingulate cortex and amygdala during a verbal fluency task .
so , while a few early imaging genetics studies have employed the polygenic risk score , use of the polygenic score approach remains to be assessed and validated in larger - scale , more robust studies , with an explicit focus on schizophrenia and with various models of the risk score calculation possible .
as highlighted in this review , initial methodological and conceptual advances in functional neuroimaging launched the epoch of imaging genetics . since then , development of genetic vulnerability maps of the brain , identifying neuroimaging intermediate phenotypes of schizophrenia and the risk variants associated with them , have become a major research industry .
while imaging genetics to date has led to an increased understanding of schizophrenia pathophysiology and potential sites of pharmacologic intervention , a new wave of imaging genetics is fueled by even further methodological and conceptual advances .
effective connectivity - modeling promises to offer causal and directional insight into brain networks and circuitry , and polygenic risk modeling promises to incorporate genetic models reflective of the polygenic complexity of the schizophrenia syndrome . | we review critical trends in imaging genetics as applied to schizophrenia research , and then discuss some future directions of the field .
a plethora of imaging genetics studies have investigated the impact of genetic variation on brain function , since the paradigm of a neuroimaging intermediate phenotype for schizophrenia first emerged .
it was initially posited that the effects of schizophrenia susceptibility genes would be more penetrant at the level of biologically based neuroimaging intermediate phenotypes than at the level of a complex and phenotypically heterogeneous psychiatric syndrome .
the results of many studies support this assumption , most of which show single genetic variants to be associated with changes in activity of localized brain regions , as determined by select cognitive controlled tasks
. from these basic studies , functional neuroimaging analysis of intermediate phenotypes has progressed to more complex and realistic models of brain dysfunction , incorporating models of functional and effective connectivity , including the modalities of psycho - physiological interaction , dynamic causal modeling , and graph theory metrics .
the genetic association approaches applied to imaging genetics have also progressed to more sophisticated multivariate effects , including incorporation of two - way and three - way epistatic interactions , and most recently polygenic risk models .
imaging genetics is a unique and powerful strategy for understanding the neural mechanisms of genetic risk for complex cns disorders at the human brain level . |
lung cancer is the most common fatal cancer worldwide and has become the leading cause of cancer deaths in korea
the risk of human cancer can be associated with environmental , occupational , and recreational exposures to carcinogens ( 2 ) .
oncogenesis is related with epigenetic changes , oncogenes , tumor suppressor genes , apoptosis , and genetic changes associated with dna repair .
there have been many investigations on the prognostic role of p53 , bcl-2 , and ki-67 expression in non - small cell lung cancer ( nsclc ) .
early reports of p53 mutation suggested a variable relationship to survival ( 3 , 4 ) .
another study showed that p53 mutations detectable in tumor tissues had been shown to be an independent marker for the poor prognosis in resectable stage i nsclc ( 5 ) .
data on bcl-2 expression from a study on nsclc patients showed positive correlations with longer survival ( 6 ) .
there were reports on an inverse relationship between bcl-2 and p53 in nsclc ( 7 , 8) .
however , another studies did not support a relevant prognositic role for p53 , bcl-2 , or ki-67 immunohistochemical markers in nsclc regardless of stage ( 9 , 10 ) .
the relationship between a positive rate for ki-67 and prognosis remains unclear ( 11 ) .
there was a report that bcl-2 expression did not influence survival in sclc ( 12 ) .
we conducted a retrospective study on the value of mutant p53 , bcl-2 , and ki-67 expressions in sclc patients from korea cancer center hospital .
all of the patients were primarily diagnosed as sclc at the department of internal medicine of korean cancer center hospital between february 1997 and december 2002 .
immunohistochemical ( ihc ) stainings for mutant p53 , bcl-2 and ki-67 expressions were performed in the 66 paraffin - embedded biopsy samples among the 75 member treatment group .
the study group included patients ( 57 males and 9 females , 61 yr mean age ) with cytologically or histopathologically diagnosed sclc .
histopathological diagnoses were done by bronchoscopic biopsy , lymph node biopsy , and percutaneous lung gun biopsy . staging procedures included physical examination , chest radiography , chest computed tomography ( ct ) scan , and bone scintigraphy .
brain ct / magnetic resonance image ( mri ) , bone marrow biopsy and other studies were optional in asymptomatic patients but mandatory in those with symptoms suggesting disseminations .
patients were staged as either limited , with disease confined to one hemithorax , or extensive , with disease beyond one hemithorax .
all patients were administered one of the three chemotherapy regimens : cisplatin and etoposide ( ep ) ; cyclophosphamide , adriamycin , and vincristine ( cav ) ; etoposide and carboplatin ( ec ) .
we established the principle that all limited disease patients would be treated by radiotherapy if the chemotherapy were effective .
response categories i.e. complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd ) were evaluated according to new response evaluation criteria in solid tumor ( recist ) guidelines ( 13 ) . the cr and pr patients
survival was defined as time lapse from the day of first chemotherapy course until the day of death .
five - micrometer thickness , paraffin - embedded , tissue sections were deparaffinized in xylene and hydrated in a graded ethanol series .
tissue sections were heated in 10 mm sodium citrate , ph 6.0 , in a microwave oven for 10 min to expose the antigens .
the streptavidinbiotin - peroxidase complex technique ( universal lsab kit , dako , glostrup , denmark ) was used for immunohistochemical stain .
the sections were incubated overnight at 4 with the three monoclonal antibodies : p53 ( 1:50 , dako , glostrup , denmark ) , bcl-2 ( 1:40 , dako ) and ki-67 ( 1:50 , zymed , san francisco , ca , u.s.a . ) .
sections were then washed with tbs and incubated with biotinylated secondary antibody for 30 min at room temperature .
after washing , the sections were incubated with peroxidase - labelled streptavidin at room temperature for 30 min .
diaminobenzidine / hydrogen peroxidase was used as a chromogen and sections were counterstained with hematoxylin .
when the cell nuclei were stained strongly with dark brown color , the cells were considered to be positive for p53 and ki-67 .
bcl-2 expression was noted in the cytoplasm . if more than 10% of the tumor cells were positively stained , it was considered as positive for p53 and bcl-2 . in the case of ki-67 ,
the labeling index was determined by scanning areas with uniformly stained cells at a low magnification , followed by counting of the cells at high power field ( 400 ) .
median values and ranges were used to calculate patient characteristics and to compare the factors . to examine factors affecting survival ,
the following variables were analyzed : age , sex , disease extent , performance status , and ki-67 , p53 , and bcl-2 expression .
each of these variables was divided into two categories as follows : age was divided by mean age , i.e. , less than 60 yr vs. more than or equal to 60 yr ; sex , male vs. female ; disease extent , limited disease ( ld ) vs. extended disease ( ed ) ; performance status ( ps ) , good ( ps 0 - 1 ) vs. poor ( ps 2 - 4 ) ; ki-67 , equal to or greater than 50% vs. less than 50% ; p53 and bcl-2 , positive vs. negative . to compare the response rate ,
survival was assessed by the kaplan - meier method and compared with log - rank test .
hazard ratio and its 95% confidence interval for each variable were estimated by cox proportional hazard model . a p - value lower than 0.05 was considered statistically significant .
patient characteristics are shown in table 1 . overall response rate for evaluable 66 patients were 72% .
there was no difference of the response rate among three regimens ( ep , 69% ; cav , 65% ; ec , 75% ; p=0.83 ) .
forty - one ( 62% ) out of the 66 patients were positive for p53 antibodies ; 25 ( 40% ) with limited disease ( ld ) and 16 ( 24% ) with extensive disease ( ed ) ( p=0.36 ) .
the response rate for the positive p53 group was 68% , that for the negative group was 84% ( p=0.15 ) .
median survival was 13 months for the positive p53 group , and 16 months for the negative group ( p=0.50 ) .
there were no significant differences in sex , age , disease extent , performance status , and survival between patients with and without mutant p53 antibodies ( table 2 ) . according to the analysis of prognostic factors which influenced chemotherapeutic response rate and survival , only the extent of the disease was significant for patient survival by univariate and multivariate analysis ( table 3 ) ( fig .
median survival for the entire group was 15 months ; 16 months for ld patients , and 10 months for ed patients ( p=0.04 ) .
we also analyzed the survival between positive and negative p53 groups in ld patients or ed patients .
in ld patients , median survival was 15 months for the positive p53 group , and 18 months for the negative group ( p=0.37 ) . in ed patients ,
median survival was 10 months for positive p53 and 12 months for negative ( p=0.87 ) .
no correlation was found between survival and p53 expression in either ld or ed patients .
for bcl-2 , 45 ( 70% ) out of 64 patients were positive ; 26 ( 40% ) with ld and 19 ( 30% ) with ed ( p=0.15 ) ( table 2 ) .
the response rate was 75% for the positive bcl-2 group , and 73% for the negative group ( p=0.72 ) .
median survival was 16 months for positive bcl-2 , and 15 months for the negative group ( p=0.46 ) .
in ld patients , median survival was 15 months for the positive bcl-2 group and 19 months for the negative group 19 months ( p=0.37 ) . in ed patients ,
median survival was 9 months for positive bcl-2 , and 12 months for negative ( p=0.16 ) .
no correlation was found between survival and bcl-2 expression in either ld or ed patients . like mutant p53
, there were no significant differences in sex , gender , disease extent , performance status , and survival between patients with and without bcl-2 expression .
thirty - two ( 49% ) out of 65 were in the above 50% group , 22 ( 34% ) with ld and 10 ( 15% ) with ed ( p=0.60 ) ( table 2 ) .
the response rate was 69% for the above 50% group , and 79% for the below group ( p=0.55 ) .
median survival was 16 months for the above 50% group , and 15 months for the below group ( p=0.21 ) .
in ld patients , median survival was 16 months for the above 50% group , and 16 months for the below group ( p=0.37 ) . in ed patients ,
median survival was 12 months for the above 50% group and 10 months for the below group ( p=0.65 ) .
no correlation was found between survival and ki-67 expression in either ld or ed patients . there were no significant differences in sex , gender , disease extent , performance status , and survival according to ki-67 expression .
the p53 protein is recognized as an important cell regulatory factor that arrests the growth of cells containing damaged dna .
a reversible arrest in the g1 phase of the cell cycle enables dna repair before dna synthesis . when appropriate repair is not possible , p53 expression may trigger apoptosis , a reversible process culminating in cell death . if normal , wild type p53 function is lost , the treatment is relatively resistant , as a result of deficiency of p53-dependent apoptosis ( 14 , 15 ) .
thus , the mutated gene loses its natural tumor suppressor function allowing damaged cells to divide unchecked and finally to become malignant cells .
some studies reported that p53 mutation had been associated with poor prognosis and shorter survival in nsclc ( 5 , 8 , 16 , 17 ) .
however , some others reported no such correlation in nsclc ( 3 , 9 , 10 , 18 ) .
some studies showed that bcl-2 expression and p53 mutation had no relation to survival in sclc ( 9 , 10 , 12 , 20 ) .
another study divided their sclc patients into limited and extensive - stage disease , after which p53-antibody positivity emerged as an independent marker of poor prognosis in ld but had no relation to survival ( 21 ) .
one study indicated that p53 played an important role as a determinant of chemosensitivity in sclc and that p53 immunostaining could be used in clinical practice to determine the presence of tumor - chemoresistance ( 22 ) .
kenichi et al . reported that patients with expression of mutant p53 protein showed lower response rate than those having p53-negative tumors and were less sensitive to anticancer drugs ( 23 ) .
the bcl-2 proto - oncogene is encoded by a 230-kb gene that gives rise to a 24- to 26-kda protein that is localized in the inner mitochondrial membrane , and , to a lesser extent , in the cell membrane ( 24 ) .
the major function of bcl-2 appears to be the inhibition of apoptosis or programmed cell death , whereas bax , bad , bak , and others promote cell death ( 25 ) .
it is well documented that bcl-2 becomes deregulated in tumor cells as a result of translocation into the immunoglobulin heavy - chain locus , and is therefore constitutively activated in follicular lymphoma ( 26 ) . in epithelial tumors ,
however , bcl-2 expression has been described in a series of solid tumors , particularly in nsclc and in breast cancer ( 27 ) .
an in vitro study showed that bcl-2 expression may be related to chemoresistance due to inhibition of drug - induced apoptosis ( 28 ) .
thus multidrug resistance is probably linked , at least in part , to high levels of bcl-2 expression .
bcl-2 blocks the cell death pathway ( apoptosis ) and is not directly associated with cell proliferation ( 29 ) .
studies examining the association of bcl-2 expression with survival in nsclc have been contradictory , with some reporting bcl-2 as an indicator of better survival ( 6 , 7 ) , while other showed no survival differences according to bcl-2 status ( 30 , 31 ) .
brdu ( bromodeoxyuridine ) , pcna ( proliferating cell nuclear antigen ) , ki-67 , and others have been used as cell proliferation markers .
pcna , a 36-kilodalton , nuclear polypeptide that is related to cell proliferation , is identical to cyclin , which is a protein that appears in the proliferative phase of cells ( 32 ) .
being synthesized during the late g1 to s phase , pcna is an auxillary protein for dna polymerase . ki-67 , a more reliable proliferating marker , reacts with nuclear antigen that is present only in proliferating cells ( g1 , s , g2 , and m phase ) , not in resting ( g0 ) cells ( 33 - 35 ) , and thereby provides a reliable method for evaluating tumor growth fraction in many malignant tumors , including lung cancer ( 36 ) .
it appears to be a useful prognostic marker of nsclc , especially in the early stage ( 37 ) .
compared to p53 and bcl-2 , the relationship between ki-67 and survival has been studied less extensively in nsclc , and especially so in sclc .
our results showed that ki-67 expression had no relation to survival in sclc . from our results ,
the apoptosis - related genes , mutant p53 and bcl-2 , and the proliferative marker , ki-67 , were found to be useful in the diagnosis of sclc patients because of their relatively high prevalence ( 38 , 39 ) .
however , as they did not show clinical significance for prognosis or survival , further investigation will be required to confirm the clinical role of these markers . | small cell lung cancer ( sclc ) is one of the most fatal cancers in humans and many factors are known to be related to its poor prognosis .
immunohistochemical ( ihc ) stainings were done on sclc specimens in order to investigate the prognostic value of the apoptosis - related gene expression and the tumor proliferative maker , and the relationships among these ihc results and patients clinical characteristics , chemoresponsiveness , and survival were analyzed .
the medical records of 107 patients were reviewed retrospectively .
ihc stainings for p53 , bcl-2 and ki-67 expressions were performed in the 66 paraffin - embedded biopsy samples .
sixty - six out of the 107 patients were evaluable for response rate and survival .
the overall response rate was 75% ( 95% confidence interval=74 - 76% ) and the median survival time was 14 months .
the median survival time of limited stage was 16 months and that of extensive stage was 10 months .
the prevalence of p53 , bcl-2 and ki-67 expression was 62% , 70% , and 49% , respectively .
there were no correlations among the immunoreactivities of p53 , bcl-2 and ki-67 with clinical stage , chemoresponsiveness or overall survival .
the clinical stage was the only prognostic factor influencing survival .
the expression rates of p53 , bcl-2 , and ki-67 were relatively high in sclc without any prognostic significance
. the exact clinical role of these markers should be defined through further investigations . |
the common cold , an acute infection properly known as cold syndrome , is the most common human illness .
the majority of cases of cold syndrome are acute infections of the upper respiratory tract , and its major cause is viral infection . from 30 to 50% of cases of cold syndrome
conventional methods of treatment use medications , such as analgesic agents and antihistamines , but these are only effective for the alleviation of symptoms , such as sneezing and runny nose . furthermore , antiviral agents , such as neuraminidase inhibitors , are believed to be effective ; however , their application is currently limited to the influenza virus [ 1 , 36 ] . recently
, chinese medicine and dietary supplements have attracted attention as effective new methods for the treatment and prevention of cold syndrome ; for example , vitamin c ; allysine , which is found in garlic ; and the extract of the natural herb echinacea [ 810 ]
. however , there have also been reports indicating that the incidence of cold syndrome is unaffected by the above treatments [ 11 , 12 ] , and thus their effectiveness has yet to be clearly demonstrated . in a previous experiment in mice
, we revealed that the oral administration of l - cystine and l - theanine ( ct ) reinforced antigen - specific antibody production after antigen stimulation ; these effects were possibly caused by the reinforcement of glutathione ( gsh ) synthesis and the humoral immune response . based on previous reports ,
the objective of this pilot study was to evaluate the effects of ct against cold syndrome in humans , using a double - blind comparative study .
this randomized , double - blind , and parallel - group comparative study with a placebo group as a reference was performed as a pilot study in accordance with the declaration of helsinki and after obtaining approval from all members of the ajinomoto co. , inc . , institutional review board ( irb ) , as organized by the executive director , the director of the quality assurance department , the general managers of research and development , and the divisional director of food business on december 17 , 2001 .
the subjects were healthy male volunteers ( for safety reasons and to avoid individual differences influenced by the estrous cycle ) who are employees of ajinomoto co. , inc .
, excluding those with a history of or a current liver or kidney disease as well as those with critical illness who were not able to be diagnosed by a clinician .
the subjects were randomly allocated into one of two groups using an allocation table : a group receiving placebo ( p ) and another group receiving cystine / theanine ( ct ) .
, we determined the number of subjects with allergy symptoms , the number of smokers , the number of subjects who caught a cold twice or more per year , and the number of subjects living with school - aged children , which are the family members most likely to catch a cold .
when the subjects registered for the study , the investigator and the physician in charge provided the informed consent form , which was discussed and approved by the irb .
the content of the study was explained in detail , and the subjects provided their written voluntary consent . according to the third national health and nutrition examination survey that took place in the usa from 1988 to 1994 , the average intake of l - cystine or l - cysteine from meals and supplements is 1000 mg / day . in males in the 51 to 70-year - old age group , the average intake among the subjects in the highest 1% of the group was 2200 mg / day .
considering the safety data described above , the applied dose in the present study was set at 700 mg / day for l - cystine to remain within the range of the average daily intake of l - cystine or l - cysteine .
the applied dose of l - theanine was calculated as 280 mg / day based on analyses in mice , which suggested that the most effective weight ratio of cystine : theanine = 5 : 2 .
glycine was used as a placebo so that the subjects received the same source of nitrogen , and the applied dose was set at 980 mg / day . during the 5-week period from january 18 , 2002 to february 21 , 2002 ,
the subjects were asked to take 2 tablets orally after breakfast and 2 tablets orally after dinner , with each tablet containing 175 mg of l - cystine plus 70 mg of l - theanine or 245 mg of glycine as a placebo ( 11 mm , table 1 ) ( a total of 4 tablets daily ; total daily consumption of cystine , theanine , and glycine = 700 mg , 280 mg , and 980 mg , resp . ) .
the onset of common cold symptoms as well as data regarding intake of the test meals during this 5-week period were recorded by the subjects on a daily basis as described below .
the subjects performed a self - evaluation of 19 symptom items ( symptoms of the common cold : runny nose , stuffy nose , sneezing , sore throat , cough , sputum , chills , fever , headache , joint pain , pain in the skin , and general malaise ; adverse events accompanying test meal ingestion : nausea , vomiting , abdominal pain , diarrhea , loss of appetite , chest pain , and skin rash ) which were graded according to 3 levels ( + : presence of symptoms , : slight symptoms , and : absence of symptoms ) .
the subjects were not restricted with regard to medical treatment during this period , such as taking medication for the common cold , but were asked to record any treatment received . in the analysis ,
the criteria used in the clinical test of anti - influenza virus agents were used as a reference [ 5 , 6 ] because the period of the trial ( january to february ) corresponds to the season in which influenza virus is most prevalent in japan .
the symptoms recorded on the data sheets were converted to scores as follows : presence of symptoms ( + ) = 2 , slight symptoms ( ) = 1 , and absence of symptoms ( ) = 0 .
the items were classified into four predominant symptoms : ( 1 ) stuffy nose / runny nose / sneezing as nasal symptoms , ( 2 ) cough / sputum / sore throat as throat symptoms , ( 3 ) chills and fever as febrile symptoms , and ( 4 ) headache / joint pain / pain in the skin as pain symptoms .
if the score for the predominant symptom was more than 3 of 6 for nasal , throat , or pain symptoms , the predominant symptom was considered to be present .
we considered febrile symptoms to be present if the score was more than 2 of 4 .
if 3 of 4 predominant symptoms were observed on the same day or if the body temperature was above 37.0c and two or more common cold symptoms ( stuffy nose / runny nose / sneezing , cough / sputum / sore throat / chills / headache / joint pain / pain in the skin / general malaise ) displayed a score of 2 , the subject was considered to have the common cold ( table 2 ) . based on this definition ,
the number of subjects with incidences of the common cold during the 5-week period of the study was counted , and the two groups were compared using fisher 's exact test .
when there was another incidence of the common cold , according to the above definition , after 3 or more days of recovery from an earlier cold , this was counted as a separate incident .
the number of affected days was also counted and was compared between the two groups using poisson regression analysis .
in addition , the average duration of the colds and the number of subjects that experienced a body temperature above 37.0c were also determined and were compared between the two groups using the t - test and fisher 's exact test , respectively .
the incidences of individual symptoms of the cold and adverse events in the evaluation of safety were counted if the score had reached 2 at least once during the 35-day period of the trial , and the number was compared between the two groups using fisher 's exact test .
all statistical analyses were performed using the statistical analysis system ( sas ) version 8.2 ( sas institute , inc .
, cary , nc , usa ) , and the results were considered as significant at p < .05 .
the total number of subjects registered in this study was 176 , and , as a result of the randomized allocation , 89 subjects were allocated into the ct group and 87 subjects were assigned to the p group .
one subject in the ct group and one subject in the p group lost their data recording sheets , and one subject in the p group withdrew the informed consent .
therefore , the final analysis was performed using data from a total of 173 subjects , consisting of 88 subjects in the ct group and 85 subjects in the p group .
table 3 shows the results of the background factor questionnaire , which was completed by the subjects prior to the study ; there were no significant differences in background factors between the two groups .
the tablet ingestion rate during the trial period indicates an approximately 90% ingestion rate in both groups , where ingestion of 70 tablets during the 5-week trial period was defined as 100% ( table 4 ) .
we counted the number of subjects in each group according to the ingestion rate , and there was no significant difference between the two groups ( p = .357 ) .
as shown in figure 1 , the incidence of the common cold in the ct group was lower than in the p group throughout the 5-week period of the trial . as shown in table 5 , the incidences of the common cold according to the definition set by our laboratory were 11.4% in the ct group and 27.1% in the p group , with that in the ct group being significantly lower than that in the p group ( p = .011 ) .
the cumulative incidences during the trial period were 10 in the ct group and 29 in the p group ( in the placebo group , four subjects had two and one subject had three separate instances of colds during the trial period ) ; the ct group demonstrated a significantly reduced number of colds than the p group ( p = .002 ) .
in addition , the cumulative numbers of days affected by the common cold were 18 days in the ct group and 59 days in the p group ; the ct group demonstrated significantly fewer days affected by the cold than the p group ( p = .002 ) .
the average duration of the colds was approximately 10% less in the ct group than that in the p group , although this difference was not significant .
the proportion of subjects with body temperatures above 37c was 18.2% in the ct group and 32.9% in the p group , with that in the ct group being significantly lower than that in the p group ( p = .036 ) .
the average duration of a body temperature above 37c was approximately 15% shorter in the ct group than in the p group , but the difference was not significant .
in addition , a poisson regression analysis was performed to look for correlations between the incidence of colds and the backgrounds of the subjects as well as to investigate any interactions between ct ingestion and the backgrounds of the subjects on the incidence of colds .
however , no significant correlations or interactions were observed between the ct ingestion and the backgrounds of the subjects or the incidence of colds in the present trial ( data not shown ) .
the results of the analyses regarding the incidences of each symptom in the trial period are shown in table 6 .
for all items except pain in the skin , the incidences of symptoms were lower in the ct group than in the p group .
chills and fever in particular , included in the febrile symptoms , displayed significant decreases in incidence in the ct group ( p = .029 and p = .003 , resp . ) .
the incidences of predominant symptoms other than febrile symptoms , such as runny nose ( p = .057 ) , sore throat ( p = .089 ) , and joint pain ( p = .069 ) , tended to be lower in the ct group than in the p group , although the differences were not statistically significant .
the number of times that drugs to treat the common cold were taken during the trial period was 11 times in the ct group and 20 times in the p group ( data not shown ) .
the incidences of diarrhea in the ct and p groups were 14.8% and 15.3% , respectively , which were not significantly different ( table 7 ) .
the present exploratory trial showed that continuous ingestion of ct significantly suppressed the incidence of the common cold compared to a placebo control group and also significantly reduced the incidence of chills and fever among individual symptoms .
investigated the safety of continuous ingestion of coq10 and asked subjects to self - evaluate the severity of common cold symptoms on a 4-level scale .
the results indicated that 24 of 85 subjects ( 28.2% ) experienced incidences of common cold symptoms .
by contrast , in a report on the prevention of upper respiratory tract infection using extract of north american ginseng containing poly - furanosyl - pyranosyl - saccharides by predy et al .
, 10 types of common cold symptoms were scored on a 4-level scale , and the sum of the scores on 2 consecutive days over 14 was considered as an incidence of the common cold . in the placebo group ,
the incidence of colds was 22.8% during the 4-month trial period . considering the above findings as well as the differences in weather conditions and evaluation methods , the 27.1% incidence of colds ( p group ) in our study indicated that the definition of the common cold and the evaluation method used in this study were valid .
limited to those under physically stressed conditions , a meta - analysis of the effects of vitamin c indicated 50% suppression of the incidence of the common cold .
in addition , in the above - mentioned study by predy et al . , ingestion of the ginseng extract was shown to reduce the incidence of the common cold by approximately 56% . moreover , the extract of the natural herb echinacea was reported to have a prophylactic efficacy of 55% or 58% on the incidence of common cold by two meta - analyses [ 18 , 19 ] .
by contrast , a study that employed continuous administration of n - acetylcysteine ( nac ) indicated a 43% reduction in the incidence of influenza - like symptoms . in the present study , the incidence of the common cold according to our definition was decreased by approximately 58% by ct ingestion .
this result indicated that ct has a cold prevention effect with an efficacy similar to that of other nutrients that are considered to possess this effect .
as previously mentioned , the majority of common colds are caused by viral infection , with rhinoviruses being the most common infectious agent .
these viruses are known to infect cells via intercellular adhesion molecule-1 ( icam-1 ) , which is a receptor on epithelial cells of the respiratory tract , and reduced gsh is known to inhibit the increased expression of icam-1 . in our previous study in mice , we showed that the administration of ct significantly increases the amount of reduced gsh .
the above findings suggest that the increase in reduced gsh due to the ingestion of ct , which suppressed the expression of the virus receptor , led to prevention of the common cold and reduced the severity of cold symptoms .
the average duration of the colds was reduced by approximately 10% , although this reduction was not statistically significant .
thirty clinical studies were compared in a meta - analysis that was employed for the vitamin c study , and it was found that the duration of the colds was reduced by 8% in adults and 13.6% in children due to the ingestion of vitamin c . taking these results into consideration , we feel that another study of about the same scale or larger than this study will reveal a clear effect of ct on the consecutive number of days that individuals are affected by the common cold . for individual symptoms of the common cold , significant reductions in febrile symptoms ( chills and fever ) were observed due to ct ingestion as well as a decreasing trend in the incidence of other predominant symptoms , for example , runny nose , sore throat , and joint pain .
riedel and maulik reported that the increase in body temperature after lipopolysaccharide stimulation can be suppressed by administering a reducing agent , and they suggested the possibility that the increase in reduced gsh in response to the reducing agent suppressed fever .
reduced gsh is known to suppress the activation of nf-b , which is an inflammatory mediator at the time of rhinoviral infection .
these findings suggest that the suppression of individual symptoms of the common cold in the present study may have been due to the anti - inflammatory effect of increased gsh caused by ct ingestion . in this trial
, there were some cases of observed symptoms but a lack of a cold during the trial .
consequently , we performed a pearson correlation coefficient analysis between the incidence of colds during the trial and each symptom .
the symptoms of chill ( r = 0.694 ) , fever ( r = 0.663 ) , and arthralgia ( r = 0.610 ) showed strong correlations with the incidence of colds .
in addition , the symptoms of cough ( r = 0.467 ) , runny nose ( r = 0.458 ) , and headache ( r = 0.451 ) showed moderate correlations with the incidence of colds .
these findings suggest that the definition of colds used in the present trial has a strong correlation with relatively severe symptoms . in evaluating the safety of this study ,
we observed a relatively high incidence of diarrhea in the ct group as an adverse event .
however , approximately the same incidence of diarrhea was observed in the p group , and therefore this effect is unlikely to be an adverse reaction specific to ct ingestion . in the present study ,
these tablets contained nondigestible substances , such as crystalline cellulose and dextrin , to maintain their shape , and these substances may have affected the digestive system .
ct , which has been shown to reinforce gsh synthesis and antibody production in mice following antigen stimulation , was used in humans , and a significant inhibitory effect on the common cold has been observed . in a clinical study in humans , miyagawa et al .
reported that oral administration of ct improves antibody production in the elderly , with decreased immune function at the time of flu vaccination .
moreover , murakami et al . reported that ct ingestion by long - distance runners before a training camp suppressed the increase in blood neutrophil counts and the decrease in lymphocyte counts observed in control subjects after training .
those results suggest that ct ingestion may improve the immunosuppression induced by aging in the elderly and by intense exercise in athletes . to determine whether this common cold inhibitory effect is due to the reinforcement of gsh synthesis and antibody production by ct ingestion
the results of the present exploratory trial showed that ct is an effective and safe drug as a food ingredient to suppress the common cold , and we anticipate its use as a drug or food ingredient for cold prevention and symptom relief in the future .
a randomized , placebo - controlled 5-week trial indicated that ct supplementation significantly reduced the incidences of colds and of fever symptoms , but had no apparent effect on the duration of colds .
these results suggest that ct supplementation may be useful for the prevention of the common cold . | the common cold is one of the most frequent illnesses caused by viral infection .
recently , we have reported that oral administration of cystine and theanine ( ct ) to mice enhanced the humoral immune response associated with antibody production .
based on this mouse study , we investigated the effects of ct supplementation on the common cold in humans as a pilot study .
a total of 176 healthy male volunteers were randomized to receive either placebo or ct ( 490 mg ) tablets twice daily for 35 days .
the incidence outcome was assessed using the definition in our laboratory based on questionnaires regarding cold symptoms .
the incidence of subjects with colds during the trial was significantly lower in the ct group than in the placebo group , although the duration of the colds was not significantly different between the groups .
these results suggest that ct supplementation may be useful for the prevention of the common cold . |
focused electron beam
induced deposition ( febid ) , which also goes
by the alternative names of focused electron beam induced processing
( febip ) or simply as electron beam induced deposition ( ebid ) , is a
promising technique for the direct writing of patterns
onto solid surfaces .
it operates at a resolution approaching 1 nm and
can be regarded as a variant of chemical vapor deposition in which
a focused beam of electrons induces decomposition in a volatile precursor ,
leading to deposition of the desired material . the deposition of metals
is commonly achieved in febid using volatile - metal - containing complexes ,
which , in addition to their high vapor pressures , are often relatively
easy to decompose because of low binding energies between the metal
atom and the surrounding ligands . while febid shows great promise ,
it also has limitations .
one problem
is the incorporation of nonmetallic impurities into metal deposits ,
which can arise for several reasons , including incomplete dissociation
of the precursor , recombination reactions on the substrate surface ,
or the electron - induced production of involatile nonmetallic species
such as carbon clusters .
another well - known problem is broadening
of the deposited structures relative to the diameter of the focused
electron beam , a process which is often ascribed to dissociative electron
attachment ( dea ) .
dea can give rise to a loss of pattern resolution
because low energy secondary electrons and electrons that are inelastically
backscattered from the substrate can attach to precursor molecules
outside of the primary excitation zone and then initiate decomposition
chemistry . as a result of these limitations and to try and find
ways of mitigating
them , it is important to understand the dea mechanisms for common
febid precursor molecules .
one such molecule is cobalt tricarbonyl
nitrosyl , co(co)3no , which has recently been used as an
alternative to co2(co)8 to deposit cobalt using
febid .
the deposition of cobalt nanostructures is particularly
interesting because of the ferromagnetic properties of this element ,
which might be exploited in devices such as nanoscale hall sensors and magnetic force microscopy tips .
the
dea mechanism for gaseous co(co)3no has recently been explored
for the first time by engmann et al .
absolute dea cross sections
were established in this work , and mechanistic information was extracted ,
including evidence that the first step involves predominantly co loss .
another recent study has investigated the electron - induced decomposition
of a nanofilm of co(co)3no on a solid substrate and employed
surface science techniques , such as x - ray photoelectron spectroscopy ,
to extract mechanistic information .
as
in the gas phase , decomposition on a surface seems to involve facile
loss of co. however , in the surface study , there was clear evidence
of formation of a nitride as the initial step , i.e.1the formation of a relatively
stable con entity is consistent with the observation that the level
of nitrogen in febid deposits derived from co(co)3no is
usually greater than carbon .
it should be noted that
the surface study by rosenberg et al . used electrons with 500 ev kinetic energy , which is much higher
than the electron energies used in the gas phase work by engmann et
al .
here , we adopt a different approach
for exploring the dea mechanism
of co(co)3no .
this involves isolating the precursor in
a liquid helium nanodroplet , where electrons of defined energy are
then subsequently added to induce chemistry .
one of the advantages
of working with helium droplets is the possibility of using the liquid
helium to rapidly quench reactions , thus making it more likely to
detect intermediate reaction products .
in addition , by increasing
the doping rate , it is straightforward to add multiple precursor molecules
to a helium nanodroplet , which makes it possible to see if the dea
mechanism is altered by clustering .
information of this type is potentially
highly significant , since , in febid , the decomposition is likely to
take place on a substrate surface and within a thin precursor film ,
rather than via individual isolated molecules in the gas phase .
thus , the current study can potentially bridge the gap between a
purely gas phase study of the dea mechanism of the co(co)3no monomer and a study of the decomposition of a thin co(co)3no film . a third advantage of working with helium nanodroplets ,
which we
also exploit here ,
is that it is possible to add another species to
the droplets to act as a model substrate on which to initiate chemistry .
in this study
we
find that the electron - induced decomposition of a single co(co)3no complex in a helium droplet has much in common with the
same molecule in the gas phase .
furthermore , the anion yields
as a function of electron energy change dramatically when switching
either to [ co(co)3no]n clusters
or to a single complex located on a c60 surface .
this suggests
that , while dea studies of the monomer are a valuable starting point ,
the dea behavior of febid precursor molecules on a solid surface will
differ substantially from the gas phase scenario .
full details of the experimental setup and the general
operating
procedure can be found elsewhere .
briefly ,
helium nanodroplets were produced by expanding high purity gaseous
helium at a stagnation pressure of 21 bar and a temperature of 9.4
k through a 5 m pinhole into a vacuum . under these conditions ,
the average number of helium atoms per droplet was in the region of
4 10 .
the expansion
was skimmed to form a collimated droplet beam , and this was then passed
through a pick - up cell containing vapor of co(co)3no ( strem
chemicals inc . , 99% stated purity ) .
the external reservoir in which
the co(co)3no was held was heated to roughly 320 k for
these experiments , and the pick - up conditions were such that acquisition
of a single co(co)3no molecule was more likely than pick - up
of two co(co)3no molecules .
experiments were also performed
where c60 molecules were added to the helium droplets immediately
after the addition of co(co)3no .
the c60 ( ses
research , 99.95% stated purity ) was heated to a temperature of 558
k and added to the droplets in a second pick - up zone .
after
picking up dopant molecules , the helium droplets passed through
a skimmer to enter another differentially pumped vacuum chamber , where
they were exposed to an electron beam of variable energy ( 0150
ev ) .
any ions produced were then extracted into a high resolution
and high repetition rate reflectron time - of - flight mass spectrometer
( tofwerk ) .
this system can be used to explore both cations and anions ,
but in this particular study , we concentrate on anions generated by
relatively low energy electrons . the electron energy was calibrated
using the anion yield data collected by engmann et al .
anion yield curves derived from clusters and from co(co)3no on c60 are hence referenced to gas phase measurements
and are , therefore , corrected for the excess energy needed to inject
electrons into liquid helium .
all anion yield curves
have been corrected for background counts and isotopic effects of
neighboring ions in the mass spectra .
figure 1 shows ion yield curves as a function of electron kinetic energy
for the eight anions detected . these range from the intact [ co(co)3no ] parent anion through a series of fragment
ions all the way down to co. all of these anions
the observation of the latter in the current
study is presumably the result of the very low temperature in a helium
droplet , which , combined with the rapid dissipation of heat through
evaporative loss of helium atoms , should
help to stabilize the anion .
nevertheless , [ co(co)3no ] is not an abundant product when compared to the anionic
fragments .
table 1 shows
relative integrated peak areas between 0 and 20 ev to enable a comparison
of ion abundances .
yields for the parent anion , [ co(co3no ] , and its fragments as a function of electron energy .
the signal / noise
ratio for the parent anion in the bottom right panel is very low ,
and so the solid line ( in red ) through the data points was generated
using data smoothing ( 15 pt fft filter ) to indicate how the yield
varies with electron energy .
to give some idea of the agreement between
the helium droplet findings and the gas phase results obtained by
engmann et al .
, raw data for the coco ion from the gas phase study have been incorporated into the upper
right panel ( shown by the data points marked in red ) .
the agreement
between the gas phase and helium droplet data for this ion is good ,
with only marginal broadening of the peak being seen in the helium
droplet experiment .
similarly , good agreement is obtained for several
other ions ( not shown here ) , and any exceptions are discussed in detail
in the main text .
there are considerable similarities
between the ion yield curves
shown in figure 1 and
those reported by engmann et al . in their gas phase studies , particularly with regard to the energy of peak ion production and
the widths of the observed resonances . however , there are also a couple
of significant differences , and it is these differences which we focus
upon here .
first , we see a marked change in the shape of the ion yield
profile for [ co(co)2no ] when compared
to the gas phase .
second , the most prominent anion seen in the helium
nanodroplet experiments is not [ co(co)2no ] , as reported by engmann et al . , but is instead [ co(co)2 ] . in all other respects , the agreement with engmann
et al .
two
clear resonances with similar
widths are seen at low energies , peaking at 1.6 and 4.3 ev .
although
two peaks were seen by engmann et al . , these were at much lower energies
( ca . 0 and 0.5 ev ) .
furthermore , in the gas phase , these two peaks
had very different widths , with the higher energy peak being far more
intense and much broader than the lower energy peak .
another major
difference is that we see a broad and monotonically increasing ion
yield above roughly 7 ev , whereas the gas phase work showed no production
of [ co(co)2no ] between 2 and 9 ev . at
least , in part , we attribute this difference in behavior to quenching
of reaction channels in helium droplets . in other words , resonances
which would lead to certain fragments in the gas phase
may be inhibited
by rapid cooling in the helium nanodroplets , giving peaks in the yield
of [ co(co)2no ] at energies that would
coincide with other reaction products if allowed to go to completion .
thus , the peak seen at 1.6 ev is close to the peaks seen for production
of [ co(co)3 ] and [ cocono ] , and thus quenching of these higher energy channels into the lower
energy [ co(co)2no ] channel might account
for this peak .
the peak at 4.3 ev overlaps with the low energy tail
of the [ cono ] resonance , perhaps indicating that
this channel is quenched on the low electron energy side of this tail ,
leading to [ co(co)2no ] production .
the
enhanced production of [ co(co)2no ] above
7 ev may be the result of fragmentation of the dimer , [ co(co)3no]2 , which shows much broader
ion yield curves ( see next section ) . the most surprising observation
for the monomer is that loss of
a single co molecule does not give the most prominent anion signal
for helium nanodroplets , in marked contrast to the gas phase case .
since the dea process with the lowest activation energy is expected
to be loss of a single co molecule , one might reasonably expect this
route to be enhanced in helium droplets relative to the gas phase
because of the quenching of secondary reactions .
instead , the combined
loss of no and a single co molecule is found to be the most probable
dea process . we have calculated the energy required to eject various
ligands from the parent anionic complex using the established quantum
chemical extrapolation scheme cbs - qb3 . in the cbs - qb3 model ,
geometries
are optimized and the zero - point energy is evaluated by vibrational
frequency calculations in the harmonic approximation using density
functional theory ( dft ) methodology .
the obtained geometries are then
used as a starting point for more accurate energy calculations , including
extrapolation to the complete basis set limit .
the results are summarized in table 2 , which shows the energy required to break
specific bonds in the parent anion at 0 k. the calculations conform
to expectation , with the channel leading to loss of a single co predicted
to have by far the lowest threshold energy , 0.65 ev .
the preferential loss of co rather than no in the gas phase is
presumably a consequence of statistical dissociation in the thermally
activated initial state of the anion .
however , the preferential ejection
of co + no rather than a single co molecule suggests that , in a helium
droplet environment , the reaction process becomes constrained by specific
dissociation dynamics .
one possibility is that this process is assisted
by a concerted rather than sequential loss of no and co from the parent
anion .
concerted dissociation dynamics in dea has been reported previously , although we are not aware of any prior examples
for metal - containing complexes .
similar observations
apply to the trimer , but the data in that case are of lesser quality
than those for the dimer .
the fragment ions detected are analogous
to those identified from the monomer but are now attached to an intact
co(co)3no unit ( which , for notational convenience , we will
often refer to simply as m in the discussion below ) .
the ion yield
profiles for these clusters are dramatically different from those
seen for the monomer . for the discussion that follows , we have assumed
that peaks corresponding to the ions [ ( m)n(x ) ] , where x is a fragment such as co or co(co)2 , derive from the neutral species ( m)n+1 .
this assumption is necessary for a simple interpretation
of the experimental data , but one should recognize that a cascade
effect from larger clusters could contribute to the observed signals .
this becomes particularly clear when looking at fragments of the form
[ ( m)nco2(x ) ] , which , for example ,
in the case of x = ( co)2no , are abundant and among the
most remarkable peaks in the mass spectra ( see figure 3 ) .
.
however , as figure 3 clearly illustrates , fragments originating from ( m)n+3 also play a visible role .
the yield curves in figure 3 show a convergence
in behavior with cluster size , suggesting that comparison with the
condensed phase is already viable at these cluster sizes .
yields for
the parent dimer anion , [ co(co)3no]2 ( here abbreviated as m2 ) , and smaller
fragments of the type [ mxnym ] as a function
of electron energy .
a 15 pt fft smoothing filter was used to generate
the solid line ( in red ) in the bottom right panel .
yields for the anions [ mnco2(co)2no ] , where m = co(co)3no and n = 1 , 2 , and 3 . a 20 pt fft smoothing
filter
was used to generate the solid line ( in red ) in the bottom right panel . the most striking difference between
the monomer ion yield plots
in figure 1 and the
dimer plots in figure 2 is that the latter are much broader and peak at far higher electron
energies ( in excess of 10 ev ) .
indeed , the anion yields arising from
the monomer and dimer have almost nothing in common .
even the reaction
probabilities are different , with the dimer no longer favoring loss
of no in preference to loss of a single co. instead , we see that the
most abundant products correspond to loss of 2co or co + no , with
nearly equal probabilities at their peak electron energies .
we still
find , however , that loss of a single co is not the most probable reaction
outcome .
the source of the very different anion yield curves
for the co(co)3no monomer relative to its oligomers is
not clear .
it is possible
that the oligomerization process has a profound impact on the electronic
structure , perhaps because the monomer units interact rather strongly .
in such cases ,
the electronic structure that must be considered is
that of the supermolecule entity , rather than the monomers . an estimate
of the dimerization energy of co(co)3no would help to establish
if this explanation is plausible , but this is beyond the scope of
the current study .
we must also consider the possibility that dissociative
ion - pair formation ( also known as dipolar dissociation ) becomes an
option for the oligomers .
an example would be the co - loss channel ,
the lowest energy dipolar dissociation channel , shown below:2since we do not know the
dimerization energy
of the neutral complex , we can not calculate an accurate threshold
energy for reaction 2 .
however , assuming a dimer binding energy of 1 ev and combining
this with the known first ionization energy of co(co)3no
( 8.33 ev ) , the calculated electron affinity
of co(co)3no extracted in the current study ( 0.75 ev ) ,
and the energy required to remove a co from the monomer anion shown
in table 2 , we obtain
an approximate energy threshold of 9.2 ev for reaction 2 .
this does not account for the tails to much
lower electron energies seen in all of the anion yield curves shown
in figure 2 , but it
does suggest that dipolar dissociation channels could come into play
at energies in the region of 10 ev . the dramatic difference
in dea behavior between the co(co)3no monomer and the dimer
is important because it shows that
the monomer is not a reliable guide to dea behavior in an agglomerated
sample such as a thin film , as would be the case in a real febid experiment .
one important consequence of the broad anion yield curves for ( co(co)3no)n clusters is that a much wider
range of electron kinetic energies can effect dea than is implied
by the quite strongly resonant behavior of the monomer .
accordingly ,
a much greater fraction of the available secondary electrons and backscattered
electrons will be able to initiate decomposition if they are able
to encounter a co(co)3no film rather than the gaseous monomer .
figure 4 shows the anion yield curves
for products derived from co(co)3no in combination with
a single c60 molecule .
the signal levels were not strong
for these anions , and so the signal - to - noise ratios seen in figure 4 are considerably
worse than those in figure 1 .
first ,
the ion yield profiles have more in common with those seen for the
[ co(co)3no]2 dimer than for the isolated monomer ,
in that the curves are very broad and peak at far higher electron
kinetic energies than for the monomer .
since c60 has a
reasonably high electron affinity ( 2.6835 0.0006 ev ) , which is comfortably in excess of that of
co(co)3no ( 0.75 ev according to cbs - qb3 calculations ) ,
low energy electrons might be expected to be preferentially injected
into the c60 electronic manifold . however , while very broad
anion yield curves have been recorded previously for c60 in helium , these peak near 56 ev .
the anion yield curves shown in figure 4 are different , suggesting that , at the very
least , there is funneling of excess energy into the co(co)3no adsorbate , which can then initiate chemistry .
however , another
possibility is ion - pair formation leading to production of c60 and the anion [ co(co)3no ] and its fragments ( dipolar dissociation ) , channels which will start
to become energetically accessible at energies roughly exceeding 9
ev .
a notable difference in the anion chemistry between the [ co(co)3no]2 dimer and the behavior of monomeric co(co)3no on c60 is that the loss of no on electron attachment
becomes a minor channel for co(co)3no on c60 .
we saw previously that , for [ co(co)3no]2 , the losses of 2co and no + co are the two most probable
reaction outcomes , with almost equal cross sections . furthermore ,
the loss of a single co molecule is still a minor channel for the
dimer .
the two most probable outcomes , with almost equal probabilities ,
are now for loss of either one or two co molecules on electron attachment
above 10 ev .
thus , when co(co)3no is subjected to electron
exposure when adsorbed on c60 , there is a far stronger
propensity to retain no than for either the isolated co(co)3no monomer or its dimer .
we are not in a position to establish
for certain why no retention
becomes more likely when co(co)3no is located on c60 . however , one possibility is that the c60 surface
might catalyze the dissociation of no to form a co
this suggestion is prompted by the observations by rosenberg et al .
on the electron - induced decomposition of co(co)3no on a
solid surface , as discussed earlier .
one can imagine that , once such dissociation has
occurred , the n and o atoms would be difficult to dislodge from the
cobalt , in contrast to molecular no
. it would be interesting to explore
whether or not this suggestion is applicable to co(co)3no on c60 , perhaps by carrying out detailed ab
initio calculations of the available reaction pathways and
how these are affected by contact with a carbonaceous surface . clearly ,
there are alternative explanations which might involve specific chemical
binding of the parent anion or a fragment to c60
. a neat
way to test this hypothesis would be to carry out similar experiments
using a metallic cluster as the model surface to see if the substrate
identity affects the dea chemistry .
the
effect of dissociative electron attachment to co(co)3no
in helium nanodroplets has been investigated for the first time .
the anion yield curves for the monomer show similarities to those
reported previously for gaseous co(co)3no . however , in
helium nanodroplets , there are three notable differences : ( 1 ) observation
of the parent anion , [ co(co)3no ] , albeit
at very low abundance , ( 2 ) different resonances in the channel producing
loss of a single co molecule , and ( 3 ) a surprising preference for
no loss rather than co loss . observations ( 1 ) and ( 2 ) can be accounted
for by quenching of certain reaction channels provided by the surrounding
liquid helium .
observation ( 3 ) seems to be an effect delivered by
constrained dynamics imposed by the liquid helium , although , at present ,
this is not fully understood .
ab initio calculations
to establish the relevant potential energy surfaces and barriers to
specific reaction channels would seem to be essential for resolving
this matter . for clusters of co(co)3no , the anion
yield curves shift
to higher electron energies and are much broader .
the reaction probabilities
also change , but the most dramatic effect is seen when co(co)3no is located on the surface of c60 .
it would be useful
for an improved understanding of the febid mechanism to understand
why the introduction of a surface induces this significant change
in branching ratios .
in particular , it will be important to establish
if the surface catalyzes the decomposition of no to form a cobalt
oxynitride unit , as previously suggested by rosenberg et al . | electron addition to cobalt tricarbonyl
nitrosyl ( co(co3no ) and its clusters has been explored
in helium nanodroplets .
anions
were formed by adding electrons with controlled energies , and reaction
products were identified by mass spectrometry .
dissociative electron
attachment ( dea ) to the co(co)3no monomer gave reaction
products similar to those reported in earlier gas phase experiments .
however , loss of no was more prevalent than loss of co , in marked
contrast to the gas phase . since the co
n bond is significantly
stronger than the co c bond , this preference for no loss must
be driven by selective reaction dynamics at low temperature . for [ co(co)3no]n clusters ,
the dea chemistry
is similar to that of the monomer , but the anion yields as a function
of electron energy show large differences , with the relatively sharp
resonances of the monomer being replaced by broad profiles peaking
at much higher electron energies . a third experiment involved dea
of co(co)3no on a c60 molecule in an attempt
to simulate the effect of a surface .
once again , broad ion yield curves
are seen , but co loss now becomes the most probable reaction channel .
the implication of these findings for understanding focused electron
beam induced deposition of cobalt is described . |
available data indicates that the prevalence of periodontal disease is very high in several african countries affecting all age groups .
libya is one of the north african country in the mediterranean region and sebha is the largest city situated in the south of the libyan arab jamahiriya and located in the middle of libyan desert .
people come to sebha from the various urban areas surrounding it and therefore it has a heterogeneous society .
information on the oral health status of the sebha population is scarce . to date , except for the report of hassan and leous , there is a paucity in publication concerning the oral health in general and the prevalence and severity of periodontal diseases in particular .
this data is stored in the who global oral data bank ( godb ) . the periodontal country profile for libya
is mentioned in the who data base , which is an extract of the 1982 - 1983 pathfinder study , conducted to assess the oral health situation in socialist people 's libyan arab jamahiriya .
it was conducted on 849 children and 269 adults from 14 sites of the following localities namely tripoli , sebha , benghazi , zwara , ajelat , and kaddah .
cpitn index was used to assess the periodontal disease and its results indicate that the periodontal disease is present from a very young age .
high prevalence of periodontal disease was the main cause of tooth loss in adulthood . among the 3544 years
ever since the completion of this study , two decades have passed and there is no new data which has been updated .
age [ 5 , 6 ] , gender [ 7 , 8 ] , toothbrushing [ 9 , 10 ] , and smoking [ 7 , 1113 ] are among the important risk factors for periodontal disease .
information of the periodontal status and additional information of risk factors can help the dental professional , to identify people in the high risk for periodontal disease and undertake strategic planning for a preventive and therapeutic treatment program . against this background
, the present study has been designed to assess the periodontal status and risk factors like age , gender , frequency of toothbrushing , and smoking of the adult population in sebha city , libya .
this study is a part of the extended project which was undertaken to evaluate the periodontal status and its determinants on a large scale amongst the population of sebha ( details are published elsewhere ) . here , data of the adult population aged 3554 years old is presented .
one who , index age group of 3544 years and other group of 4554 years which are representative of adult population was considered . for sampling , the who guidelines ( 1997 ) were followed .
4 sites were identified and 50 subjects per site for each of the 2 age groups were selected . an additional 10 subjects each were added to compensate for any non - participation and hence the sample was 480 ( 4 2 60 ) .
the survey was conducted at the primary health centre ( phc ) in al - jadeed , al - mahdia , gurda , and sukkara .
the medical officer posted at the phc was responsible to collect the participants for the study in the respective areas .
people from household , offices , and those visiting the phc comprised the study sample .
the participant was first interviewed to collect general information and data about the oral hygiene practices and smoking habits .
the participant was asked simple questions about the oral hygiene aids used ( toothbrush and others ) , use of toothpaste ( yes , no ) , frequency of toothbrushing ( once , twice , > twice ) , current smoker ( yes , no ) , and frequency of smoking ( 5 , 610 , 1120 , > 20/per day ) .
the main outcome measures of cpi are presence of gingival bleeding on gentle probing , dental calculus , and probing periodontal pocket depth ( ppd ) : 4 - 5 or 6 mm .
the index teeth were 16 , 17 , 11 , 26 , 27 , and 36 , 37 , 31 , 46 , 47 . when two index teeth were considered in a sextant , the tooth with the highest score was recorded .
the overall cpi score of the participant represented the value of the highest recorded code for that individual .
the examination was performed by a single , trained , calibrated examiner assisted by a recording clerk , using a cpi probe and mouth mirror . during calibration 10% of the sample was reexamined and kappa value of 0.8 was obtained which indicates a good intra - examiner consistency .
all the subjects were well educated and signed the informed consent form . the ethical board , scientific committee of the faculty of dentistry , sebha university , sebha , libya approved the protocol of the study .
statistical analysis was done with the help of a statistical package for social sciences ( spss version 16 ) .
chi - square test and anova were used to compare between the groups at 5% level of significance .
the variables were age , gender , frequency of toothbrushing , and smoking habit , which were compared with the cpi codes .
the sample size calculated was 480 , but the survey could be completed among 452 people , of which 266 ( 58.84% ) were females and 186 ( 41.16% ) were males .
249 ( 55.08% ) were in the age group of 3544 years and 203 ( 44.92% ) in 4554 years , respectively .
overall , 344 ( 76.15% ) reported use of toothbrush and paste , 45 ( 9.96% ) used finger , and 63 ( 13.93% ) used siwak . 225 ( 49.78% ) brushed once daily , 96 ( 21.23% ) brushed twice , and 21 ( 4.64% ) brushed more than twice .
19 ( 4.2% ) reported to have smoked 510 cigarettes , while 12 ( 2.65% ) smoked 1020/per day and 9 ( 1.95% ) smoked more than 20 .
majority , 52.65% ( n = 238 ) , were detected with shallow pockets ( cpi code 3 ) followed by 30.08% ( n = 136 ) with calculus ( code 2 ) , 12.17% ( n = 55 ) had deep pockets ( code 4 ) , 3.31% ( n = 15 ) had bleeding ( code 1 ) , and only 1.33% ( n = 6 ) were healthy ( code 0 ) .
figure 1 shows the distribution of the study sample according to the cpi codes .
table 1 shows the distribution of cpi codes in relation to the different variables considered in the study .
age , gender , current smoking status , and frequency of toothbrushing showed statistically significant difference with cpi codes .
table 2 shows that for 3544 years , the mean number of sextant for calculus was significantly higher as compared to the shallow pockets which was higher in 4554 years age group .
the mean ( sd ) cpi score for the entire sample was 2.71 ( 0.77 ) .
the mean cpi score was 2.57 ( 0.79 ) and 2.89 ( 0.72 ) for 3544 years and for 4554 years , respectively .
the present study is the first population - based survey among adult population in sebha in the recent times after the pathfinder survey conducted in 1982 - 1983 .
thorough training and calibration of the examiner ensured reliable recording of the cpi ( community periodontal index ) .
the major advantages of the cpi are simplicity , speed , reproducibility , and international uniformity .
although this index has certain shortcomings when used as a stand - alone means of assessing the extent and severity of periodontal disease it has been widely used for descriptive periodontal epidemiologic studies . in addition , the cpi data has been used for surveillance of periodontal health at country and intercountry levels . for the age group 3544 years , the most frequently observed condition was shallow pockets of 4 - 5 mm among 47.39% followed by calculus in 38.15% .
deep pockets of more than 6 mm were found in 8.84% of the subjects .
figure 2 shows the comparison of periodontal status for 3544 years old from emro countries with the present study . for the countries like sudan , iran , egypt , morocco , and libya shallow pockets
. however , calculus ( code 2 ) as the highest score was reported for countries like syrian arab rep . ,
the highest percentage of healthy periodontium ( code 0 ) was reported for saudi arabia ( 20% ) .
34% libyans and 26% sudanese were among the ones to have high percentage of deep pockets ( code 4 ) .
in a recent study among sudanese , 36.1% had healthy periodontal tissues , 10.9% bleeding , 42.0% calculus , 8.5% 4 - 5 mm periodontal pocketing , 0.7% periodontal pocketing of 6 mm , and 1.8% excluded sextants .
the sudanese have better periodontal health as compared to the present study population . when compared with the 1982 - 1983 pathfinder survey in libya , a high percentage of people had shallow pockets and deep pockets versus the present study where percentage of shallow pockets and calculus was more . even after 20 years
, there is no improvement in the percentage of healthy periodontium ( 0% versus 1.33% in the present study ) but the prevalence of severe form of periodontitis ( deep pockets > 6 mm ) has been reduced from 34% to 12% in the present study .
it is widely assumed that symptoms of periodontal diseases escalates with age . in the present study ,
percentage of subjects with healthy periodontium , bleeding , and calculus was comparatively more in the 3544 years age group , while signs of periodontal destruction , that is , shallow pockets , and deep pockets were on a higher side among 4554 years old ( figure 1 ) . in the present study , there was a significant statistical difference observed between the cpi codes and gender .
the percentage calculation showed that the percentage of healthy periodontium and bleeding were higher among females and percentage of deep pockets were more prevalent among males .
this may be because the females give more importance to aesthetics and have the habit of toothbrushing and seeking dental care regularly .
smoking can cause a number of changes within the periodontium which can predispose an individual to the progression of periodontal disease . in a study ,
5.5% of smokers had healthy periodontium compared to 44.2% among non - smokers and the periodontal condition of the smokers was very poor compared with that of non - smokers and this difference was statistically significant .
literature indicates that predominantly males , due to their smoking habit , have shown more susceptibility to periodontal diseases .
the smokers in the present study were all males ; significant difference in periodontal status was observed with the current smoking status , but when the data was stratified according to the frequency of smoking no statistically significant difference was found with the cpi codes .
in the present study , 21.23% people brushed twice daily in comparison to 53% sudanese and 32% chinese .
lang et al . showed that frequent tooth brushing and flossing as well as regular dental attendance were found to be significantly associated with lower plaque , gingivitis , and calculus scores .
studies have reported that in saudi arabia , 65% urban population used siwak on a daily basis .
miswak ( siwak ) is known to have antibacterial effect on pathogens causing periodontal disease and caries .
this study shows that more than half of the adult population in sebha are detected with the signs of destructive periodontitis .
if this trend continues , in the coming years the severity of periodontal disease is bound to increase enormously . in view of the potential association of periodontal disease with systemic disease ( atherosclerosis , myocardial infarction , stroke , pneumonia , diabetes mellitus , and pregnancy ) , this current health situation is of public health significance especially among adults and needs urgent attention .
hence , the study results can be utilized to identify individuals at risk of periodontal disease and to target population level interventions . | the present study was aimed at assessing the periodontal status and risk factors like age , gender , tooth brushing habit , and smoking among the adult population of sebha city , libya .
452 adults , aged 3554 years , comprised the study sample .
266 ( 58.84% ) were females and 186 ( 41.15% ) were males .
data was collected by interview and clinical examination using cpi of cpitn index .
chi - square test and anova were used for statistical analysis at 5% level of significance .
results indicate that 76.32% used toothbrush and paste ; 8.84% were current smokers and were all males .
majority , 52.65% were , detected with shallow pockets followed by 30.08% with calculus , 12.17% had deep pockets , 3.31% had bleeding , and only 1.33% were healthy .
age , gender , current smoking status and frequency of tooth brushing showed statistically significant difference with cpi codes .
health professionals can utilize this data to identify individuals at risk and to target population level interventions . |
malignant cells infiltrate the subarachnoid space and cerebral pia mater , causing meningeal irritation and cranial nerve symptoms .
mc is frequently diagnosed in patients with breast cancer , lung cancer , lymphoma or leukemia as well as tumors of the central nervous system ( cns ) , mostly medulloblastoma .
intrathecal chemotherapy is effective in patients with mc from lymphoma , leukemia and breast cancer .
however , intrathecal treatment requires invasive procedures , preferably repeated lumber puncture , and limits the use of chemotherapy agents due to the cerebrospinal damage .
a 60-year - old postmenopausal woman was referred to our hospital due to the tumor in the right breast , in april 2008 .
she was diagnosed with stage iib breast cancer ( ct3n0m0 , invasive lobular carcinoma ) , and received 12 cycles of weekly paclitaxel 80 mg / m , with fec ( 5-fluorouracil 500 mg / m , epirubicin 100 mg / m , cyclophosphamide 500 mg / m ) as a neoadjuvant chemotherapy ( nac ) .
pathological examination showed the breast cancer to be node - negative , estrogen receptor - positive ( 99% ) , progesterone receptor - negative , and human epidermal growth factor receptor type 2 ( her2)-negative
, she developed a tongue deviation ; magnetic resonance imaging ( mri ) showed multiple leptomeningeal metastases ( fig .
serum cancer antigen 15 - 3 ( ca15 - 3 ) and carcinoembryonic antigen ( cea ) levels increased from 7.0 iu / ml and 0.3 ng / ml to 96.0 iu / ml and 3.5 ng / ml , respectively .
cerebrospinal fluid ( csf ) analysis was not performed ; mc was diagnosed through positive imaging and neurological symptoms . based on the diagnosis of mc , she received capecitabine monotherapy ( 2,400 mg / day ) . only 3 weeks later , her tongue deviation disappeared .
since she had started receiving capecitabine , ca15 - 3 and cea levels gradually reduced ( fig .
in america and european countries , breast cancer is most frequently associated with mc among extra - cns solid malignancies .
about 25% of patients with breast cancer develop mc , usually late in the course of their metastatic disease . on the other hand , in japan , gastric cancer ( 55% ) and lung cancer ( 30% )
are highly associated with mc , but mc from breast cancer is extremely rare ( 1.7% ) .
there are no distinct reasons for this difference , which may be attributed to the lower disease rate of breast cancer itself , or the lower incidence of invasive lobular carcinoma of all histologic subtypes of breast cancer in japan .
gauthier et al . reported a high incidence ( 28% ) of invasive lobular carcinoma in mc from breast cancer [ 2 , 7 ] . by hematogenous spread or direct extension from bone metastasis or brain metastasis
the most common sign is multiple cranial nerve palsies , but headache , backache , polyradiculopathies , incontinence , confusional state , lower motor neuron weakness and sensory abnormalities may be the principal manifestations [ 4 , 8 ] .
although mc from breast cancer shows the best prognosis among mc from various malignancies , median overall survival is no more than 6 months following diagnosis of mc from breast cancer .
treatment of mc relies mainly on intrathecally and/or systemically delivered chemotherapy , combined with other treatment modalities , such as irradiation or debulking surgery of meningeal metastases .
there are no standard chemotherapy regimens for mc because neither prospective nor retrospective studies have shown any anticancer drugs to be highly effective against mc .
there are reports of breast cancer - derived mc that responded to hormonal manipulation alone [ 2 , 8 ] , and some cases that demonstrate the efficacy of intrathecal trastuzumab against mc from her2-positive breast cancer [ 10 , 11 ] .
the present case was hormone - resistant and her2-negative breast cancer , which limited options for chemotherapy .
there is only 1 case report describing the efficacy of capecitabine monotherapy against mc from breast cancer .
capecitabine is an oral prodrug of fluorouracil used in the palliative treatment of metastatic breast cancer that is resistant to taxane and anthracycline chemotherapy .
animal studies demonstrate that capecitabine and its metabolites cross the blood - brain barrier in limited quantities . a study in humans using a fluoropyrimidine similar to capecitabine found that fluorouracil concentrations in csf were only 13% of plasma concentrations .
we do not know the mechanism of response to capecitabine in our case , but a similar rapid and durable response to capecitabine was reported in a patient with refractory brain metastasis from breast cancer .
it is possible that diffuse mc in our case disrupted the blood - csf barrier and allowed a higher fluorouracil concentration in the csf .
since most patients with mc from breast cancer have various prior therapies and poor performance status , capecitabine monotherapy is a feasible approach in the treatment of mc from breast cancer . | a 62-year - old woman with breast cancer received neoadjuvant chemotherapy followed by breast - conserving surgery and sentinel node biopsy . during adjuvant endocrine therapy with aromatase inhibitor
, she developed multiple bone metastases .
thereafter , she received tamoxifen and zoledronate therapy . in may 2011
, she developed a tongue deviation and was diagnosed as having meningeal carcinomatosis .
the tongue deviation disappeared 3 weeks after taking capecitabine ( 2,400 mg / day ) .
magnetic resonance imaging of the brain showed regression of meningeal carcinomatosis .
levels of tumor markers cea and ca15 - 3 changed from 96.0 iu / ml and 3.5 ng / ml to 47.0 iu / ml and 1.5 ng / ml , respectively .
progression - free survival with capecitabine monotherapy was 5 months . |
a replication - deficient recombinant adenovirus expressing human hsp72 was constructed using the adeasy system ( qbiogene , carlsbad , ca , usa ) .
the cdna for hsp72 ( approved gene symbol hspa1a ) was amplified by reverse transcription - polymerase chain reaction ( pcr ) using total rna extracted from hela cells that were previously heat shocked at 42 for 30 minutes .
the amplified product was cloned into the pshuttle - cmv vector and verified by sequencing .
the vector was then transfomed into escherichia coli strain bj5183 together with the padeasy-1 vector that contains the entire adenovirus serotype 5 genome except the e1 and e3 regions .
the recombinant adenoviral constructs were selected and transfected into the hek-293 packaging cell line to produce viral particles .
viral titers were determined by limiting dilution on 293 cells and the absence of the e1a gene in the viral constructs was confirmed by pcr . the rat retinal ganglion cell line , rgc-5 ,
neeraj agarwal ( university of north texas health science center , fort worth , tx , usa ) .
rgc-5 cells were maintained in medium containing dulbecco 's modified eagle 's medium ( life technologies , rockville , md , usa ) , 10% fetal bovine serum , and antibiotics at 37 in a humidified 5% co2 - 95% air atmosphere .
rgc-5 cells grown to 70% confluence in a 24-well plate were transduced with a recombinant adenovirus expressing hsp72 for 2 hours at a multiplicity of infection ( moi ) of 5 to 50 plaque forming units ( pfu ) per cell .
forty - eight hours after transduction , adherent cells were lysed with 0.4 ml of 1 laemmli sample buffer and boiled for 5 minutes .
the protein samples were resolved by sodium dodesyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane .
the membrane was blocked with 5% skim milk overnight , incubated for 2 hours with anti - hsp72 antibody ( stressgen , victoria , canada ) , and reacted for 2 hours with peroxidase - conjugated anti - mouse antibody ( amersham , buckinghamshire , uk ) , followed by being probed with a chemiluminescence kit ( amersham ) .
rgc-5 cells grown in a chamber slide were transduced for 2 hours at an moi of 20 pfu per cell .
forty - eight hours post - transduction , the cells were fixed with 4% paraformaldehyde , permeabilized with 0.5% triton x-100 , and blocked with 2% bovine serum albumin .
the cells were then incubated for 2 hours with anti - hsp72 antibody , reacted for 2 hours with alexa fluor 568-conjugated goat anti - mouse igg ( molecular probes , eugene , or , usa ) , and counterstained with 4',6-diamidino-2-phenylindole .
rgc-5 cells were dispensed in a 96 well plate at a concentration of 1 10 cells per well and transduced for 2 hours at an moi of 5 and 20 pfu .
the cells were washed , replenished with 100 l of media containing 5 to 20 m etoposide and cultured further .
after 48 hours , 10 ul of reagent for used in the cytotoxicity assay ( wst-8 ; dojindo , rockville , md , usa ) per well was added , and the absorbance at 450 nm was read by an automatic microplate reader after 1 hour of incubation .
a replication - deficient recombinant adenovirus expressing human hsp72 was constructed using the adeasy system ( qbiogene , carlsbad , ca , usa ) .
the cdna for hsp72 ( approved gene symbol hspa1a ) was amplified by reverse transcription - polymerase chain reaction ( pcr ) using total rna extracted from hela cells that were previously heat shocked at 42 for 30 minutes .
the amplified product was cloned into the pshuttle - cmv vector and verified by sequencing .
the vector was then transfomed into escherichia coli strain bj5183 together with the padeasy-1 vector that contains the entire adenovirus serotype 5 genome except the e1 and e3 regions .
the recombinant adenoviral constructs were selected and transfected into the hek-293 packaging cell line to produce viral particles .
viral titers were determined by limiting dilution on 293 cells and the absence of the e1a gene in the viral constructs was confirmed by pcr .
the rat retinal ganglion cell line , rgc-5 , was originally provided as a gift from dr .
neeraj agarwal ( university of north texas health science center , fort worth , tx , usa ) .
rgc-5 cells were maintained in medium containing dulbecco 's modified eagle 's medium ( life technologies , rockville , md , usa ) , 10% fetal bovine serum , and antibiotics at 37 in a humidified 5% co2 - 95% air atmosphere .
rgc-5 cells grown to 70% confluence in a 24-well plate were transduced with a recombinant adenovirus expressing hsp72 for 2 hours at a multiplicity of infection ( moi ) of 5 to 50 plaque forming units ( pfu ) per cell .
forty - eight hours after transduction , adherent cells were lysed with 0.4 ml of 1 laemmli sample buffer and boiled for 5 minutes .
the protein samples were resolved by sodium dodesyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane .
the membrane was blocked with 5% skim milk overnight , incubated for 2 hours with anti - hsp72 antibody ( stressgen , victoria , canada ) , and reacted for 2 hours with peroxidase - conjugated anti - mouse antibody ( amersham , buckinghamshire , uk ) , followed by being probed with a chemiluminescence kit ( amersham ) .
rgc-5 cells grown in a chamber slide were transduced for 2 hours at an moi of 20 pfu per cell .
forty - eight hours post - transduction , the cells were fixed with 4% paraformaldehyde , permeabilized with 0.5% triton x-100 , and blocked with 2% bovine serum albumin .
the cells were then incubated for 2 hours with anti - hsp72 antibody , reacted for 2 hours with alexa fluor 568-conjugated goat anti - mouse igg ( molecular probes , eugene , or , usa ) , and counterstained with 4',6-diamidino-2-phenylindole .
rgc-5 cells were dispensed in a 96 well plate at a concentration of 1 10 cells per well and transduced for 2 hours at an moi of 5 and 20 pfu .
the cells were washed , replenished with 100 l of media containing 5 to 20 m etoposide and cultured further .
after 48 hours , 10 ul of reagent for used in the cytotoxicity assay ( wst-8 ; dojindo , rockville , md , usa ) per well was added , and the absorbance at 450 nm was read by an automatic microplate reader after 1 hour of incubation .
to express hsp72 exogenously in rgc-5 cells that are not easily transfected by conventional methods , the cells were transduced with a recombinant adenovirus expressing human hsp72 .
western blot analysis showed that protein bands of 72 kda corresponding to hsp72 were clearly detected in lysates of cells transduced with a recombinant adenovirus expressing human hsp72 .
the intensities of the bands were found to be in proportion to the degree of viral transduction . in untransduced control cells ,
immunocytochemistry yielded consistent results showing that cytoplasmic staining for characteristic hsp72 expression could be detected exclusively in transduced cells but not in untransduced cells , confirming the adenovirus - mediated hsp72 gene expression ( fig .
1b ) . to examine the effect of hsp72 on the death of rgc-5 cells caused by apoptosis ,
the cells were treated with etoposide immediately after viral transduction . compared with control cells ( fig .
2a ) , early cell death was seen in a small proportion of cells in the culture without serum ( fig .
2b ) , and the degree of cell death was accelerated by the treatment of etoposide ( fig .
. however , the etoposide - induced cell death appeared to be blocked by hsp72 expression , as observed by the enhanced viability of cells that were transduced immediately before etoposide treatment ( fig .
to confirm the effect of hsp72 on the survival of rgc-5 cells , a cell proliferation assay was performed on cells treated with etoposide following viral transduction ( fig .
the rate of viable cells was reduced by 44.8 6.7% and 80.8 9.2% relative to control cells at 5 and 20 m etoposide , respectively .
viral transduction itself might be toxic to cells because cell viability was slightly decreased when hsp72 was expressed in control cells .
the hsp72 expression , however , seemed to be protective to cells undergoing apoptosis in that the rate of cell death in the cultures exposed to etoposide was progressively reduced as the degree of viral transduction increased . at the highest level of hsp72 expression ,
the number of viable cells recovered was increased by 24.0 3.1% and 94.1 10.3% over that in the control cultures at 5 and 20 m etoposide , respectively .
in this study , we addressed whether hsp72 can protect rgc-5 cells from apoptotic cell death .
rgc-5 cells are the only immortalized rgc cell line established to date and are used widely in glaucoma research , although it is uncertain whether the cell line is of mouse or rat origin . to investigate directly the effect of hsp72 on the death of rgc-5 cells , it is necessary to express the protein exogenously in the cells .
to overcome this obstacle , we constructed a recombinant adenovirus in which a human hsp72 cdna is under the control of the human cytomegalovirus promoter for its constitutive expression and demonstrated that the viral vector was successful in both transducing rgc-5 cells and expressing its transgene inside the cells .
we showed that the degree of cell death by etoposide was inversely proportional to the degree of viral transduction in the cells .
etoposide , an inhibitor of topoisomerase ii , is a well - known apoptosis - inducing reagent .
therefore , our results present direct evidence for the cytoprotective ability of hsp72 against apoptotic cell death .
the protective role of heat shock was first described in light - damaged rat retina and hsp72 protein was identified as the entity primarily responsible for the response .
a growing body of evidence has accumulated that induction of hsp72 is essential for neuronal tolerance to a wide variety of stressful conditions including ischemic or excitotoxic insults , seizure , oxidative stress , and apoptotic stimuli .
the present study demonstrates that hsp72 expression itself is sufficient to protect rgc-5 cells from apoptotic cell death .
apoptosis , or programmed cell death , of rgcs is one of the earlier signs of the pathogenesis of glaucoma . because inhibition of rgc death or a halt of its progression is the key to glaucoma treatments , the ability of hsp72 to intervene in the process of apoptosis is attractive as a novel therapeutic intervention in glaucoma .
gene therapy , which is a practical alternative to current therapies that are limited to reduction of elevated intraocular pressure , is an approach by which hsp72 could be useful in a clinical context .
recent studies reported that hsp72 induction by zinc or geranylgeranylacetone administration increased rgc survival in a rat glaucoma model , suggesting that hsp72 could be therapeutic for glaucoma .
the eye may perhaps be the first organ for which gene therapy is used routinely in a clinical setting because it has advantages such as well - defined anatomy with small size , translucent media allowing excellent visual localization , and easy accessibility by which therapeutic agents can be delivered to the vicinity of cells with a particular defect .
the replication - deficient adenovirus generated in this study is a sophisticated gene delivery tool and such viral - based vectors are expected to have enormous potential as a mammalian expression system for gene therapy .
recently , it has been reported that hsp72 can be identified in the extracellular compartments and the extracellular hsp72 acts as a cytokine that induces inflammation and modulates the innate immune response [ 17 - 20 ] .
the dual role of hsp72 as a molecular chaperone and cytokine suggests that fine tuning its expression may be a potential route for a novel therapeutic approach to glaucoma and other neurodegenerative diseases . | purposeto assess whether the expression of heat shock protein 72 ( hsp72 ) protects rat retinal ganglion cells ( rgc-5 ) from apoptotic cell death.methodshsp72 expression in rgc-5 cells transduced with replication - deficient recombinant adenovirus was analyzed by western blot analysis and immunofluorescence .
the effect of hsp72 expression on etoposide - induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay.resultswestern blot analysis and immunofluorescence clearly showed adenovirus - mediated hsp72 expression in rgc-5 cells .
treatment with etoposide resulted in the death of a proportion of the cells by apoptosis .
however , this apoptotic cell death was significantly reduced in cells expressing hsp72 , with the reduction in cell death correlating to the level of hsp72 expression.conclusionsover-expression of hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death , suggesting that fine - tuning its expression may be an effective neuroprotective approach in retinal degenerative disease . |
the inferior alveolar ( ian ) and lingual nerves can be injured by many dental or maxillofacial surgical procedures involving the mandible ( third molar extraction , placement of endosseous implants , excision , osteotomy ) . due to compression , stretching , or laceration of the alveolar or lingual nerves during surgical steps , some patients complain of sensory disturbances such as pain , paresthesia , dysesthesia and hypoesthesia .
these sensory disturbances , which may involve the chin , lower lip , gums , and tongue , are unpleasant conditions that often cause litigation . the masseter inhibitory reflex ( mir ) , also called exteroceptive suppression , is the most used neurophysiological tool for investigating function of the third trigeminal division and mandibular nerves .
it consists of a reflex inhibition of the jaw - closing muscles elicited by peri- or intraoral electrical stimulations .
mir comprises an early and a late phase of suppression in the ipsilateral and contralateral masseter muscles .
these silent periods are mediated by non - nociceptive a - beta afferents through oligosynaptic ( sp1 ) and polysynaptic ( sp2 ) circuits in the brainstem .
our aim was to assess the mir sensitivity and specificity in patients with iatrogenic damage to the ian or lingual nerves , and thus to understand to what extent mir testing may be used for legal purposes .
in a blinded , controlled study , we recorded the mir in 160 consecutive patients ( 49 f , 111 m ; mean age 44.4 13.5 years ) who underwent dental or surgical procedures and thereafter reported sensory disturbances in the territory of the mandibular ( ian or lingual ) nerves ( third molar extraction : 81 ; dental implants : 37 ; mandibular surgery : 22 ; multiple procedures : 20 ) .
all patients were clinically stabilised ; the time elapsing between injury and our examination ranged between 2 months and 9 years . diagnosis of iatrogenic lesion to the mandibular nerves was based on clinical history and examination .
two physicians independently assessed the patients . only patients with a concordant diagnosis of mandibular nerve lesion
patients were clinically examined for negative ( tactile , pinprick , and thermal hypoesthesia ) and positive symptoms ( pain , dysesthesias , mechanical allodynia , and pinprick hyperalgesia ) .
the mir was recorded according to the recommendations of the international federation of clinical neurophysiology ( ifcn ) .
briefly , subjects were instructed to clench their teeth at maximum strength with the aid of auditory feedback .
emg signals were recorded through surface electrodes from the masseter muscles bilaterally ( active electrode over the lower third of the muscle belly and reference electrode about 2 cm below the angle of the mandible ) .
the mental nerve was stimulated transcutaneously with the cathode over the mental foramen and the anode 1 cm laterally .
the lingual nerve was stimulated through two adhesive surface electrodes attached 1 cm on the lateral margin of the tongue ( fig . 1 ) .
while the subjects were biting in the intercuspal position an electrical square - wave pulse ( 0.1 ms ) was delivered .
stimulus intensity was adjusted to 2.5 times the reflex threshold on the unaffected side ( 1545 ma ) and kept equal on both sides .
in each condition ( mental and lingual stimulations ) , eight trials per side were recorded .
two of the authors , blind to the side of damage , took the measurements.fig .
1masseter inhibitory reflex ( mir ) in patients with iatrogenic damage to the mandibular nerves . left column inferior alveolar dental nerve testing .
top schematic drawings showing cathode ( ) and anode ( + ) electrodes position for stimulation of the mental nerve ( a ) and tongue ( b ) .
midand bottom traces early ( sp1 ) and late ( sp2 ) components of the mir after contralateral and affected side stimulations in two representative subjects .
note that on the affected side the sp1 response is slightly delayed after mental stimulation and it is almost completely absent after tongue stimulation masseter inhibitory reflex ( mir ) in patients with iatrogenic damage to the mandibular nerves . left column inferior alveolar dental nerve testing .
top schematic drawings showing cathode ( ) and anode ( + ) electrodes position for stimulation of the mental nerve ( a ) and tongue ( b ) .
midand bottom traces early ( sp1 ) and late ( sp2 ) components of the mir after contralateral and affected side stimulations in two representative subjects .
note that on the affected side the sp1 response is slightly delayed after mental stimulation and it is almost completely absent after tongue stimulation to evaluate the diagnostic accuracy of mir , we chose to measure only the early sp1 component .
the short - latency response sp1 is far more accurate than the long - latency response sp2 , because it is supplied by fewer reflex afferents .
additionally , due to the higher number of synapses in the reflex circuit , the late sp2 component is comparatively unstable and is strongly modulated by suprasegmental influences , including psychological factors [ 3 , 57 ] .
because sp1 data had a normal distribution but unequal variances , we analyzed the latency difference between normal and affected side with the t test with welch s correction .
the reflex was considered abnormal when absent or when the sp1 latency difference between normal and affected side was greater than 1.2 ms . to calculate sensitivity and specificity
possible associations between pain and reflex abnormality and between pain and sensory deficits were evaluated by chi square test .
r correlation coefficient was used to assess correlation between the delay since injury ( months ) and the severity of nerve damage ( latency difference between affected and contralateral side ) .
the patients had damage to the ian ( n = 109 ) , the lingual ( n = 35 ) , or both nerves ( n = 16 ) . on the unaffected side ,
the reflex latency after stimulation of the mental nerve was within the normal range found in 100 healthy subjects in the same laboratory and similar to normal values reported in the literature [ 69 ] .
the mean latency was longer after lingual than mental stimulation ( table 1 ) .
both after mental and lingual stimulation , the latency was significantly longer on the affected than the normal side ( p < 0.0001 ) ( fig . 1 ; table 1).table 1latency of the masseter inhibitory reflex ( mean sd ) in 160 patients with iatrogenic damage to the mandibular nervesstimulation siteaffectedcontralateralp*inferior alveolar nerve ( n = 125)12.0 1.311.3 0.8<0.0001lingual nerve
( n = 51)14.3 2.612.9 1.7<0.0001p * t test with welch s correction latency of the masseter inhibitory reflex ( mean sd ) in 160 patients with iatrogenic damage to the mandibular nerves p * t test with welch s correction concerning the overall diagnostic accuracy , the mir was abnormal in 90 nerves and normal in 86 on the affected side whereas on the contralateral side it was abnormal in 2 and normal in 174 , which resulted in a strong association ( p < 0.0001 ; fisher s exact test ) , with 51% sensitivity ( ci : 0.4350.587 ) and 99% specificity ( ci : 0.9590.999 ) .
we did not find a significant correlation between the delay since injury and the severity of nerve damage as assessed by the side asymmetry between affected and contralateral side ( p > 0.10 ; r = 0.1483 ; spearman s correlation coefficient ) .
there was no significant association between pain and reflex abnormalities or between pain and sensory deficits ( p > 0.10 ) .
our study in a large cohort of patients now shows the diagnostic accuracy of the mir , a standard neurophysiological tool , in demonstrating iatrogenic damage to the mandibular nerves .
we found that the mir had 99% specificity and 51% sensitivity , i.e. mir testing reliably demonstrates nerve damage beyond doubt , whereas the finding of a normal mir is by no means sufficient to exclude nerve damage .
probably the dysfunction of a small number of nerve fibres , insufficient to produce a mir abnormality , may still engender important sensory disturbances .
we propose that mir testing , when used for legal purposes , be considered reliable in one direction only , i.e. abnormality does prove nerve damage , normality does not disprove it .
although it would be reasonable to expect some degree of recovery with time , because of spontaneous healing and reinnervation , we found no correlation between the delay since injury and mir abnormality .
this is probably due to the characteristics of our patient sample : all patients were examined when at least 2 months had elapsed from injury ( i.e. when no further healing is expected to occur ) and 146 out of 160 were examined beyond 4 months ( i.e. when , given the short length of the lingual and inferior dental nerves , the process of reinnervation was completed ) .
given the importance of this aspect , however , we are now planning a study in the acute phase after injury .
other neurophysiological tests may also assess mandibular nerve function : trigeminal - evoked potentials , nerve conduction study , and blink reflex .
although some authors used trigeminal somatosensory - evoked potentials to study ian injuries after surgical procedures , the reliability of these signals has been questioned because , rather than reflecting genuine brain activity , probably they result from volume - conducted muscle signals , as they disappear in the curarized subject [ 10 , 11 ] .
consistently , the ifcn recently recommended to investigate the trigeminal function with reflex rather than evoked potential studies .
the ian nerve conduction study is a reliable method , but it is undeniably invasive ( the recording needle - electrode is inserted below the zygomatic arc to a depth of about 4.5 cm ) . unlike the above techniques , the blink reflex after mental or lingual stimulation seems a promising alternative to the mir .
it has been widely studied in patients with orofacial pains or trigeminal neuropathy and , in combination with the ian conduction study , showed sensitivity ( 59% ) and specificity ( 100% ) values very similar to those we found in our patients .
the patients with iatrogenic damage after dental procedures , however , still represent a comparatively small sample [ 13 , 14 ] .
damage to the lingual nerve after third molar extraction can also be tested with an interesting method , very similar to ours ( even though the main measure is not the reflex latency ) .
also in this case , the number of patients is still too small to test diagnostic accuracy . whereas most patients had hypoesthesia , less than one - third had pain , probably because mechanical injury mainly damages large myelinated , non - nociceptive fibres , and tends to spare small nociceptive fibres .
accordingly , because mir is mediated by non - nociceptive a fibres , and it does not provide any information on nociceptive pathways , we found that mir abnormalities were unrelated to pain .
alternatively , the complexity of pain mechanisms at orofacial level simply makes it unreasonable to expect a direct correlation between pain and number of damaged primary afferents .
in particular , we can not exclude that plastic changes in the central nervous system contributed to the sensory disturbances reported by our patients . according to this hypothesis ,
sensory disturbances could be triggered by the nerve damage and persist after nerve recovery due to central mechanisms such as central sensitization . | iatrogenic injury of the inferior alveolar or lingual nerves frequently leads to legal actions for damage and compensation for personal suffering .
the masseter inhibitory reflex ( mir ) is the most used neurophysiological tool for the functional assessment of the trigeminal mandibular division .
aiming at measuring the mir sensitivity and specificity , we recorded this reflex after mental and tongue stimulations in a controlled , blinded study in 160 consecutive patients with sensory disturbances following dental procedures .
the mir latency was longer on the affected than the contralateral side ( p < 0.0001 ) .
the overall specificity and sensitivity were 99 and 51% .
our findings indicate that mir testing , showing an almost absolute specificity , reliably demonstrates nerve damage beyond doubt , whereas the relatively low sensitivity makes the finding of a normal mir by no means sufficient to exclude nerve damage .
probably , the dysfunction of a small number of nerve fibres , insufficient to produce a mir abnormality , may still engender important sensory disturbances .
we propose that mir testing , when used for legal purposes , be considered reliable in one direction only , i.e. abnormality does prove nerve damage , normality does not disprove it . |
human brucellosis remains the most common zoonotic disease worldwide , with more than 500,000 new cases annually 1 .
it is caused by gram - negative bacteria , brucella spp . and is transmissible to humans through direct contact with infected animals , consumption of dairy products , or inhalation of aerosols 2 .
its main clinical signs are fever , headache , anorexia , fatigue , arthritis , hepatosplenomegaly , and neurological signs 2 .
the disease represents serious consequences for public health by long treatment , slow recovery and possible serious sequelae in the locomotive and nervous system 2 . although brucellosis has been eradicated in many northern european countries , in australia , new zealand , and canada due to the implementation of national surveillance program and vaccination of livestock , it is still hyperendemic in the mediterranean basin , middle east , southwest asia and parts of latin america 1,3 . in turkey , brucellosis is common , especially in east and southeast anatolia regions 4,5 . among high - risk patients in the eastern part of turkey ,
seropositivity has been reported to be as high as 27.2% 6 , but there have been no extensive studies done on the identification of brucella species in this hyperendemic part of anatolia .
the genus brucella is an intracellular bacterial pathogen that infects host macrophage cells . in consequence ,
specialized agents that are able to penetrate the macrophages and function within their cytoplasm are required for the treatment of brucellosis 2 .
therefore , a limited number of antibiotics are effective against these organisms . in 1986 , the who has released recommendations for use of doxycycline , combined with either rifampin or streptomycin for treating human brucellosis 7 .
although this recommendation is still in function and brucella isolates are generally considered susceptible to the recommended by the who antibiotics , sporadic cases of a kind of antibiotic resistance have been reported 8,9 .
up until 2006 , in vitro antimicrobial suspectibility testing of brucella spp is not standardised and not generally recommended due to risk of laboratory - acquired infection and requirement of biological safety level 3 precautions , so there are few studies on this issue in the literature 8 - 16 .
furthermore in vitro susceptibilities of these antibiotics may change over time and from one geographical region to another 17,18 .
the side - effects of drug combination schemes , and the high incidence of relapses and therapeutic failures , have led to the investigation of new drugs to treat the disease .
fluoroquinolones , macrolides and tigecycline ( tig ) , a member of a new class of antimicrobials , the glycylcyclines , may serve as alternative drug choices 12 - 16 .
this study aimed to find the most common brucella species in this endemic region of turkey since strategies for disease control and eradication derive primarily from the epidemiological characteristics of the disease and to determine the in vitro antimicrobial susceptibilities of these strains to commonly used anti - brucella agents and a possible new alternative tigecycline .
bacterial strains : 56 brucella isolates were collected prospectively between 2008 - 2009 from blood ( 45 ) , synovial fluid ( 8) , bone marrow ( 2 ) , and cerebrospinal fluid ( 1 ) cultures of patients with acute brucellosis who were admitted to van education and research hospital and the hospital of the medical faculty of van yuzuncu yil university ( van , turkey ) .
identification methods : identification of species was made on the basis of the requirement of co2 for growth , production of urease and h2s , sensitivity to the dyes basic fuchsine and thionine ( at final concentrations of 20 - 40 g / ml ) , and agglutination with monospecific antisera for a and m antigens 19 .
the strains were stored in skim milk at -40c and subcultured twice before the susceptibility tests .
antimicrobial susceptibility testing : minimum inhibitory concentration ( mic ) of doxycycline ( dox ) , rifampin ( rif ) , streptomycin ( str ) , tigecycline ( tig ) and trimethoprim - sulfamethoxazole ( tmp - smz ) were determined by e - test ( biomerieux , sweden ) method on mueller - hinton agar ( oxoid , basingstoke , uk ) supplemented with 5% sheep blood and interpreted after 48 hours of incubation at ambient air .
mueller - hinton agar supplemented with 5% sheep 's blood was inoculated with suspensions of the test organism equivalent 0.5 mcfarland turbidity , and e - test strips were applied onto culture plates .
the mic was interpreted as the value at which the inhibition zone intercepted the scale on the e - test strip . mic50 and mic90 levels defined as the lowest concentration of the antibiotic at which 50% and 90% of the isolates inhibited , respectively .
the clinical laboratory standarts institute ( clsi ; formerly the nccls ) breakpoints for tmp - smz , str , dox were employed for the results .
three brucella reference strains ( b. abortus 544 , b. melitensis 16 m , and b. suis 1330 ) were used as controls for identification , biotyping and antimicrobial susceptibility testing .
in addition to these brucella reference strains , esherichia coli atcc 25922 , staphylococcus aureus atcc 29213 were also used as the quality control strain for susceptibility testing .
all isolates were identified as b. melitensis . in vitro activities of dox , str , rif , tmp - smz , and tig against these isolates were evaluated .
the mic values of dox , str and tmp - smz interpreted according to the clsi criteria for potential bioterrorism agents and interpretive criteria for slow growing bacteria ( haemophilus ) has been used to evaluate the results of mics of tig .
the mic50 and mic90 values of relevant antibiotics are shown in table 1 . according to mic90 , dox ( 0.064 g / ml )
was found to be the most active agent , followed by tig ( 0.094 g / ml ) , tmp - smz ( 0.125 g / ml ) , str ( 1 g / ml ) and rif ( 2 g / ml ) respectively . all isolates were found to be sensitive to dox , str and tmp - smz .
the mic values of tig interpreted according to the clsi criteria for slow growing bacteria , has shown ranges below the breakpoints for sensitivity determination .
brucellosis is endemic in turkey and approximately 10,000 cases of human brucellosis are reported annually 5 .
brucellosis and its complications are still serious public health concern in eastern anatolia . although the diagnosis of brucellosis can be made only by the isolation of causative agent ; brucella spp .
are difficult to isolate and the procedures are time consuming and expensive 8,20 . moreover , brucella spp .
are so highly infectious that the attempts at isolation and identification of brucella from clinical specimens are not routinely performed 8,20 - 22 .
therefore , the epidemiology of brucellosis has not been extensively studied , and limited data are available about the prevalence and species most commonly encountered in eastern anatolia .
this is the first study which identifies brucella species and their susceptibility pattern in this region .
our findings are in accordance with the previous reports from different regions of turkey , mediterranean and south america basin which have revealed that human brucellosis is almost exclusively caused by b.melitensis , accounting for 99% of total cases 8 - 16,22 - 25 . in this present study
, we also performed in vitro susceptibilities of b.melitensis against commonly used antimicrobials and a novel compound tigecycline .
antimicrobial susceptibility testing for brucella spp is not generally recommended for routine microbiology laboratories except in life - threatening organ involvement , and in case of treatment failure and relapse 21 . another problem with such testing is the lack of standardization .
methods for mic determination are described for potential bioterrorism agents including brucella species by the clsi .
the breakpoints used for interpretation as susceptible were as follows : tet / dox 1 g / ml , tmp - smz 2 g / ml , and str 8 g / ml according to the the clsi interpretive criteria 26 . in vitro efficacy of antibiotics against brucella spp .
has usually been based on the determination of mic values by micro broth dilution , agar dilution , and e - test methods 20 .
e - test method was found to be reliable , reproducible , less labor - intensive , less time - consuming , and more practical than the broth micro dilution method 11,24,27 .
e - test could be performed on two different culture media : the mueller - hinton agar plates widely used for antibiotic susceptibility testing and the brucella agar plates commonly used in the laboratory as brucella growth medium .
although no significant differences were observed between two culture media , we preferred the mueller - hinton agar plate in this study because clearer inhibition zones are visible and the calibrated carrier strip indicating the mic can be more easily read 25 .
dox has become the most commonly prescribed tetracycline derivative in the treatment of brucella infections because of its superior pharmacokinetic features 28 . in the present study , among the tested antibacterial agents ,
dox was found to have the lowest mic50 and mic90 values which is consistent with previous reports 8,10,11,22 - 24,27,29 .
conversely in a mexican study , lopez - merino et al . found the mic values for tet were higher than in brucella strains isolated in turkey 9 which demonstrates the antibiotic susceptibility patterns of brucella strains appear to vary geographically .
another drug of choice in the treatment regimen of brucellosis is rif and it was found to be the only antibiotic with increased activity in acidic environmental conditions 27 . in our study , the highest mic values were determined for rif among the studied antimicrobials . as mic values of rif in previous studies
were reported to range from 0.047 to 4 g / ml , its values confirmed again by our findings 8,10 - 12,22 - 25 .
these findings should be taken into consideration for the potential emergence of rif resistance of brucella spp . in the region .
another concern for rif using widespread in the long treatment regimens like brucellosis may cause an increase in rif resistance in m. tuberculosis because both brucellosis and tuberculosis can simultaneously exist in the same countries in many parts of the world 32 .
furthermore experimental studies suggested that the development of mycobacterial resistance to rif may lead to development of resistance to other antimicrobials as well 32 .
the resistance rate of rif against m. tuberculosis was reported as 15 - 58% in turkey 33 .
tmp - smz containing regimens is considered to be suitable oral regimens that may be of significantly lower cost than traditional combinations in certain developing countries and mostly prescribed in brucellosis for children and pregnant women 2 . in our study
mic50 and mic90 values for tmp - smz were lower than those previously observed in turkey 8,10,11 and conforming the results of kilic et al . 16 . in vitro tmp - smz resistance rate
however , significant rates of tmp - smz resistance have been reported in the world 31,34 .
although streptomycin is known to be one of the most active agent against brucellosis , its adverse effects , such as ototoxicity , nephrotoxicity , and parenteral administration , preclude its wider use 24,29 . in our study susceptibility to str
this is one of the few studies which , determines the in vitro activity of tig , a new glycylcycline compound , against brucella strains .
we found that tig was more effective than rif , tmp - smz and str but was not as effective as dox .
reported tig was more effective than rif , sxt , str , and dox 8 . also kilic et al .
found tig had the least mic50 and mic90 values compared to tet , and fluoroquinolones against brucella strains isolated in central anatolia 13 . these are in contrast with our findings and might be due to the strain specific susceptibility . as mic50 and mic90 values of tig in these two previous studies
were reported to be 0.064 and 0.125 g / ml respectively , values of them confirmed again by our findings . although tig has similar properties to tet , it has been reported that it is more potent than tet 35,36 .
therefore , pappas et al . suggested replacing dox with more potent tig might increase efficacy and reduce treatment duration 37 . on the other hand , parenteral administration of tig
, the conservation of tig because of promising results of its use in the treatment of multiresistant bacterial infections , and overall cost were considered as limitations of such a therapy 12 . in conclusion , there is no significantly important resistance problem for classically recommended antibiotics targeted to brucella species in turkey , but antibiotic susceptibility patterns of brucella spp . appear to vary geographically .
the results of this in vitro study suggest tig as a therapeutic option in the treatment of brucellosis .
clinical trials are warranted to assess the real therapeutic potential of tig in human brucellosis , particularly in countries with higher prevalence of antibiotic resistance . | purpose : brucellosis is a worldwide zoonotic disease and still constitutes a major public health problem . in the study we claimed to identify brucella species from clinical samples of patients with active brucellosis from van region of eastern anatolia and to determine in vitro antimicrobial susceptibilities of these strains to commonly used anti - brucella agents and a possible new alternative tigecycline.materials and methods :
a total of 56 brucella isolates were enrolled the study and the identification of the isolates were based on conventional methods . in vitro activities of antimicrobials were evaluated by the e test method.results : all isolates were identified as b. melitensis .
mic90 values of doxycycline , streptomycin , rifampin , trimethoprim - sulfamethoxazole and tigecycline were 0.064 mg / l , 1 mg / l , 2 mg / l , 0.125 mg / l and 0.094 mg / l , respectively .
tigecycline had low mic50 and mic90 values against all b. melitensis strains ; the highest mic observed was 0.25 g / ml.conclusion : our data suggest that tigecycline can be a therapeutic alternative option for the treatment of brucellosis . |
people living with cancer experience a variety of symptoms - such as pain , worry , depression , diarrhea , cough , shortness of breath , nausea , weakness , fatigue , fever , and confusion .
lack of care results in untreated symptoms that hamper an individual 's ability to continue his or her activities of daily living .
treatment of symptoms relieves suffering and often improves the rate of recovery and quality of life .
ward and gatwood(us ) identified barriers like fear of addiction , beliefs that good patients do not complain about pain , and concerns about side effects borneman et al . ( uk )
pnina has reported three types of barriers to manage pain ; those were an addiction to medication , saving pain medicine to use when the pain gets worse , and pain is a sign that the disease was getting worse .
yeom and heidrich(2009 , usa ) identified three possible barriers to symptom management ; they were : negative beliefs about managing symptoms , perceived negative attitudes of healthcare providers , and difficulties in communicating about symptoms .
barriers to symptom management may lead to poorer self - care of symptoms that can result in lower levels of psychosocial quality of life
. objectives of the study were to :
assess the symptoms among cancer patientsidentify the perceived barriers to symptom management among cancer patientsfind the association between perceived barriers and study variables of client such as age , education , income , type of cancer , stage of cancer , duration of illness , and treatment .
assess the symptoms among cancer patients identify the perceived barriers to symptom management among cancer patients find the association between perceived barriers and study variables of client such as age , education , income , type of cancer , stage of cancer , duration of illness , and treatment .
one of the main objectives was to estimate the barriers perceived by the cancer patients .
the sample size for this study was calculated based on the pilot study result . for 95% confidence limits , the allowable error of 1.5% and a design effect of 2 for cluster sampling , a sample size of 768 would be sufficient to estimate the rate at 5% level of significance .
formula used for the calculation of sample size was : = 7.8 ( barrier score ) total = 384 .
objectives of the study were to :
assess the symptoms among cancer patientsidentify the perceived barriers to symptom management among cancer patientsfind the association between perceived barriers and study variables of client such as age , education , income , type of cancer , stage of cancer , duration of illness , and treatment .
assess the symptoms among cancer patients identify the perceived barriers to symptom management among cancer patients find the association between perceived barriers and study variables of client such as age , education , income , type of cancer , stage of cancer , duration of illness , and treatment .
one of the main objectives was to estimate the barriers perceived by the cancer patients .
the sample size for this study was calculated based on the pilot study result . for 95% confidence limits , the allowable error of 1.5% and a design effect of 2 for cluster sampling , a sample size of 768 would be sufficient to estimate the rate at 5% level of significance .
formula used for the calculation of sample size was : = 7.8 ( barrier score ) total = 384 .
an exploratory survey was done among 768 cancer inpatients aged above 30 years and diagnosed to be in third or above stages of cancer of breast / cervix / head and neck / gastro intestinal tract / lung / colorectal cancer and have undergone radiotherapy or chemotherapy or surgery or combination of them in selected reputed cancer hospitals of karnataka .
reputed cancer hospitals all over karnataka were selected by purposive sampling , and each hospital was considered as a stratum . then proportionate sample from each stratum was selected for the study .
data were collected from cancer patients of various hospitals using pre - tested structured interview technique after obtaining permission from the respective hospital administrators .
the objective and study protocol were explained to the study participants , and written consent was taken .
the objective and study protocol were explained to the study participants , and written consent was taken .
the data from 768 samples were collected and analyzed based on the objectives of the study . out of 768
, majority of the cancer patients ( 30.2% ) belonged to the age group of 5160 years , most of them were females ( 57.2% ) , belonged to hindu religion ( 87.9% ) and 94.5% were married [ table 1 ] .
the majority of the cancer patients ( 39.2% ) were primary educated [ figure 1 ] .
most of the affected patients were housewives ( 49% ) , their monthly income was between rs .
2501 and 5000 ( 51.3% ) , and 33.1% of the patients had taken combination therapy [ table 2 ] .
most of them availed modern medical treatment from the hospital after final diagnosis ( 80.3% ) and 57.7% of the samples were in the third stage of cancer [ table 3 ] . around
40% of the patients suffered with head and neck cancer and most ( 76.2% ) of the patients were suffering from the illness for < 6 months [ figures 2 and 3 ] .
frequency and percentage distribution of cancer patients based on socio - demographic characteristics such as age , gender , marital status , type of family ( n=768 ) educational status of cancer patients frequency and percentage distribution of cancer patients based on employment , income , and type of treatment ( n=768 ) frequency and percentage distribution of cancer patients based on treatment modality , duration of treatment and stage of cancer ( n=768 ) types of cancers .
git : gastro intestinal tract duration of illness among cancer patients during survey , patients reported various symptoms .
major symptoms are depicted in figure 4 . majority of the patients ( 96.5% ) had tiredness , 96.4% had disturbed sleep , 94.5% had worry , 90.4% of the patients reported that they lost their appetite after diagnosis , 85.7% patients felt irritable , 83% of patients had feeling of nervousness , 77% had pain and 63.3% of the patients lost their weight after diagnosis , 60.7% had anxiety and 57.6% had depression .
self - reported symptoms the barriers were categorized into : communication , personal , professional , financial and misconceptional barriers .
the response for all items are graded on a four - point scale ( 4 = very much , 3
= quite a bit , 2 = somewhat , 1 = a little , and 0 = not at all ) .
majority of the patients ( 36.1% ) did not know how to express symptoms since the majority had only primary education , they were facing language problems while communicating with the health care personnel ( 45.8% ) .
90.6% had a fear of consequences of treatment , and 96.9% had fear about disease progression .
most ( 58.8% ) of the patients had a fear of further hospitalization , and there were no professional barriers identified except overcrowding in the cancer clinic ( 50% ) [ table 4 ] .
about 89.3% had lack of resources in the family , 99% of the samples expressed that the treatment is very expensive , 72% of samples were not able to afford nutritional supplements , 82.6% of them reported inability to afford the cost of medicine and to pay for health care services ( 83.1% ) [ table 5 ] .
majority of the samples had some misconception that is , increasing pain signifies disease in progress ( 92.7% ) , medicine to control pain may weaken the immune system ( 89.9% ) , pain is inevitable for cancer patients ( 78.5% ) , and 77.1% of samples reported that the anxiety or depression are expected after diagnosis of cancer [ table 6 ] .
frequency and percentage on communication , medication , personal and professional barriers as presented by cancer patients ( n=768 ) frequency and percentage of financial barriers as presented by cancer patients ( n=768 ) frequency and percentage of misconceptions among cancer patients ( n=768 ) data further showed that for total barriers score among cancer patients from all categories , the mean was 44.86 and standard deviation 19.38 [ table 7 ] .
median and interquartile range of each type of barriers identified among cancer patients are presented in table 8 .
mean and sd of total score of perceived barriers among cancer patients ( n=768 ) median and iqr of all types of barriers among cancer patients ( n=768 ) one - way anova test was used to test the association between the mean score of barriers perceived by the cancer patients and study variables such as age , gender , education , income , type and duration of illness .
result showed that as education and income increases , barriers score decreases , which is found to be statistically significant ( education : f = 13.556 , p= 0.001 and income : f = 16.338 , p= 0.001 ) [ table 9 ] .
comparison of mean barrier scores according to socio - demographic and disease characteristics ( n=768 )
majority of the patients ( 96.5% ) had tiredness , 96.4% had disturbed sleep , 94.5% had worry , 90.4% of the patients reported that they lost their appetite after diagnosis , 85.7% patients felt irritable , 83% of patients had feeling of nervousness , 77% had pain and 63.3% of the patients lost their weight after diagnosis , 60.7% had anxiety and 57.6% had depression .
the barriers were categorized into : communication , personal , professional , financial and misconceptional barriers .
the response for all items are graded on a four - point scale ( 4 = very much , 3
= quite a bit , 2 = somewhat , 1 = a little , and 0 = not at all ) .
majority of the patients ( 36.1% ) did not know how to express symptoms since the majority had only primary education , they were facing language problems while communicating with the health care personnel ( 45.8% ) .
90.6% had a fear of consequences of treatment , and 96.9% had fear about disease progression .
most ( 58.8% ) of the patients had a fear of further hospitalization , and there were no professional barriers identified except overcrowding in the cancer clinic ( 50% ) [ table 4 ] .
about 89.3% had lack of resources in the family , 99% of the samples expressed that the treatment is very expensive , 72% of samples were not able to afford nutritional supplements , 82.6% of them reported inability to afford the cost of medicine and to pay for health care services ( 83.1% ) [ table 5 ] .
majority of the samples had some misconception that is , increasing pain signifies disease in progress ( 92.7% ) , medicine to control pain may weaken the immune system ( 89.9% ) , pain is inevitable for cancer patients ( 78.5% ) , and 77.1% of samples reported that the anxiety or depression are expected after diagnosis of cancer [ table 6 ] .
frequency and percentage on communication , medication , personal and professional barriers as presented by cancer patients ( n=768 ) frequency and percentage of financial barriers as presented by cancer patients ( n=768 ) frequency and percentage of misconceptions among cancer patients ( n=768 ) data further showed that for total barriers score among cancer patients from all categories , the mean was 44.86 and standard deviation 19.38 [ table 7 ] .
median and interquartile range of each type of barriers identified among cancer patients are presented in table 8 .
mean and sd of total score of perceived barriers among cancer patients ( n=768 ) median and iqr of all types of barriers among cancer patients ( n=768 )
one - way anova test was used to test the association between the mean score of barriers perceived by the cancer patients and study variables such as age , gender , education , income , type and duration of illness .
result showed that as education and income increases , barriers score decreases , which is found to be statistically significant ( education : f = 13.556 , p= 0.001 and income : f = 16.338 , p= 0.001 ) [ table 9 ] .
comparison of mean barrier scores according to socio - demographic and disease characteristics ( n=768 )
the study findings were in conformity with a study done by naveh et al . who reported that 66% of the patients had severe pain and iyer et al . who found that more than 90% of the patients expressed pain , fatigue and loss of appetite
fatigue , loss of appetite , shortness of breath and pain have a significant negative impact on patient reported disease specific health related quality of life in advanced non - small - cell lung cancer patients .
reported in their study that most of the cancer patients had pain ( 87.5% ) , lack of energy ( 77.7% ) , feeling sad ( 75.9% ) , feeling of drowsiness ( 72.3% ) and worry ( 69.6% ) .
reported some of the symptoms experienced by patients in their study : 48% of cancer patients had pain within the previous 24 h and they were depressed ( 41.8% ) , worried ( 38.2% ) , frightened ( 30.9% ) and angry ( 18.2% ) due to disease - related symptoms .
the present study showed that barriers to symptoms management were : worry about side effect ( 71.4% ) , fear of addiction ( 23.1% ) and pain medication harm the immune system ( 89.0% ) .
, edrington et al . and ward et al . in their studies ; which were : concern about medication and its side - effects , fear of addiction and beliefs that good patients do not complain about pain and disease progression .
found that pain medications harm the immune system and yates et al . reported that almost 71% believed it is easy to become addicted to pain relieving medication .
since this study was confined to a single state , the findings can not be generalized to nationwidecancer patients who gave consent for participation only were interviewedpermitted hospitals only were selected for the study .
since this study
was confined to a single state , the findings can not be generalized to nationwide cancer patients who gave consent for participation only were interviewed permitted hospitals only were selected for the study .
since this study was confined to a single state , the findings can not be generalized to nationwidecancer patients who gave consent for participation only were interviewedpermitted hospitals only were selected for the study .
since this study
was confined to a single state , the findings can not be generalized to nationwide cancer patients who gave consent for participation only were interviewed permitted hospitals only were selected for the study .
the management of cancer pain is a critical issue in the care of patients with cancer .
there is a need to develop measures for effective management of symptoms and to reduce the barriers . | background : people living with cancer experience wide variety of symptoms .
if symptoms are not managed well , it may hamper an individual 's ability to continue his or her activities of daily life .
treatment of symptoms relieves suffering and improves the rate of recovery as well as the quality of life.objectives:to assess the symptoms of suffering among cancer patients and to identify the perceived barriers to their symptom management.materials and methods : a cross - sectional study was carried out among 768 cancer patients selected by stratified sampling with a proportionate selection from each stratum .
data were collected from cancer patients by interview technique using structured validated questionnaire.results:majority of the samples ( 30.2% ) belonged to the age group of 5160 years , most of them were diagnosed with head and neck cancer ( 40.1% ) and 57.7% had stage iii disease .
the majority of the patients studied had pain ( 77% ) , tiredness ( 96.5% ) , disturbed sleep ( 96.4% ) , weight loss ( 63.3% ) , and irritability ( 85.7% ) .
most of the patients had lack of appetite ( 89.4% ) , feeling of sadness ( 96.6% ) , worry ( 94.5% ) , and feeling of nervousness ( 82.8% ) .
majority of the patients had some misconception regarding symptoms , that is , increasing pain signifies disease progression ( 92.7% ) , medicine to control pain may weaken the immune system ( 89.9% ) and pain is inevitable for cancer patients ( 78.5% ) .
seventy - seven percent of samples reported that the anxiety or depression is expected after the diagnosis of cancer.conclusion:this study provides an overview of symptoms among cancer patients and barriers experienced by them . |
the classic account , made by dandy & blackfan in 1914 * , where autopsies showed severe hypertentorial hydrocephalus , cystic dilatation of the fourth ventricle , small vermis , removal of the cerebelar hemispheres and absence of the roof of the fourth ventricle , thickening and opacity of the pia - arachnoid cisternae of the skull base and dilatation of the aqueduct1 .
the dandy - walker syndrome ( sdw ) is a non - familial syndrome characterized by cystic dilatation of the fourth ventricle and aplasia or partial or total atrophy of the cerebelar vermis .
occur other brain malformations such as agenesis of the corpus callosum , heteropsias , lissencephaly , stenosis of the aqueduct of sylvius
2
3 .
gardner et al * * proposed that the sdw , along with other syndromes ( arnold - chiari malformation , cerebelar arachnid cyst and syringomyelia ) were manifestations of the same disease1 .
some studies show an incidence of approximately 70% of relationship between the sdw and systemic abnormalities1 .
little is known about congenital malformations of the posterior pit structures , their genetic alterations have been mapped to chromosome 3q4
5 , but the gene is not located exactly , but it is known that the basis of the development process of the structures of the pit later is nature for human cerebelar malformations5 .
it is also known that structures cerebelar growth early embryo in the period until the early post - natal , this event would cerebellum susceptible to a wide spectrum of disorders of development6 .
the pathogenesis of this syndrome is controversial , but the most accepted theory is that the leaf development of the foramina of magendie and lushka during the fourth month of fetal life , leading to bulging cystic fourth ventricle .
new theories have proposed that the sdw result from a failure to develop the roof of the hindbrain , taking this as a cause teratogenic effect1 .
some studies suggest that the use of warfarin in the long run would be responsible for the development of sdw in 1 - 2% of exposed fetuses7 .
, showed no relationship between degree of hydrocephalus and the size of the cyst in the posterior pit , vermis or attenuation of the permeability of the fourth ventricle and hydrocephalus in some cases was absent1 .
the sdw is an entity of heterogenic hyperplasia of the cerebelar vermis and is a newly identified gene associated with the connection with x - hprt8 also relates to the basal ganglia disease .
clinically there may be moderate delayed psychomotor development , microcephaly , hypotonia , but the predominant symptoms refers to hydrocephalus , usually in the first two years of life , this however can be ignored , appearing late ( first or second decade of life)2
3
4
5
6
7
8
9 .
some ocular abnormalities are described in the sdw , as coloboma corioretinian10 , nystagmus11
12
13 . there may be mental retardation ( 50% ) , spasticity ( instead of hypotonia ) , seizures , vomiting , all depending on the degree of cerebelar malformation3 . in patients with vermis fissures with two conformations and almost normal brain functions are also almost normal without association with other malformations . in patients with severe malformations of the cerebellum , vermis with only one or no crack , it is common to severe mental retardation and other malformations of the central nervous system , such as agenesis of the corpus callosum
. it is divided , so the sdw into two large groups according to the previous malformations for determining the prognosis intellectual12
13 .
it is reported in the literature of the coexistence of large facial coetaneous hemangioma with sdw14 .
other syndromes where coexist brain and ocular malformations are reported as neuhauser syndrome ( mmmm - megalocornea , macrocephaly , mental and motor retardation ) , which are found cortical atrophy , enlargement of the fourth ventricle , hyperplasia of the corpus callosum .
, there is need for magnetic resonance images of good quality axial view of the cerebelar vermis and t2 images12 .
the neuroradiological findings are characteristic , such as cystic dilatation of the fourth ventricle and changes in the cerebelar vermis2 , besides others already mentioned .
the bilateral sensorineural deafness can be part of the clinical picture of the syndrome and we describe a case whose patient underwent implantation cochlear by presenting profound bilateral deafness .
the aim of this paper is to describe a case of female patient , 13 years with a diagnosis of this syndrome and bilateral hearing loss underwent cochlear implant surgery under local anesthesia and sedation .
cgs , 13 years old female was referred to the ent department of otolaryngology institute of parana with a diagnosis of dandy - walker syndrome for ent evaluation for bilateral hearing loss with no response to the use of hearing aids .
the patient performs follow up with neurology since birth , with a history of gestational rubella , cataracts in both eye and neurological surgery prior hydrocephalus , absence of parental consanguinity .
child with imaging exam compatible with dandy - walker syndrome with posterior pit collection and communication with the fourth ventricle , associated with signs of hyperplasia of the cerebelar hemispheres and vermis .
was not possible to detect brainstem evoked potentials bilaterally . in the absence of otoacoustic emission response bilaterally mri of the brain showing cerebelar vermis hyperplasia associated with mega cistern magna , communicating with the fourth ventricle ( figures 1 and 2 ) .
the fluid spaces of the cochlea are normal thickness , ranging from 1.3 to 1.7 mm the right and 1.4 to 1.7 mm left .
the cochlear nerves have been identified and normal thickness of 0.6 mm ( figure 3 ) .
surgery was performed under local anesthesia and sedation by retroauricular access , creation of niche outdoor unit , closed mastoidectomy , posterior tympanotomy , chocleostomy , inserting the internal components and telemetry . all steps performed uneventfully .
the implant was activated 1 month after surgery with excellent sound perception at the time of activation .
the patient has excellent levels of discrimination with less need for lip reading and excellent response to therapy with spectacular mend the quality of speech .
no doubt the results bring impact on quality of life of patients while providing better social integration .
the field of cochlear implants is growing rapidly due to improved quality of the implants , surgery less invasive , and wider dissemination of this type of treatment of deafness .
the surgery is now much faster and less invasive than some years ago , with smaller incisions and less morbidity for the patient .
the patient even though the syndrome dandy - walker does not present significant deficit in neuropsychomotor development , fulfilling the criteria established in the literature for the performance of cochlear implant surgery .
there are already cases of patients with the same syndrome who underwent cochlear implant surgery , complications described in the literature21 and with good results despite co morbidities . in our facility
this type of surgical anesthesia brings morbidity / anesthetic besides a minor post - operative recovery and faster lower hospital costs when compared with general anesthesia and were perfect forward from the patient .
we believe that the presence of dandy - walker syndrome can not be considered a contraindication to the performance of cochlear implant surgery , and there were no surgical complications due to neurological disorders with very favorable results for the patient who exhibits excellent discrimination . | summary
introduction : dandy walker syndrome is a congenital abnormality in the central nervous system , characterized by a deficiency in the development of middle cerebelar structures , cystic dilatation of the posterior pit communicating with the fourth ventricle and upward shift of the transverse sinuses , tentorium and dyes . among the clinical signs are occipital protuberances , a progressive increase of the skull , bowing before the fontanels , papilledema , ataxia , gait disturbances , nystagmus , and intellectual impairment .
objectives : to describe a case of female patient , 13 years old with a diagnosis of this syndrome and bilateral hearing loss underwent cochlear implant surgery under local anesthesia and sedation .
case report : cgs , 13 years old female was referred to the otolaryngological department of otolaryngology institute of parana with a diagnosis of dandy - walker syndrome for otolaryngological evaluation for bilateral hearing loss with no response to the use of hearing aids .
final comments : the field of cochlear implants is growing rapidly .
we believe that the presence of dandy - walker syndrome can not be considered a contraindication to the performance of cochlear implant surgery , and there were no surgical complications due to neurological disorders with very favorable results for the patient who exhibits excellent discrimination .
it has less need for lip reading with improvement in speech quality . |
for patients of congenital adrenal hyperplasia ( cah ) with ambiguous genitalia ( ag ) , the decision of gender assignment and sex of rearing can be complex , especially if the genitalia are highly virilized . while assigning gender , apart from karyotype and prenatal androgen exposure , sociocultural influences , and parental expectations play a role .
there have been debates on the role of gender in shaping an individual 's mode of functioning nature ( pre- and post - natal androgen exposure , genetics ) versus nurture ( sex of rearing , parenting style ) .
however , sex of rearing has been considered a promising predictor of an individual 's gender identity .
since gender is conventionally divided as being either a male or a female , unacceptability can arise if it lies in - between these two points .
being labeled queer or a transgender can become a major source of distress for the patients and their family .
one - third of 46 xx patients with disorders of sexual differentiation ( dsd ) tend to experience gender dissonance with their biological sex .
most of the 46 xx cah patients raised as females tend to retain their chromosomal gender identity throughout their lives . however , one study reported that 10 out of 217 females , diagnosed with cah and reared as women , showed some form of gender dissonance or identity confusion .
the difficulty in assigning gender particularly arises if the genitalia of the new - born patient is highly virilized .
a literature review assessed studies on gender dysphoria and gender confusion in patients with 46 xx cah reared as females .
hence , the authors concluded the need for assignment of female gender identity in cah patients even in the face of highly virilized genitalia ( prader stage iv or v ) .
however , there have been reports of successful male gender assignment in patients with 46 xx cah and highly virilized genitalia , who have demonstrated adequate social and sexual functioning as males .
these reports have challenged the notion of typically relying on internal genitalia ( presence of uterus and ovaries ) while assigning gender in these patients .
thus the decision to rear 46xxcah patients as male or female needs to be based on several factors .
first , it helps gain insight about the distress experienced by the patient and their family .
second , it would help in understanding the impact of their distress on different facets of their lives .
hence , this study aims to gain insight into the psychosocial functioning , gender rearing , and gender dysphoria in patients of 46 xx cah in indian setup .
this is a cross - sectional study in which patients of 46 xx cah were enrolled .
a diagnosis of cah was made on the basis of history and examination , cytogenetic and hormonal analysis .
diagnosis of salt - wasting cah ( sw - cah ) was made on the basis of genital ambiguity with history of diarrhea / vomiting in infancy with electrolyte imbalance , hypokalemia , and elevated 17-hydroxyprogesterone ( ohp ) levels .
diagnosis of simple virilizing cah ( sv - cah ) was made on the basis of the presence of ag / clitoromegaly with elevated 17 ohp levels without a history of salt wasting .
patients who presented at puberty with features of irregular periods , acne , hirsutism , and raised 17 ohp levels , a diagnosis of nonclassical cah ( nc - cah ) was made .
a semi - structured interview proforma was used to elicit history and psychosocial background of the patients .
all the 22 patients were screened for gender dysphoria using :
parent report gender identity questionnaire ( pr - giq ) for children is a parent report assessment of problems in the gender identity development of children aged 2.512 years . for this purpose , parents are asked to 16 questions that cover a range of sex - typical behaviors of each gender .
a mean score of 2.87 or below for boys and 2.67 or below for girls indicates the presence of gender dysphoria .
specificity rate is 95% for the controls , and sensitivity rate for the probands is 86.8%gender identity / gender dysphoria questionnaire for adolescents and adults ( gidaa ) is an assessment tool used on adolescents and adults to assess gender dysphoria
it displays a strong discriminant validity of 0.97 ( cronbach alpha coefficient ) with 90.4% sensitivity for the gender identity patients and specificity of 99.7% for the controls .
parent report gender identity questionnaire ( pr - giq ) for children is a parent report assessment of problems in the gender identity development of children aged 2.512 years . for this purpose ,
parents are asked to 16 questions that cover a range of sex - typical behaviors of each gender .
a mean score of 2.87 or below for boys and 2.67 or below for girls indicates the presence of gender dysphoria .
specificity rate is 95% for the controls , and sensitivity rate for the probands is 86.8% gender identity / gender dysphoria questionnaire for adolescents and adults ( gidaa ) is an assessment tool used on adolescents and adults to assess gender dysphoria
it displays a strong discriminant validity of 0.97 ( cronbach alpha coefficient ) with 90.4% sensitivity for the gender identity patients and specificity of 99.7% for the controls . to assess gender dysphoria , pr - giq was administered on the parents of patients who were 12 years of age and below .
the male version of these tests was used on patients reared as males and female version for those reared as females .
since the patients were fluent in hindi , the two questionnaires - pr - giq for children and gidaa were translated in hindi and back translated .
there were 11 patients with sw - cah and 8 patients with sv - cah and 3 with nc - cah . clinical profile and diagnosis of patients among the 19 patients reared as females , most were well - adjusted as females and
two patients showed marginally low scores for gender dysphoria by the tests applied [ table 2 ] .
first patient , c.4 , aged 6 years old , was being reared as a female and expressed her desire to be a boy .
since her score on pr - giq was marginally on the lower side , draw - a - person - test ( dapt ) was performed on her to ascertain the extent of dissonance in her gender identity .
dapt is a projective test in which a subject is asked to draw a figure of anyone and any gender they like .
dapt has been extensively used to assess the cognitive and psychological characteristics of children and adults . according to the projective school of thought
, drawings are assumed to be symbolic representations of the things in the unconscious that is usually suppressed . in general , drawing tests require no prior training and are often used as icebreakers as they help to reduce the anxiety of the child .
our patient drew a figure of a 5-year - old boy named muni . on questioning , muni was the name of the patient 's cousin she liked playing with . since it was an opposite sex figure , it may point toward ambivalence or conflict regarding her sexual identity .
although , the validity of this test is often questioned as its interpretations are subjective and qualitative in nature .
however , atypical drawings can be seen as strong indicators of maladjustment in an individual .
gender dysphoria test findings the second patient c.16 , reared as a female in whom gender dysphoria was found by the tests applied , was aged 22 .
though she was reared as a female , she wished to live as a male .
on further enquiry , the patient showed depressive symptoms with inadequate sleep , reduced hunger , weight loss , and she also exhibited a very low self - esteem and body image disturbances .
she was assessed by a psychiatrist , following which she was diagnosed with sub - syndromal depression under remission .
she was advised to take psychiatric treatment to improve her condition . among the three patients reared as males , genitalia were prader grade iii or v [ table 1 ] .
gender dysphoria was found in none at the time of this study [ table 2 ] . among them , the first patient , c.20 , aged 32 , had successfully undergone gender reassignment as a female 2 years before this study was conducted and at present is living as a woman
. therefore female version of gender dysphoria score was applied . the second patient , c.21 , aged 12 , sought clearance for panhysterectomy before entering puberty .
the third patient , c.22 , aged 8 years , was satisfied with his male gender identity and had clearly stated to remain a boy .
he had been admitted 2 years back for feminizing genitoplasty after counseling his parents about his chromosomal sex being female
. however , the patient refused to undergo the surgery as he was completely satisfied in being a boy .
majority of patients reared as females ( 17 out of 19 ) were well - adjusted .
out of the patients reared as female , two patients , c.16 and c.4 showed gender dysphoria by the scales applied .
patients c.16 and c.4 , aged 22 and 6 , reared as girls , were unsatisfied with their female gender identity .
they like to dress themselves as males ( pants and shirt ) and enjoy male associated activities .
mother of patient c.4 reported scolding the patient when she referred to herself as a boy .
however , research has indicated that some behaviors of girls with cah can be tomboyish , hence , it may not indicate a dissonance in the core gender identity . in a study
, a prenatally hyperandrogenized sample of young girls with cah did not show any masculinization of gender identity .
the drawing of an opposite sex figure on dapt by the 6 year old c.4 also does not necessarily point toward gender dysphoria or emotional issues .
if the child 's drawing is neither typically male nor female , it may point towards some maladjustment .
hence , for patient c.4 , surgical procedures have been postponed until she attains puberty when she will be reassessed .
since she does not meet the criteria for gender identity disorder in childhood according to guidelines in icd-10 , her behavior can be best viewed under the lens of being tomboyish .
due to familial and social pressure , the second patient c.16 with gender dysphoria , aged 22 , has not disclosed the dissonance in her gender identity to anyone .
she has attempted to harm herself physically in the past due to being teased by her peers . as revealed by the patient 's sister , she disliked her body and often doubted herself .
this may have contributed to the patient not having any social circle , inadequate sleep cycle with anxiety and reduced hunger .
the stress of her medical condition may have further led her to develop depression and low self - esteem . since depression and self - esteem
are negatively correlated to each other , a psychiatric assessment and psychological support were sought . due to the aforementioned complexities , her feminizing surgery has been postponed . in a study of 70 cah patients ( both 46 xx and xy ) ,
46 xx cah patients scored higher on depression and anxiety scores accompanied with a poor quality of life as compared to controls .
conversely , psychological distress , self - harming behavior , and suicidal tendencies were assessed by a group of researchers in different subgroups of dsd using standardized tests .
eleven 46 xx cah patients , reared as females , were found to be less psychologically distressed with lower self - harming behavior than the rest of the subgroups .
sw - cah patients were more distressed than patients with simple virilizing cah . in our study ,
the psychoneuroendocrinological causative factors behind a typical and an atypical gender identity are still poorly understood .
the exact roots of gender identity issues in patients with cah are difficult to ascertain .
it can perhaps be best understood as an interplay of prenatal androgenization , sex of rearing , and the sociocultural context . for patient c.4 , her tomboyish behavior at the age of 6 years ,
can be explained by the dominance of androgenizing hormones and interaction with her immediate peer circle .
patient c.16 , being 22 years old , also did not meet the criteria for gidaa according to icd-10 .
her gender confusion and psychological distress had more of societal and psychological underpinnings than the sole effect of hormones . on a social level , she liked playing cricket and staying out late at night , which is usually allowed in the case of boys . on a personal level
her psychiatric consultation and counseling revealed high levels of anxiety and rumination regarding her future and financial independence . at present , she accompanies her father to work and is thinking of pursuing the same career line .
done to ascertain the role of hormones to explain the masculine preferences and behavior of cah patients , sw - cah patients were compared with sv - cah patients on their gender - interests and roles .
hence , gender dysphoria and confusion can not be solely attributed to psychosocial variables . finding a suitable partner , sexual functioning and subfertility are some of the many sources that cause distress to the patients and their families .
long - term exposure to negative conditions can compromise the coping mechanisms of these individuals that can take the form of mental illness and/or complete loss of self - confidence .
unacceptability , hostility , teasing , and discrimination from others can severely hamper an individual in academia , intimate relations , and occupation .
on the other hand involving the patient and family and ensuring support can go a long way in making them well - adjusted individuals .
our study included 19 46 xx cah patients reared as females who had clitoromegaly and/or ag .
there were five patients in the preschool age ( c.1 , 2 , 3 , 4 and 5 ) .
mother of patient c.3 ( aged 5 years ) seeks ways to help her daughter from drawing unwanted attention by not having to use the school washroom by using disposable absorbent undergarments .
such alternatives are used by parents to avoid stigma and shame . in light of such problems ,
mother of patient c.3 is presently under treatment for depression and anxiety . due to stigma and shame associated with ag ,
parents of patients c.3 and c.5 have decided to get their children operated for genitoplasty at another center despite being reassured that surgery at a later age proves to be more beneficial for the patient .
there were 7 post pre - school patients in the prepubertal age group ( c.6 , 7 , 8 , 9 , 10 , 11 and 12 ) . due to being toilet - trained
they are being followed up for adequacy of medical treatment , height , growth , and counseling .
the main concerns in this age were delayed menarche , delayed breast development , irregular periods , the proper dose of medication to ensure optimal growth and height , acne , and hirsutism .
both the patients , c.13 and c.14 , aged 14 , were reared as females .
they did not show signs of gender dysphoria and seemed well - adjusted apart from the abovementioned concerns .
since , rapport could not be established with patient c.13 during assessment as she had an extremely shy nature , pr - giq was administered on her elder sister to ascertain gender dysphoria that was not found .
there were five patients who were above 18 years of age ( c.15 , 16 , 17 , 18 , and 19 ) .
patient c.18 , aged 29 , mature and financially independent , approached us with the issue of bisexuality .
as in the case of cah patients , along with a tomboyish personality , it is not uncommon to find their sexual preferences to be less heterosexual .
being in a similar situation , patient c.19 , aged 31 , was concerned about her masculine
she was advised by her family to learn to be more feminine otherwise it may pose a threat to finding a suitable marriage partner for her . at present , she is awaiting surgery before getting married .
patient c.17 , aged 28 , educated and financially independent , is well - adjustment to her long - term intimate partner who is supportive despite knowing about her condition and they plan to settle down in the near future .
patient c.15 , aged 21 , did not show any signs of gender dissonance and/or psychological maladjustments .
three patients c.20 , c.21 , and c.22 , aged 8 , 12 , and 32 , respectively , were reared as males .
patient c.20 , at present 32 years old , was born with ag and had been reared as a male for the first 17 years of her life .
she was brought to medical attention by her parents at 17 years of age when she began her menstrual cycles .
after investigations , the parents were counseled about the patient 's sex being chromosomally female .
however , the patient 's father insisted on raising her as a boy , following which , a panhysterectomy was performed without the patient 's involvement .
postsurgery , she was prescribed testosterone injections for the development of male secondary sexual characteristics . due to her genital ambiguity and fear of being ridiculed ,
therefore , in spite of belonging to a financially stable family , she remains uneducated .
soon afterward , the patient realized her discomfort in being a male , but felt sexually attracted to them .
she felt comfortable in being a woman , liked dressing up like a female , and felt a strong sense of purpose in doing female - associated activities such as stitching and embroidering clothes .
as a result , she decided to live her life as a female and thus , discontinued her testosterone injections .
consequently , she had also strongly expressed her wish to become a female , following which , her gender reassignment was performed at the age of 30 years .
she underwent feminizing genitoplasty despite little parental support . at present , she is satisfied with her new female gender identity and did not show gender dysphoria on the female version of gidaa .
however , due to unacceptability from the society and her father , in particular , she has been ostracized from attending familial and sociocultural events .
initially , she admitted feeling anxious and suffering from low self - esteem due to body image issues .
however , with the passage of time and due to hormone replacement therapy with psychological support , she has become comfortable with herself . at present , she seeks a partner for marriage .
patient c.21 , aged 12 years ( prepubertal ) , was brought by his family for masculinizing genital surgery .
a psychiatry consultation with psychological assessment for the patient and family is being sought before acceptance could be given for the same as it involves panhysterectomy followed by lifelong testosterone replacement .
although , to rear a phenotypically female cah patient as a boy is an uncommon event according to the guidelines and standard , with the help of surgical procedures and hormone replacement , normal secondary sexual characteristics can be achieved .
a recent study has shown increased psychiatric morbidity in cah men . to further facilitate the process of psychological adjustment and a good quality of life , social support is required .
both c.21 and c.22 , aged 12 and 8 years , respectively , are satisfied with their male gender identity and their families are supportive of this decision . they have been reared as boys since birth .
fifth stage of ego identity versus role confusion in the psychosocial theory by erik erikson elucidates the effects of dissonance in gender identity during the transition from childhood to adolescence . during this stage ,
a child begins to examine and understand the roles he / she will play as adults .
they explore two crucial identities in depth personal and occupational , and begin to form a personal identity based on the outcome of their experiences .
nevertheless , a failure to establish this sense of personal identity can lead to identity confusion and inadequacy .
thus , a failure to establish and conform to a socially - accepted gender identity can cause psychological maladjustment .
out of the 19 patients reared as females , majority were well - adjusted and showed no gender dysphoria .
two patients revealed gender dysphoria as indicated by their marginally low scores on the gender dysphoria assessment .
however , in view of current literature and the age groups of the patients , behavior of the 6-year - old patient can be best understood as being tomboyish and in the 22-year - old can be explained by the dominance of psychosocial factors and not hormones alone . among the three patients reared as males , two prepubertal were satisfied with their male gender identity .
however , since they are prepubertal , they are in follow - up , it remains to be seen how they progress in future .
the third patient , aged 32 , had gender dysphoria when reared as a male that resolved when feminizing surgery was done , and gender was reassigned as a female .
thus , gender assignment in 46 xx cah and reassignment are guided by several factors that include the degree of virilization of genitalia , gender orientation , patient involvement , parental , and sociocultural influences .
| background : in congenital adrenal hyperplasia ( cah ) with ambiguous genitalia , assigning gender of rearing can be complex , especially if genitalia is highly virilized . apart from karyotype , prenatal androgen exposure , patient 's gender orientation , sociocultural , and parental influences play a role .
the aim of this study was to assess gender dysphoria and psychosocial issues in patients of cah raised as males and females.materials and methods : this is a cross - sectional study that includes patients ( old and new ) with cah who were treated by us in the last 6 months .
a semi - structured interview proforma was used to elicit history and psychosocial background of the patients .
the clinical and biochemical details were noted . for psychological analysis ,
patients were screened for gender dysphoria using parent report gender identity questionnaire for children < 12 years and gender identity / gender dysphoria questionnaire for adolescents and adults.results:we analyzed 22 46 xx cah patients among which , 3 were reared as males and 19 as females . among the 19 patients reared as females , 17 patients showed no gender dysphoria .
two patients revealed gender dysphoria as indicated by their marginally low scores on the gender dysphoria assessment .
however , in view of current literature and the age groups of the patients , behavior of the 6-year - old patient can be best understood as being tomboyish .
gender dysphoria in the 22-year - old can be explained by the dominance of psychosocial factors and not hormones alone . among the three patients reared as males , two prepubertal were satisfied with their male gender identity . the third patient , aged 32 years , had gender dysphoria when reared as a male that resolved when gender was reassigned as female and feminizing surgery was done.conclusion:gender assignment in 46 xx cah is guided by factors such as degree of virilization of genitalia , gender orientation , patient involvement , sociocultural , and parental influences . |
pyomyositis is charactarized by abscesses in the skeletal muscles of mainly the pelvis and lower extremities and is rarely seen in temperate regions . due to an increase in immunocompromised patients the incidence of pyomyositis is rising in the western world .
we describe a case of a young man with tenderness of his muscles , caused by multiple abscesses and swine flu .
a 31 year old , otherwise healthy young man presented to our emergency department with muscle ache of his legs , a sore throat and fever .
physical examination showed subfebrile temperature , pain of both upper legs and buttocks , but no local inflammation or swelling was noticed .
laboratory examination showed slightly elevated levels of coreactive protein ( crp ) , white blood count ( wbc ) and creatine kinase ( ck ) .
swine flu the patient was admitted to the department of internal medicine and therefore oseltamivir treatment was started .
however this treatment rendered no success , since the pain increased and spread to both legs and arms .
the subsequent ct - scan showed multiple abscesses ( fig . 1 , 2 ) in the psoas muscle , in both legs and arms .
all wound and blood cultures showed staphylococcus aureus , mrsa and pv negative . our patient was also treated with intravenous flucloxacilline and rifampicine .
pyomyositis was first described in 1885 by scriba as an infectious disease of mainly the skeletal muscles ( 4 ) .
it can be seen in all stages of life , but most frequent in adolescents , with a male : female ratio of 3:1 .
the incidence of pyomyositis in temperate regions is increasing , due to an increase of predisposing factors , such as immunocompromised patients , e.g. due to hiv , diabetes , malignancies and chronical diseases .
preceding trauma of the affected muscles , by for example fierce exercise , has also been proposed as an important predisposing factor of pyomyositis .
the etiology of pyomyositis however is still unknown ( 2,5,6,7 ) staphylococcus aureus is cultured in 70 - 95 % of the cases , which often is an mrsa strand .
other microorganisms cultured are streptococcus sp , escherichia coli and mycobacterium tuberculosis . cultures and gram - stains are therefore very important to guide proper antibiotic treatment .
( 1,2,3 ) there are three stages of pyomyositis ; stage i is characterized by vague symptoms ; sore muscles , cramping , low - grade fever and discomfort .
most patients only seek medical help in stage ii , because of formation of abscesses in the muscles , approximately 10 - 21 days after the onset of the disease .
if not treated properly this will proceed into stage iii , with serious illness , metastatic abscesses , sepsis , necrosis of the muscles , multiorgan failure and higher mortality .
( 7,8 ) diagnosing pyomyositis may be challenging , due to the vague presentation in stage i.
when the symptoms become more outspoken , apart from history and physical examination , imaging studies are usefull .
mri is the diagnostic tool of choice , because it can detect affected muscles in an early stage .
also more abscesses can be found at unexpected sites , especially when the patient is not responding to treatment .
but , as with our patient , it is of great importance to scan the entire body to detect other abscesses in unexpected regions , if a patient does not respond adequately to the initial treatment .
stage i can be treated with adequate antibiotic therapy alone . in case of abscess formation proper surgical or radiologic drainage of all abscesses
the duration of administration of antibiotics is usually 7 - 10 days , but needs to be adjusted regarding extensiveness of the disease ; number of abscesses , osteomyelitis and immune status of the patient .
( 1 ) if treated properly , most patients heal without additional complications and low mortality rates have been described , up to 1.5% .
( 1 ) the presentation of our patient is very extraordinary , because of the extent of the disease and the abundance of abscesses .
an explanation for the severity of the ilness might be found in the preceding or simultaneous swine flu infection .
myositis due to influenza and even rhabdomyolysis due to h1n1infection have been described ( 9,10 ) .
we consider muscletrauma due to the viral infection to be a predisposing factor for the extensiveness of the disease in this case .
we conclude that pyomyositis can be widespread in patients , which is sometimes hard to diagnose , but should be thought off , especially in times of an influenza pandemic . | pyomyositis is a bacterial infectious disease of the large skeletal muscles , mostly seen in tropical regions .
a case with such a multitude of abscesses has never been described in the western world , nor following an h1n1 infection .
we report of a 31 year old man who presented himself with complaints of muscle pain and fever .
his complaints were attributed to a proven h1n1 infection . however , despite proper therapy his condition worsened .
he had developed multiple abscesses in both arms and legs .
after surgical and radiologic drainage and antibiotic treatment our patient healed without any symptoms .
pyomositis is related with immuno - compromisation .
our patient might have been immunocompromised due to the h1n1 infection .
rhabdomyolysis following influenza has been described before , however a relation with h1n1 never was .
imaging studies help detect and confirm the diagnosis . when missed
, serious complications may arise . |
accessory spleen ( supernumerary spleen , splenule , or splenunculus ) is a focus healthy of splenic tissue separated from the main body of the spleen .
we present a case of acute torsion of a previously diagnosed huge accessory spleen in a young patient .
a 20-year - old woman was admitted to our department with a 5-day history of left - sided abdominal pain accompanied by loss of appetite and a single vomit .
it was first found on ultra sound in the left low quadrant of the abdomen and measured 8.3 cm .
three years later , a routine ultrasound showed enlargement of the accessory spleen to 12 by 7 cm . until this episode , she was asymptomatic .
physical examination revealed a soft abdomen and a tender palpable mass in the left side of her abdomen .
abdominal computed tomographic angiography ( cta ) demonstrated a huge non - enhancing mass in left abdomen measured 17 cm in the largest diameter ( fig .
a twisted vascular pedicle was attached to the lower pole of a small native spleen in its normal anatomical location .
the patient underwent a small midline laparotomy . on exploration , an ischemic accessory huge spleen on a long twisted pedicle
accessory spleens are found in approximately 10% of population and are typically approximately 1 cm in diameter .
the most frequent locations are posteromedial to the spleen ( 22% ) ; anterolateral to the upper pole of the left kidney ; and lateral , posterior , and superior to the tail of the pancreas .
there are few reports describing torsion of accessory spleen presenting as an acute abdomen , most of them in childhood , but also a few in elderly patients .
torsion of an accessory spleen does not relate to the time of diagnosis , size and localization of the spleen or the age of the patient .
torsion of an accessory spleen should be considered in the differential diagnosis of acute abdomen .
the question as whether or not to perform a splenectomy for an asymptomatic known accessory spleen is not well answered in the literature .
however , a prophylactic splenectomy should be considered for a known accessory spleen that increases in size during follow up or has a reasonable potential for complications .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request . | introductionaccessory spleen is a rare condition .
torsion of accessory spleen can lead to acute abdomen.presentation of casewe describe a young woman with an acute abdomen caused by torsion of accessory spleen . abdominal computed tomography angiography ( cta )
demonstrated an ischemic giant accessory spleen with a twisted vascular pedicle .
an emergency laparotomy was performed with resection of the infarcted accessory spleen.discussionaccessory spleen is a rare and asymptomatic condition .
torsion of accessory spleen is also uncommon .
abdominal pain is the main symptom .
cta is effective in reaching a diagnosis .
definitive treatment of an acute abdomen due to accessory splenic torsion is emergency accessory splenectomy.conclusionelective accessory splenectomy should be recommended for known giant accessory spleen to prevent complications in future . |
a 39-year - old male patient presented with a slow - growing , painless , scrotal enlargement with a soft consistency growing over a 2-year duration .
recently , another 2 cm sized , ovoid lesion had been newly palpated on the contralateral spermatic cord area .
his medical history revealed no evidence of previous urinary tract infection , tuberculosis , abdomino - pelvic surgery , or scrotal trauma . on physical examination ,
non - tender , elastic , freely movable masses were palpated at the lower right scrotal base and contralateral inguinal area , which was adjacent to the left spermatic cord .
testicular tumor markers ( alpha - fetoprotein , beta - chorionic gonadotropin , and lactate dehydrogenase ) and hormones were within the normal limits .
gray scale ultrasonography with high frequency transducer showed an oval - shaped , homogenous , hypoechoic , well - encapsulated solid mass with scattered internal thin septa - like structures without evidence of calcification or communication with the testis parenchyma in the caudal scrotum .
the tumor on the spermatic cord area showed a very similar ultrasonographic appearance ( fig .
the color flow doppler sonography showed no intratumoral vascular signals , suggesting that they were not typical solid masses . on the pelvic mri ,
the masses showed homogenous , low signal intensity on t1-weighted images and intermediate signal intensity on t2-weighted images .
furthermore , they were located within the subcutaneous layer and sharply demarcated from surrounding structures .
gadolinium - enhanced t1-weighted images showed peripheral rim enhancement without significant internal enhancement , suggestive of a cystic structure encapsulated within a thin fibrous capsule ( fig .
an incidental cystic mass was also detected at the medial portion of the right upper thigh .
the scrotal mass was a 55 cm sized cystic mass without any connection with the adjacent testis , filled compactly with a brownish soft , pastelike material on gross inspection .
the cystic wall showed no solid portion without evidence of rupture or secondary foreign body - type granulomatous reaction or abscess ( fig .
the inguinal mass was easily separated from the layers of the spermatic cord , and it also contained brownish sebaceous material in the cyst .
there were no teratomatous components or adnexal structures such as sebaceous glands , hair follicles , or sweat glands within the cystic lumen .
microscopic evaluation demonstrated a cystic structure , lined by keratinized squamous epithelium with laminated keratin materials in the lumen ( fig .
an epidermoid cyst is the most common benign simple epithelial cyst without malignant potential , most often found on the scalp , face , trunk , and back .
it is characterized histologically by a cyst lining of stratified squamous cells and loosely packed lamellae of keratin debris , cholesterol , and water without teratomatous elements or skin appendages in the stromal tissue .
the histogenesis of the epidermoid cyst is not precisely known , but there are different theories about the embryonic origin of this lesion : ( i ) they arise from ectopic cutaneous tissue due to dislocation of tissue into a neighboring area ; ( ii ) they are end results of monolayer teratoma from germ cells ; ( iii ) they occur due to traumatic implantation of epidermal tissue into the dermis and subcutis . however , in the case of the extratesticular scrotal epidermoid cyst , they are believed to be an abnormal closure or associated degenerative process of the median raphe and urethral groove .
scrotal ultrasonography is usually the first and the most important imaging modality to investigate scrotal and inguinal pathologic conditions , and it can reliably differentiate extratesticular from intratesticular ones , as well as cystic from solid ones , with very high accuracy .
the sonographic appearance of an epidermal cyst varies from an anechoic lesion to a hyperechoic , solid appearing mass , depending on its content . although there are no specific pathognomonic findings to diagnose the epidermoid cysts accurately , they have most often appeared with well demarcated hypoechoic masses with multiple scattered reflectors from the keratinous debris and posterior sound enhancement without internal vascular signals on the color flow doppler sonography .
some cases show specific manifestations of an onionskin appearance of concentric , swirling rings of alternating dense echogenicity and sonolucency .
this is pathologically well correlated with alternating layers of laminating keratin debris and loosely scattered desquamated squamous cells . in other cases , the keratinized debris primarily collected centrally , producing a centrally dense echogenic focus that has been described as having the appearance of a " target " of a " bull's - eye " as the keratinized debris primarily collects centrally , producing a central echogenic focus .
dynamic - enhanced mr imaging is very precise in showing the pelvis structures and frequently gives additional useful information to help to characterize the mass .
epidermoid cysts have usually shown as thin - walled cysts with hypointensity on t1-weighted images and hyperintensity on t2-weighted images .
epidermoid cysts have usually shown internal heterogenous intensity on t1- and t2-weighted images due to inner dense keratinous materials .
gadolinium - enhanced images usually show no enhancement with or without a peripheral enhancement of the cystic wall
. tumors of the spermatic cord and paratesticular tissue are uncommon and mostly benign . the most common benign paratesticular tumor is mesothelioma of the epididymis , closely followed by lipoma , leiomyoma , fibroma , and hemangioma .
primary malignant epithelial tumors of the paratesticular region are exceedingly rare and are mainly known to be rhabdomyosarcoma .
complete local excision is considered the treatment of choice for an epidermoid cyst because there have been no reported cases of malignant transformation or distant metastasis in the literature , in contrast with dermoid cysts , which have malignant potential .
in addition , cross - sectional imaging studies such as scrotal sonography and mri are unable to differentiate a benign from a malignant mass accurately , so complete local excision with intraoperative frozen section biopsy should be done when malignancy is suspected . | an extratesticular scrotal epidermoid cyst is a relatively very rare condition , and an epidermoid cyst arising from the spermatic cord area is extremely rare .
we report a case of multiple epidermoid cysts arising from the extratesticular scrotum , spermatic cord , and lower extremities . to our best knowledge , concomitant occurrence of these lesions
has not been reported previously in the literature . |
we studied 29 eyes of 25 patients with exudative armd complicated by rap of variable onset that were identified from january , 2007 to january , 2011 .
the diagnosis of rap was based on angiographic evidence ( fa and icga ) of an intraretinal neovascular complex connected with the retinal circulation , the patients were subjected to one of the three vasogenic stages described by yannuzzi et al . (
stage i : intraretinal neovascularization , stage ii : subretinal neovascularization with or without ped , and stage iii : cnv with rca ) .
all patients were older than 53 years and exhibited unique presentations of armd , such as soft or exudative drusen , retinal pigment epithelium ( rpe ) pigmentation and geographic atrophy .
none of the patients had other neovascular maculopathy that may be complicated by chorio - retinal anastomosis , such as infectious or inflammatory chorioretinal disease , angioid streaks , hereditary retinal disorders or trauma .
we performed a complete ophthalmologic examination including fa ( digital retinal camera cx-1 ; canon , tokyo , japan ) , dynamic icga ( hra2-kt-02972 ; heidelberg engineering , heidelberg , germany ) and oct ( spectral oct slo ; a division of opko health , toronto , canada ) in all 29 eyes .
a total of 29 eyes of 25 patients diagnosed with rap were examined in this study .
the patients were all of asian descent , including 10 ( 40% ) men and 15 ( 60% ) women .
the mean age of the patients was 65.5 7.7 years ( range , 53 to 81 years ) .
the best - corrected visual acuity ( bcva ) of the patients at initial and final visits were 0.74 0.31 and 0.57 0.17 , respectively ( table 1 ) .
based on the fa angiograms , all eyes demonstrated an ill - defined leakage which produced an indiscreet continuous zone within and beneath the retina .
a hyperreflective focal area inside the neuroretinal layers , corresponding to the intraretinal vascular complex seen in fa and icga , was detected in all oct scans .
oct revealed drusen in 29 eyes ( 100% ) ; out of these 29 eyes , 24 eyes had inner retinal cyst . in 18 eyes the inner retinal cysts were accompanied by the outer retinal cystoid change , whereas in eight eyes there were no outer retinal cystoid changes .
fibrovascular pigment epithelium detachment ( fvped ) which was observed in 23 eyes ( 79% ) , 18 of which also had outer retinal cystoids change .
no patient had outer retinal cystoid change or subretinal detachment ( srd ) without other accompanying findings .
of the 11 eyes evidencing srd , all 11 also displayed fvped , and nine eyes ( 82% ) also had ped .
bcva at initial and final visit of each of the 6 groups according to the oct findings were also analyzed ( table 2 ) .
drusen are formed by the deposition of lipofuscin ( a type of retinal waste product ) and by the separation of rpe from its underlying basement membrane .
drusen are characterized by hyperreflective mounds beneath the rpe layer on oct that , at large sizes , may displace retinal tissue . in our experience , drusen is present in 100% of cases previously diagnosed as rap ; yet it is almost always accompanied by the other findings observed in our study including elevation and detachment of different layers of retina ( fig .
inner retinal cyst formation is often present with cystoid macular edema and is represented by round , minimally reflective ( darker ) spaces within the neurosensory retina .
large cysts may be able to expand to the entire thickness of the retina from the retinal pigment epithelium / choriocapillaris ( the anterior boundary of neurosensory retina ) . in this study ,
four of the patients were initially diagnosed with unilateral rap but later on rap developed in the fellow eyes . in these patients ,
the first common finding in eyes without rap was inner retinal cysts , which were present even when cnv was not detected ( fig .
2 ) . along with neovascularization , disruption of the normal outer retinal and inner choroidal anatomy result in fibrovascular proliferation along with the accumulation of fluid in the subretinal pigmented epithelium ( sub - rpe ) space , the subretinal space , and within the retina . in our study ,
79% of the eyes with rap showed fvped accompanied by retinal and subretinal tissue thickening ( fig .
are cystic lesions confined to the retina 's outer nuclear layer ; they are usually located next to a pre - retinal fibrotic area or an area of retinal atrophy .
fa reveals minute dots of fluorescence which correspond to leakages , typically in the late phase . in this study , a majority of the patients had cystoid changes in the outer retinal layer than orcs where hyperreflective rings surrounding cystic cavities were absent on oct when advanced .
however , 66% of the patients demonstrated such alterations in the outer retinal layer which was sufficient to be considered meaningful ( fig .
subretinal fluid appears as a non - reflective ( dark ) space between the posterior boundary of the neurosensory retina and the retinal pigment epithelium / choriocapillaris reflection on oct .
apart from intraretinal edema , the nonreflective area is not present within the neurosensory retina .
the neurosensory retina overlaying the area of subretinal fluid is typically not thickened on oct images except when the subretinal fluid has been chronic ( fig .
detached rpe is slightly more reflective where increased reflectivity of the rpe band severely shadows reflections from the choroid .
hemorrhagic rpe detachments are distinguished by a moderately reflective layer directly beneath the detached rpe .
fibrovascular rpe detachments demonstrate moderate reflectivity throughout the entire sub - rpe space . in this study , even in cases without clearly demonstrated cnv , ped on oct were accompanied by fvped and/or srd in all patients , which implicates the presence of cnv at some level .
cnv may have been masked by fluids , hemorrhages or space - occupying lesions ( fig .
drusen are formed by the deposition of lipofuscin ( a type of retinal waste product ) and by the separation of rpe from its underlying basement membrane .
drusen are characterized by hyperreflective mounds beneath the rpe layer on oct that , at large sizes , may displace retinal tissue . in our experience , drusen is present in 100% of cases previously diagnosed as rap ; yet it is almost always accompanied by the other findings observed in our study including elevation and detachment of different layers of retina ( fig .
inner retinal cyst formation is often present with cystoid macular edema and is represented by round , minimally reflective ( darker ) spaces within the neurosensory retina .
large cysts may be able to expand to the entire thickness of the retina from the retinal pigment epithelium / choriocapillaris ( the anterior boundary of neurosensory retina ) . in this study ,
four of the patients were initially diagnosed with unilateral rap but later on rap developed in the fellow eyes . in these patients ,
the first common finding in eyes without rap was inner retinal cysts , which were present even when cnv was not detected ( fig .
along with neovascularization , disruption of the normal outer retinal and inner choroidal anatomy result in fibrovascular proliferation along with the accumulation of fluid in the subretinal pigmented epithelium ( sub - rpe ) space , the subretinal space , and within the retina . in our study ,
79% of the eyes with rap showed fvped accompanied by retinal and subretinal tissue thickening ( fig .
outer retinal cysts ( orcs ) are cystic lesions confined to the retina 's outer nuclear layer ; they are usually located next to a pre - retinal fibrotic area or an area of retinal atrophy .
fa reveals minute dots of fluorescence which correspond to leakages , typically in the late phase . in this study , a majority of the patients had cystoid changes in the outer retinal layer than orcs where hyperreflective rings surrounding cystic cavities were absent on oct when advanced .
however , 66% of the patients demonstrated such alterations in the outer retinal layer which was sufficient to be considered meaningful ( fig .
subretinal fluid appears as a non - reflective ( dark ) space between the posterior boundary of the neurosensory retina and the retinal pigment epithelium / choriocapillaris reflection on oct .
apart from intraretinal edema , the nonreflective area is not present within the neurosensory retina .
the neurosensory retina overlaying the area of subretinal fluid is typically not thickened on oct images except when the subretinal fluid has been chronic ( fig .
detached rpe is slightly more reflective where increased reflectivity of the rpe band severely shadows reflections from the choroid .
hemorrhagic rpe detachments are distinguished by a moderately reflective layer directly beneath the detached rpe .
fibrovascular rpe detachments demonstrate moderate reflectivity throughout the entire sub - rpe space . in this study , even in cases without clearly demonstrated cnv , ped on oct were accompanied by fvped and/or srd in all patients , which implicates the presence of cnv at some level .
cnv may have been masked by fluids , hemorrhages or space - occupying lesions ( fig .
rap has emerged as an increasingly acknowledged variant of neovascular armd [ 4,14 - 16 ] .
branches of new vessels extend into the subretinal space resulting in rpe detachments and anastomosis with choroidal neovascular complexes .
fa often results in angiographic presentations of occult or minimally classic cnv in patients with rap .
when large ped are presence , it had been difficult to reveal in rap lesion by fa .
cnv is known to originate from the choroid , then ped is formed and the fluid leaks into the retina
. however , rap originates where leakage in the retina occurs , and ped is subsequently formed .
this may be why it is difficult to detect the exact location where rap lies with fa , especially in the earliest stages of the disease with ped in rap lesions , due to the presence of intraretinal fluid .
icga is a useful tool in determining the nature of neovascularization , which appears as indistinct hyperfluorescence in fa , because it establishes the relationship between rap and retinal circulation [ 3,10 - 12 ] .
however , both fa and icga are tools that give us information by two dimension image .
employed stratus oct evaluation and revealed cnv with a small surface area beneath circular and irregular rap lesions in focal or multifocal rap , which provided better perspectives for understanding this disease .
thus , we believe that , investigating the non - cnv lesions that commonly accompany rap lesions with spectral domain oct may be helpful in the diagnosis and consideration of subsequent treatment .
we investigated lesions that may occur alone or in combination in patients with rap , and did so independently from the classification and staging proposed by yannuzzi et al . since the true nature of rap lesions had not been clearly identified .
drusen , hyperfluorescent , ill - defined confluent lesions on fa which are tiny yellow or white accumulations of extracellular material that build up in bruch 's membrane
. the presence of a few small drusen is normal with advancing age ( > 40 years old ) , especially in patients with progressing armd [ 18 - 20 ] .
drusen are typically found easily in patients with rap ; this may not be a diagnostic finding ; but it may be helpful in locating other lesions and which layers of they lie within , since drusen usually appear in combination with others findings including srd and ped .
the biomicroscopic location of rap lesion is difficult to assess because it is usually masked by intraretinal proliferations including edema and hemorrhages . in the present study , intraretinal serous edema
inner retinal cysts are lesions typically accompanied by cavitation and the subsequent retinal swelling caused by accumulation of intraretinal fluid , and their layer of origin can be difficult to detect fa and/or icga [ 6,7 - 9 ] . although oct is an excellent tool for diagnosis of the retinal alterations surrounding rap lesions , it is limited in its ability to distinguish true inner retinal cysts from pseudocystic cavities . in the present study , whether inner retinal cysts found in rap lesions were true cysts or pseudocystic , they were the second most commonly found lesion , after drusen ( table 2 ) .
moreover , regardless of their size , cnv appeared to develop subsequently and adjacent to inner retinal cysts , as a manifestation of the progression to rap .
along with the neovascularization of new vessels growing from the intraretinal complex of rap , scar tissue called fibrovascular proliferation as well as fvped may form on the surface of the retina .
the fibrovascular tissue may distort the overlying rpe and outer retinal barrier leading to the spread of new vessels into subretinal space or into the neurosensory retina , causing serous detachment of the retina .
on fa , fvped appears stippled hyperfluorescence within irregularly elevated ped , which shows leakage of an undetermined source , also in a stippled pattern .
all of the cases with fvped found in our study were complicated by srd and/or ped .
orcs are a new type of cystic structure recently identified in patients with amd , and can be differentiated from intraretinal exudates and cystoid cavities lying in the outer plexiform layer by their surrounding hyperreflective rings .
reported that tubulations in the outer retinal layer result from sublethal injury to the photoreceptors and disruption of the rpe layer . when outer retinal cystoid changes are observed in patients with rap , vision- threatening conditions
serous detachments of the rpe appear as optically clear areas between the rpe and the choriocapillaris , and a detachment can be single or , multiple , dome - shaped , or bilobed .
when rap reaches the subretinal space and merges with the rpe , a serous detachment of the rpe will develop .
srd demonstrates leakage of fluorescein in the corresponding area on fa and subsequent subretinal concretions may be indicative of spontaneously resolved previous serous detachments .
the progression of rap on oct is characterized by the presence of intraretinal ( well - defined confluent hyporeflective ) spaces or subretinal fluid ( a well - defined , hyporeflective space between the neurosensory foveal retina and other highly reflective bands corresponding to the rpe ) .
ped appears to be a dome - shaped , optically empty space lined by a highly reflective layer of rpe .
serous ped can often be found in the macular area where cnv lies at the margin or within the ped , and on icga , it can be revealed as a variable , minimal blockage of normal choroidal vessels usually evident in the midphase .
sato et al . suggested that the low height of small ped may indicate that the detached rpe and bruch 's membrane are connected by cnv . in a comparison of bcva
, patients with fvped and ped showed relatively lower visual acuity than patients with inner retinal cyst , outer retinal cystoid change and srd at initial visit .
moreover , those groups with lower initial bcva did not show improved bcva at final visit and still showed lower levels of visual acuity regardless of the conventional treatment of rap ( table 2 ) .
however , analyzing the bcva of patients in this study may not be meaningful , since the six oct findings appeared in combinations of at least two findings , with drusen present in all cases .
however , it may be used as a reference point to evaluate whether more aggressive treatment should be pursued based on the type of oct finding .
thus , the course of treatment for rap patients who have fvped and ped as demonstrated by oct should be carefully considered , as they tend to show lower bcva than patients with other findings . in conclusion
, we analyzed oct findings of rap patients and revealed six different types of lesions distinct to rap .
these findings may be a useful tool in diagnosis , elucidating the clinical course of the disease , choosing an appropriate course of treatment and predicting prognosis .
in such lesions , oct provides more accurate information than fa or high speed icga , and it can be preferable to other diagnostic tools in understanding rap . moreover , both inner retinal cysts and fvped commonly occur in patients with rap , yet only inner retinal cysts as an early finding , may be a predictive of development to rap , since fvped is more likely to appear in more advanced stages
. limitations of this study include its retrospective nature , the relatively small sample size , short follow - up period and lack of anatomic landmarks in scanning and analyzing oct .
further large , prospective studies based on sd - oct technology with long follow - up periods may be necessary to discover more features of rap that can support our findings of this study . | purposeto identify the unique pathologic findings of retinal angiomatous proliferation ( rap ) in optical coherence tomography ( oct).methodsretrospectively , 29 eyes of 25 patients with age - related macular degeneration and complicated rap were analyzed .
all 29 eyes had choroidal neovascularization ( cnv ) in the area of pigment epithelial detachment ( ped ) or adjacent to it , which was visible with fluorescein angiography or indocyanine green angiography .
cross - sectional images were obtained by oct scanning through the cnv lesions.resultssix distinctive findings of oct included drusen ( 100% ) , inner retinal cyst ( 80% ) , outer retinal cyst ( 68% ) , fibrovascular ped ( 84% ) , serous retinal detachment ( 40% ) , and ped ( 68%).conclusionsthrough analysis of oct findings , we revealed six different types of lesions distinctive of rap which may provide helpful diagnostic information for subsequent treatment and predicting the prognosis of rap . |
one hundred and eighteen postmenopausal women ( no menstruation for 12 months ) , aged 5174 years and receiving standard u.k .
care for type 2 diabetes ( including statin therapy , established for 12 months and glycemic target of hba1c of 7.0% ) were recruited through general practitioners , specialist diabetes clinics , and local media advertisements ( fig .
exclusion criteria included use of hormone replacement therapy ( within 6 months ) , known allergy to the study foods , history of vascular disease or cancer , and poor diabetes control or raised bp at screening ( as outlined in fig .
all study investigations were conducted according to the principles expressed in the declaration of helsinki and the study was approved by the norfolk research ethics committee .
subjects were randomized to placebo ( n = 59 ) or flavonoid - enriched ( n = 59 ) chocolate daily for 1 year .
treatment allocation was stratified according to age , bmi , years since menopause , insulin use , and patient characteristics are shown in table 1 .
baseline characteristics of the 93 postmenopausal patients with type 2 diabetes who completed the 1-year flavonoid intervention the daily intake of 27 g flavonoid - enriched chocolate ( conceptualized by the research team and formulated and produced by barry callebaut [ lebbeke - wieze , belgium ] specifically for the trial ) provided 90 mg epicatechin ( 850 mg total flavan-3-ols ) and 100 mg isoflavones ( aglycone equivalents ) , the optimal dose for biological efficacy on the basis of published findings from previous short - term trials ( 18,19 ) .
flavonoid extracts were purchased ( flavan-3-ols and acticoa cocoa [ barry callebaut ] ; isoflavones and soylife40 [ frutarom , veenendaal , the netherlands ] ) , and intervention and placebo chocolates were produced by barry callebaut , which were matched for macronutrient content , appearance , and taste . batch - to - batch variability and effect of storage on flavonoid
content were independently determined using high - performance liquid chromatography ( hplc ) ( 20 ) .
chocolate ( 13.5 g ) was consumed twice daily ( lunchtime and evening ) to maintain circulating levels of metabolites , given our knowledge of the half - life of the compounds ( 21 ) .
subjects were advised to exchange the study chocolate with foods of similar nutritional content ( total daily intake provided 152 kcal , 10 g fat , and 13 g carbohydrate ) .
adherence to intervention was determined by counting returned wrappers and objectively assessed through quantification of 24-h urinary total epicatechin ( sum of epicatechin , 3-methyl epicatechin , 4-methyl epicatechin , epicatechin-3-sulfate , and epicatechin - methyl - sulfate ) and isoflavone ( daidzein , genistein , and equol ) excretion levels at 6 and 12 months determined by hplc with mass spectrometric ( ms ) detection after enzyme hydrolysis using established methods .
intra - assay coefficients of variation ( cvs ) for total epicatechin , daidzein , genistein , and equol levels were 7.7 , 6.3 , 8.0 , and 8.1% , respectively , and interassay cvs were 9.1 , 11.0 , 14.1 , and 8.6% , respectively .
one week preceding and during the 1-year trial , habitual diet and exercise routines were maintained , with the exception of restricting the intake of specific flavonoid - rich foods ( a list of which was provided ) .
four - day diet diaries were used to assess habitual dietary intake at baseline , 6 months , and 12 months ( supplementary table 1 ) , and adherence to study protocol was monitored by research staff between study assessments .
additionally , for 24 h before study assessments , subjects were asked to refrain from caffeine , alcohol , and strenuous exercise .
each volunteer consumed a low - flavonoid meal the evening before each assessment followed by an overnight fast ( > 10 h ) . at baseline and 12 months , vascular measures were made at the research facility , including 2-h ambulatory bp monitoring ( spacelabs healthcare , issaquah , wa ) , with recordings every 10 min .
1c ) , with levels of fasting glucose and lipoproteins ( total cholesterol , hdl - cholesterol [ hdl - c ] , and triglycerides ) measured photometrically using a clinical chemistry autoanalyzer ( architect c systems autoanalyzer ; abbott laboratories , abbott park , il ) .
fasting hba1c was assessed by hplc ( a. menarini diagnostics , florence , italy ) .
ldl - cholesterol ( ldl - c ) was determined using the friedewald equation ( 22 ) .
anthropometric measures ( body weight and height ) were taken in duplicate , according to standardized protocols . from banked plasma , insulin concentrations
were assessed using routine methods ( insulin elisa ; mercodia , uppsala , sweden ) .
the intra - assay coefficients of variation were 2.90% ( hba1c ) , 2.82% ( glucose ) , 1.24% ( total cholesterol ) , 3.01% ( hdl - c ) , 1.43% ( triglycerides ) , and 3.84% ( insulin ) .
insulin resistance and insulin sensitivity were calculated using the homeostasis model assessment of insulin resistance ( homa - ir ) equation , homa - ir = glucose insulin/22.5 ( 23 ) , and the quantitative insulin sensitivity index ( quicki ) ( 24 ) equation , quicki = 1/(log [ fasting insulin ] + log [ fasting glucose ] ) , respectively .
homa - ir , quicki , glucose , and insulin data are reported for n = 45 ( in both groups ) , triglycerides levels for n = 45 flavonoid and n = 44 placebo subjects , and ldl - c levels for n = 45 flavonoid and n = 42 placebo subjects .
ten - year estimated risks for coronary heart disease ( chd ) , fatal chd , stroke , and fatal stroke were calculated using the uk prospective diabetes study ( ukpds ) risk engine ( version 2.0 , www.dtu.ox.ac.uk/ukpds_trial/index.php ; diabetes trials unit , university of oxford , oxford , u.k . ) .
the ukpds risk engine includes age , diabetes duration , sex , presence of atrial fibrillation , ethnicity , smoking status , hba1c ( % ) , systolic bp ( mmhg ) , total cholesterol ( mmol / l ) , and hdl - c ( mmol / l ) .
the last observation carried forward was used to interpolate incomplete data ( n = 3 observations for hba1c , cholesterol , and hdl - c ) .
mol / l ) and 12 months ( 12 mol / l ) , differences in patient characteristics were assessed using student independent - sample t test ( continuous data ) and pearson test ( categorical data ) .
data are presented for those completing the 1-year intervention per protocol , and the primary test of effect was univariate ancova analysis ( intervention group as fixed factor [ flavonoid / placebo ] versus change in variable [ pre ( 0 mol / l)/post ( 12 mol / l ) ] , with baseline value of variable as covariate ) .
further sensitivity analysis was conducted to exclude for low compliers ( returning 70% wrappers ) , those with annual change in bmi of greater than or equal to 5 kg / m , and one volunteer with atrial fibrillation ( n = 5 flavonoid ; n = 4 placebo ) .
, new york , ny ) , and statistical significance was defined as p < 0.05 .
one hundred and eighteen postmenopausal women ( no menstruation for 12 months ) , aged 5174 years and receiving standard u.k .
care for type 2 diabetes ( including statin therapy , established for 12 months and glycemic target of hba1c of 7.0% ) were recruited through general practitioners , specialist diabetes clinics , and local media advertisements ( fig .
exclusion criteria included use of hormone replacement therapy ( within 6 months ) , known allergy to the study foods , history of vascular disease or cancer , and poor diabetes control or raised bp at screening ( as outlined in fig .
all study investigations were conducted according to the principles expressed in the declaration of helsinki and the study was approved by the norfolk research ethics committee .
subjects were randomized to placebo ( n = 59 ) or flavonoid - enriched ( n = 59 ) chocolate daily for 1 year .
treatment allocation was stratified according to age , bmi , years since menopause , insulin use , and patient characteristics are shown in table 1 .
baseline characteristics of the 93 postmenopausal patients with type 2 diabetes who completed the 1-year flavonoid intervention the daily intake of 27 g flavonoid - enriched chocolate ( conceptualized by the research team and formulated and produced by barry callebaut [ lebbeke - wieze , belgium ] specifically for the trial ) provided 90 mg epicatechin ( 850 mg total flavan-3-ols ) and 100 mg isoflavones ( aglycone equivalents ) , the optimal dose for biological efficacy on the basis of published findings from previous short - term trials ( 18,19 ) .
flavonoid extracts were purchased ( flavan-3-ols and acticoa cocoa [ barry callebaut ] ; isoflavones and soylife40 [ frutarom , veenendaal , the netherlands ] ) , and intervention and placebo chocolates were produced by barry callebaut , which were matched for macronutrient content , appearance , and taste .
batch - to - batch variability and effect of storage on flavonoid content were independently determined using high - performance liquid chromatography ( hplc ) ( 20 ) .
chocolate ( 13.5 g ) was consumed twice daily ( lunchtime and evening ) to maintain circulating levels of metabolites , given our knowledge of the half - life of the compounds ( 21 ) .
subjects were advised to exchange the study chocolate with foods of similar nutritional content ( total daily intake provided 152 kcal , 10 g fat , and 13 g carbohydrate ) .
adherence to intervention was determined by counting returned wrappers and objectively assessed through quantification of 24-h urinary total epicatechin ( sum of epicatechin , 3-methyl epicatechin , 4-methyl epicatechin , epicatechin-3-sulfate , and epicatechin - methyl - sulfate ) and isoflavone ( daidzein , genistein , and equol ) excretion levels at 6 and 12 months determined by hplc with mass spectrometric ( ms ) detection after enzyme hydrolysis using established methods .
intra - assay coefficients of variation ( cvs ) for total epicatechin , daidzein , genistein , and equol levels were 7.7 , 6.3 , 8.0 , and 8.1% , respectively , and interassay cvs were 9.1 , 11.0 , 14.1 , and 8.6% , respectively .
one week preceding and during the 1-year trial , habitual diet and exercise routines were maintained , with the exception of restricting the intake of specific flavonoid - rich foods ( a list of which was provided ) .
four - day diet diaries were used to assess habitual dietary intake at baseline , 6 months , and 12 months ( supplementary table 1 ) , and adherence to study protocol was monitored by research staff between study assessments .
additionally , for 24 h before study assessments , subjects were asked to refrain from caffeine , alcohol , and strenuous exercise .
each volunteer consumed a low - flavonoid meal the evening before each assessment followed by an overnight fast ( > 10 h ) . at baseline and 12 months , vascular measures were made at the research facility , including 2-h ambulatory bp monitoring ( spacelabs healthcare , issaquah , wa ) , with recordings every 10 min .
1c ) , with levels of fasting glucose and lipoproteins ( total cholesterol , hdl - cholesterol [ hdl - c ] , and triglycerides ) measured photometrically using a clinical chemistry autoanalyzer ( architect c systems autoanalyzer ; abbott laboratories , abbott park , il ) .
fasting hba1c was assessed by hplc ( a. menarini diagnostics , florence , italy ) .
ldl - cholesterol ( ldl - c ) was determined using the friedewald equation ( 22 ) .
anthropometric measures ( body weight and height ) were taken in duplicate , according to standardized protocols . from banked plasma , insulin concentrations
were assessed using routine methods ( insulin elisa ; mercodia , uppsala , sweden ) .
the intra - assay coefficients of variation were 2.90% ( hba1c ) , 2.82% ( glucose ) , 1.24% ( total cholesterol ) , 3.01% ( hdl - c ) , 1.43% ( triglycerides ) , and 3.84% ( insulin ) .
insulin resistance and insulin sensitivity were calculated using the homeostasis model assessment of insulin resistance ( homa - ir ) equation , homa - ir = glucose insulin/22.5 ( 23 ) , and the quantitative insulin sensitivity index ( quicki ) ( 24 ) equation , quicki = 1/(log [ fasting insulin ] + log [ fasting glucose ] ) , respectively .
homa - ir , quicki , glucose , and insulin data are reported for n = 45 ( in both groups ) , triglycerides levels for n = 45 flavonoid and n = 44 placebo subjects , and ldl - c levels for n = 45 flavonoid and n = 42 placebo subjects .
ten - year estimated risks for coronary heart disease ( chd ) , fatal chd , stroke , and fatal stroke were calculated using the uk prospective diabetes study ( ukpds ) risk engine ( version 2.0 , www.dtu.ox.ac.uk/ukpds_trial/index.php ; diabetes trials unit , university of oxford , oxford , u.k . ) .
the ukpds risk engine includes age , diabetes duration , sex , presence of atrial fibrillation , ethnicity , smoking status , hba1c ( % ) , systolic bp ( mmhg ) , total cholesterol ( mmol / l ) , and hdl - c ( mmol / l ) .
the last observation carried forward was used to interpolate incomplete data ( n = 3 observations for hba1c , cholesterol , and hdl - c ) .
mol / l ) and 12 months ( 12 mol / l ) , differences in patient characteristics were assessed using student independent - sample t test ( continuous data ) and pearson test ( categorical data ) .
data are presented for those completing the 1-year intervention per protocol , and the primary test of effect was univariate ancova analysis ( intervention group as fixed factor [ flavonoid / placebo ] versus change in variable [ pre ( 0 mol / l)/post ( 12 mol / l ) ] , with baseline value of variable as covariate ) .
further sensitivity analysis was conducted to exclude for low compliers ( returning 70% wrappers ) , those with annual change in bmi of greater than or equal to 5 kg / m , and one volunteer with atrial fibrillation ( n = 5 flavonoid ; n = 4 placebo ) .
, new york , ny ) , and statistical significance was defined as p < 0.05 .
ninety three patients completed the trial , and subject characteristics and key measures of diabetes control were similar between groups at baseline ( table 1 ) ; age ranged from 51 to 74 years , and bmi was 21.557.9 kg / m .
there were 25 withdrawals during the trial ( 21% of those randomized ) , with almost equal numbers in both groups ( fig .
there were no differences in baseline characteristics of the 118 subjects randomized to treatment ( n = 118 in total ; n = 59 in flavonoid and placebo group ) ( data not shown ) . in the 93 participants who completed the study ,
the mean 1-year adherence to intervention treatment was high , with 91.3 and 91.6% of chocolate bars consumed for the flavonoid and placebo groups , respectively ( determined by wrapper returns ) .
adherence in the flavonoid group was confirmed by the sustained increases in total epicatechin and isoflavone levels in 24-h urine at 6 and 12 months ( total epicatechin 148.5 12.3 and 154.1 11.1 mol / day ; specifically 3-methyl - epicatechin 6.4 0.6 and 7.0 0.6 mol / day , 4-methyl - epicatechin 1.7 0.2 and 1.5 0.2 mol / day , epicatechin-3-sulfate 52.7 4.8 and 58.6 5.1 mol / day , epicatechin - methyl - sulfate 75.5 7.1 and 73.1 5.6 mol / day , daidzein 77.5 4.9 and 75.2 5.3 mol / day , respectively , in hydrolyzed samples at 6 and 12 months ) .
thirty six percent of the flavonoid group were equol producers , determined according to standard methods ( 25 ) .
the intervention attenuated insulin resistance ( 0.3 0.2 homa - ir flavonoid group , 0.4 0.2 placebo group ; p = 0.004 ) and improved insulin sensitivity , effects that were driven by a significant decrease in insulin levels ( table 2 ) .
we also observed significant changes in lipoprotein status : total - c : hdl - c ratio ( flavonoid 0.2 0.1 , placebo 0.0 0.6 ; p = 0.01 ) , ldl - c levels ( flavonoid 0.1 0.1
mmol / l , placebo 0.1 0.1 mmol / l ; p = 0.04 ) , and hdl - c : ldl - c ratio ( flavonoid 0.06 0.0 , placebo 0.03 0.0 ; p = 0.003 ) ( table 2 ) .
the intervention also had a significant impact on estimated percent chd risk , with risk attenuated in the intervention group relative to placebo for chd ( 0.1 0.3 vs. 1.1 0.3% , respectively ; p = 0.02 ) and a trend toward a reduction in fatal chd ( p = 0.05 ) ( table 2 ) .
the intervention had no effect on bp or glycemic control ( hba1c and glucose levels ) ( table 2 ) .
effect of 1-year combined intervention with flavan-3-ols and isoflavones on biomarkers of cvd risk in medicated postmenopausal patients with type 2 diabetes in sensitivity analyses , a similar magnitude of effect of the flavonoid intervention on homa - ir and insulin ( fig .
2 ) and estimated 10-year risk of chd ( flavonoid 0.0 0.4% , placebo 1.2 0.4% ; p = 0.02 ) was observed .
moreover , stronger statistical associations were observed for the effects on estimated 10-year risk of fatal chd ( flavonoid 0.3 0.3% , placebo 1.1 0.3% ; p = 0.04 ) and lipoprotein status : total c : hdl - c ratio ( flavonoid 0.2 0.1 , placebo 0.0 0.1 ; p = 0.004 ) and ldl - c ( flavonoid 0.1 0.1
mmol / l , placebo 0.1 0.1 mmol / l ; p = 0.03 ) .
p < 0.05 , * * p = 0.01 ; n = 42 flavonoid , n = 42 placebo .
in this 1-year , randomized , placebo - controlled intervention trial , a combined intake of flavan-3-ols and isoflavones significantly improved lipoprotein status and markers of insulin sensitivity and attenuated the estimated 10-year risk of chd in postmenopausal women receiving standard therapy for type 2 diabetes . to our knowledge , this is the only long - term flavonoid trial that has been conducted in medicated postmenopausal patients with type 2 diabetes , and these data highlight the additional benefit of dietary flavonoids to standard drug therapy in managing cvd risk in these patients .
all patients were receiving statin therapy ( 40 mg simvastatin or 10 mg atorvastatin ) as part of standard medical care , but flavonoid intervention resulted in further improvements in lipoprotein status , specifically significant reductions in ldl - c and total - c : hdl - c ratio .
the observed reduction of 0.16 mmol / l ldl - c is consistent with data from the meta - analysis of short term ( maximum 12 weeks ) cocoa ( 0.15 mmol / l ) ( 26 ) and soy isoflavone ( 0.13
mmol / l ) ( 14 ) intervention trials in healthy participants and with the magnitude of change observed in the few short - term trials on soy isoflavones ( 16,17 ) or chocolate ( 15 ) in type 2 diabetic patients .
our findings do not suggest any additive effect of our combined intervention on lipoprotein status in this medicated group .
a 1.0 mmol / l reduction in ldl - c has been associated with a 21% decrease in vascular events in patients with type 2 diabetes ( 27 ) , suggesting that long - term flavonoid intervention may be associated with a 3.4% reduction in vascular events .
these effects are also supported by in vitro work , suggesting that flavan-3-ols increase apolipoprotein a1 and decrease apolipoprotein b production as a result of upregulation of sterol regulatory element binding proteins and an increase in ldl receptor activity ( 28 ) .
likewise , isoflavones have been shown to decrease apolipoprotein b secretion in hepg2 cells through multiple mechanisms and may augment the efficacy of statins ( 29 ) .
we also observed a significant reduction in insulin resistance ( homa - ir ) and improvements in insulin sensitivity ( quicki ) that were driven by a significant decrease in insulin concentrations .
these findings are clinically important , as insulin resistance is not only a key determinant of the metabolic syndrome but is also associated with increased arterial stiffening ( 30 ) and risk of cardiovascular events even in individuals with no diabetes ( 31 ) .
our recent meta - analysis of short - term cocoa / chocolate trials ( 4 months duration ) also showed that intervention improved insulin resistance ( homa - ir 0.67 [ 95% ci 0.98 , 0.36 ] ) due to significant reductions in serum insulin , although only 3 of the 42 studies were in patients with type 2 diabetes ( a. cassidy , personal communication ) .
however another recent meta - analysis observed no effect of soy isoflavones on measures of glycemic control ( 32 ) ; although only 3 of the 24 trials were in type 2 diabetic patients , there was wide variability in source and dose of soy isoflavones fed , and few studies reported changes in homa - ir . in another review , only soy isoflavones and genistein exerted beneficial effects on homa - ir ( 14 ) .
there are supporting underlying mechanisms for these effects ; in vitro , flavan-3-ols , isoflavones , and their metabolites enhanced insulin - stimulated glucose uptake in adipocytes through increased expression of glut4 , and improved insulin secretory function of pancreatic -cells ( 10,33 ) .
our combined intervention attenuated the ukpds - estimated , 10-year chd risk as a result of beneficial effects on lipoprotein status .
the ukpds algorithm incorporates additional risk factors for diabetes ( i.e. , hba1c and duration of diabetes ) beyond traditional risk factors , and , although validation studies have identified a tendency to overestimate chd risk ( 34 ) , it is still considered a useful tool ( 35 ) to compliment other chd risk assessments . over half of our study population
was on antihypertensive medication , and this may have influenced our lack of effect on bp . in the few previous short - term studies with type 2 diabetic patients , flavan-3-ols alone
did not improve bp ( 15,36 ) , although in one study , isoflavones reduced bp in patients with type 2 diabetes who were not on medication for hypertension ( 37 ) .
this is in contrast to flavonoid trials in participants without type 2 diabetes , where systolic bp and diastolic bp were reduced by short - term flavan-3-ol ( by 4.5 and 2.5 mmhg , respectively ) ( 38 ) and isoflavone intake ( by 2.5 and 1.5 mmhg , respectively ) ( 39 ) .
the dropout rate during the intervention was high ( 21% ) , with the most frequent reason for participant withdrawal ( in both groups ) attributable to palatability ( fig .
1a ) ; however , these numbers are comparable to other nutrition trials in type 2 diabetic patients ( 40 ) , and as observed in other studies , a small number of patients had changes in bp medication during the 1-year trial period ( n = 10 ) .
our findings also relate specifically to a combined flavonoid intervention given to postmenopausal women receiving standard diabetes therapy ( including lipid - lowering medication ) , and further studies are now required to determine the relative influence of each flavonoid subclass ( flavan-3-ols and isoflavones ) on biomarkers of cvd risk in this population and to examine whether similar effects are observed in male patients or in other patient groups .
finally , although the flavonoid intake in our trial could potentially be attained through high intake of dietary flavonoid , an emerging range of flavonoid - rich functional foods may offer additional opportunities to consume flavonoids at optimal levels . despite these limitations ,
our trial is the longest intervention to date to assess the effects of flavonoids on cvd risk factors in a population of medicated , postmenopausal patients with type 2 diabetes and benefits from objective assessment of compliance .
this pragmatic 1-year trial provides evidence to suggest that the intake of flavonoids results in sustained improvements in lipid profile and insulin sensitivity and an attenuation of estimated chd risk , highlighting the additional benefit of flavonoids to standard drug therapy in managing cvd risk in patients with type 2 diabetes .
our study is the only combined flavonoid trial and the longest flavan-3-ol intervention to date , and our findings have potential clinical relevance .
long - term studies are now required to determine whether these effects are restricted to populations of medicated postmenopausal women with established type 2 diabetes and to determine whether chronic intake of flavan-3-ols or isoflavones is as effective when consumed independently .
| objectiveto assess the effect of dietary flavonoids on cardiovascular disease ( cvd ) risk in postmenopausal women with type 2 diabetes on established statin and hypoglycemic therapy.research design and methodsdespite being medicated , patients with type 2 diabetes have elevated cvd risk , particularly postmenopausal women . although dietary flavonoids have been shown to reduce cvd risk factors in healthy participants , no long - term trials have examined the additional benefits of flavonoids to cvd risk in medicated postmenopausal women with type 2 diabetes .
we conducted a parallel - design , placebo - controlled trial with type 2 diabetic patients randomized to consume 27 g / day ( split dose ) flavonoid - enriched chocolate ( containing 850 mg flavan-3-ols [ 90 mg epicatechin ] and 100 mg isoflavones [ aglycone equivalents)]/day ) or matched placebo for 1 year.resultsninety-three patients completed the trial , and adherence was high ( flavonoid 91.3% ; placebo 91.6% ) .
compared with the placebo group , the combined flavonoid intervention resulted in a significant reduction in estimated peripheral insulin resistance ( homeostasis model assessment of insulin resistance [ homa - ir ] 0.3 0.2 ; p = 0.004 ) and improvement in insulin sensitivity ( quantitative insulin sensitivity index [ quicki ] 0.003 0.00 ; p = 0.04 ) as a result of a significant decrease in insulin levels ( 0.8 0.5 mu / l ; p = 0.02 ) .
significant reductions in total cholesterol : hdl - cholesterol ( hdl - c ) ratio ( 0.2 0.1 ; p = 0.01 ) and ldl - cholesterol ( ldl - c ) ( 0.1 0.1 mmol / l ; p = 0.04 ) were also observed .
estimated 10-year total coronary heart disease risk ( derived from uk prospective diabetes study algorithm ) was attenuated after flavonoid intervention ( flavonoid + 0.1 0.3 vs. placebo 1.1 0.3 ; p = 0.02 ) .
no effect on blood pressure , hba1c , or glucose was observed.conclusionsone-year intervention with flavan-3-ols and isoflavones improved biomarkers of cvd risk , highlighting the additional benefit of flavonoids to standard drug therapy in managing cvd risk in postmenopausal type 2 diabetic patients . |
the combination of external beam radiotherapy ( ebrt ) , chemotherapy and brachytherapy ( bt ) is the standard treatment of locally advanced cervical cancer .
image - guided adaptive brachytherapy ( igabt ) has been widely adopted during the last years [ 1 , 2 ] and guidelines have been published for treatment planning and dose reporting [ 3 , 4 ] . the main advantage is dose - volume optimization , allowing adjustments to the classical pear - shaped isodose patterns according to tumor regression during treatment and residual disease at the time of bt .
literature shows that combined ebrt and bt ( especially igabt ) is curative for many patients with especially high local control rates [ 57 ] .
however , regional recurrences do occur in the pelvic lymph nodes ( plnn ) within and adjacent to radiotherapy fields .
most regional recurrences include a component of marginal failure , suggesting a deficit in target volume coverage and dose delivered to suspicious nodes . from earlier publications we know that brachytherapy contributes to the pelvic nodal dose [ 10 , 11 ] , but so far there is no data reported for mri guided igabt .
the purpose of this study was to determine the dose contributions from image guided adaptive brachytherapy ( igabt ) to individual suspicious plnn in cervical cancer patients .
data was collected in two cancer centers , university of pittsburgh cancer institute ( upci ) and university medical center utrecht ( umcu ) .
we performed a combined analysis of igabt data in two institutes ; university of pittsburgh cancer institute ( upci , usa ) and university medical center utrecht ( umcu , the netherlands ) .
twenty - seven patients with international federation of gynecology and obstetrics ( figo ) stage ib to iiib cervical cancer and enlarged plnn were treated between january 2007 and december 2010 with igabt high dose rate ( hdr ) applications .
the patients received ebrt to the pelvis or combined pelvic and para - aortic region to 45 gy in 25 fractions .
the dose to suspicious plnn was delivered with simultaneous integrated boosts ( 55 gy in 25 fractions ) .
brachytherapy was scheduled once a week during the fourth or fifth week of treatment , and twice a week after completion of the ebrt .
computed tomography ( ct)/magnetic resonance imaging ( mri ) compatible tandem - ring applicators ( nucletron , an elekta company , elekta ab , stockholm , sweden ) were used with mri - based planning for the first bt fraction followed by ct - based planning for subsequent treatments [ 6 , 12 ] .
the tandem length ranged from 40 to 60 mm , the ring- tandem angle was 45 to 60 , and ring diameter was 26 - 34 mm .
all patients were treated with 5 hdr fractions of 5.0 - 6.0 gy per fraction ( reference dose ) depending on the stage of disease and response to treatment .
planning aim was to cover 90% of the hr - ctv volume with the reference dose .
treatment planning optimization was performed using a combination of graphical and manual tools . for every patient , body mass index ( bmi )
fifteen patients with figo stage ibi to iva cervical cancer and enlarged plnn were treated between may 2009 and september 2011 .
treatment consisted of ebrt to the clinical target volume as described by van de bunt et al . combined with 3d image - guided intracavitary or combined intracavitary / interstitial pulsed dose rate ( pdr ) bt applications .
brachytherapy consisted of two pdr applications of 32 pulses of 0.6 gy ( 2 19.2 gy planning aim ) with the first insertion after 22 ebrt fractions , and the second one after completion of ebrt .
brachytherapy was performed using the utrecht interstitial applicator , a ct / mr compatible tandem - ovoid applicator that allows additional interstitial needle placement ( nucletron , an elekta company , elekta ab , stockholm , sweden ) .
for the first bt fraction , only the intracavitary part of the applicator was used . in six of the fifteen patients ,
the dose to 90% of the hr - ctv ( d90 hr - ctv ) of the first application did not meet the planning aim . in this group
the number of needles and their position and depth of insertion were based on dosimetric and spatial shortcomings of the first application .
after bt , all patients received an additional 3d - conformal ebrt boost of 10 to 14 gy in 5 to 7 fractions to suspicious plnn , taking into account the dose contribution of bt .
also for these patients , bmi was calculated . at upci , all 27 patients had mri examination ( ge signa hd excite 1.5 t , ge health care , milwaukee , wi , usa )
; t2-weighted sequences with 3.5 mm slice thickness for the first bt fraction . for the following four fractions ,
a ct examination was performed ( ge lightspeed ultra-64 slice per rotation , ge health care , milwaukee , wi , usa ) with 2.5 mm slice thickness .
ct images were acquired without intravenous contrast , but with 60 ml diluted contrast injected into the bladder .
hr - ctv and organs at risk ( oar ) were contoured on an eclipse workstation for each fraction , according to the gec estro recommendations [ 3 , 4 ] .
thereafter , they were transferred to the nucletron plato brachytherapy planning system ( version 14.3 , nucletron , an elekta company , elekta ab , stockholm , sweden ) for three - dimensional planning . at umcu ,
all fifteen patients had mri examination for each application ( gyroscan nt intera 1.5 t , philips medical systems , best , the netherlands ) based on the institutional protocol , which includes t2-weighted turbo spin - echo images with 3 or 4.5 mm slice thickness , and a balanced steady - state free precession scan with a slice thickness of 1.5 mm for applicator reconstruction purposes .
hr - ctv and oar were contoured in volumetool according to gec estro recommendations [ 3 , 4 ] .
contours were transferred into the plato brachytherapy planning system , and applicator reconstruction and treatment planning was performed .
pre - treatment mri or pet - ct information was used to guide identification of residual plnn on bt scans . in case of complete response of the enlarged plnn ,
pelvic lymph nodes contours were generated by a specifically trained resident and verified by a radiation oncologist experienced in gynecological tumors in upci and umcu , respectively .
the dose contributions ( d90 ) to each individual plnn were calculated from dose - volume histograms ( dvh ) for each fraction ; both as absolute physical dose and as percentage of the reference dose .
the doses were expressed as biologically equivalent dose in 2 gy fractions ( eqd2 ) using lq - model ( with / = 10 for tumor , / = 3 for oar and half time of repair of 1.5 hours ) and were added for all fractions .
furthermore , the d90 for the hr - ctvs were determined in eqd2 . in order to get an estimate of the influence of radiobiology
, the pdr data were recalculated as if given by hdr in 4 fractions of 7 gy and scaled for oar constraints ( clinically used hdr schedule in our institute ) . for every patient
total reference air kerma divided by the reference dose was considered as an indicator of the volume encompassed by the 100% isodose .
the contoured plnn were grouped by three physicians into anatomical nodal regions , based on radiation therapy oncology group ( rtog ) definitions of pelvic nodal groups for intensity - modulated radiation therapy ( imrt ) and other published literature [ 1820 ] .
the common iliac region ( ci ) covers the area around the common iliac vessels from the aortic to iliac bifurcation .
the internal iliac region ( ii ) is defined as an area of 7 mm around and along the internal iliac vessels , excluding plnn ventrally to external iliac vessels . from the point where the internal and external iliac vessels spread to a distance of more than 7 mm ,
the space around and along external iliac artery is called external iliac region ( ei ) . the obturator region
twenty - seven patients with international federation of gynecology and obstetrics ( figo ) stage ib to iiib cervical cancer and enlarged plnn were treated between january 2007 and december 2010 with igabt high dose rate ( hdr ) applications .
the patients received ebrt to the pelvis or combined pelvic and para - aortic region to 45 gy in 25 fractions .
the dose to suspicious plnn was delivered with simultaneous integrated boosts ( 55 gy in 25 fractions ) .
brachytherapy was scheduled once a week during the fourth or fifth week of treatment , and twice a week after completion of the ebrt .
computed tomography ( ct)/magnetic resonance imaging ( mri ) compatible tandem - ring applicators ( nucletron , an elekta company , elekta ab , stockholm , sweden ) were used with mri - based planning for the first bt fraction followed by ct - based planning for subsequent treatments [ 6 , 12 ] .
the tandem length ranged from 40 to 60 mm , the ring- tandem angle was 45 to 60 , and ring diameter was 26 - 34 mm .
all patients were treated with 5 hdr fractions of 5.0 - 6.0 gy per fraction ( reference dose ) depending on the stage of disease and response to treatment .
planning aim was to cover 90% of the hr - ctv volume with the reference dose .
treatment planning optimization was performed using a combination of graphical and manual tools . for every patient , body mass index ( bmi )
fifteen patients with figo stage ibi to iva cervical cancer and enlarged plnn were treated between may 2009 and september 2011 .
treatment consisted of ebrt to the clinical target volume as described by van de bunt et al .
combined with 3d image - guided intracavitary or combined intracavitary / interstitial pulsed dose rate ( pdr ) bt applications .
brachytherapy consisted of two pdr applications of 32 pulses of 0.6 gy ( 2 19.2 gy planning aim ) with the first insertion after 22 ebrt fractions , and the second one after completion of ebrt .
brachytherapy was performed using the utrecht interstitial applicator , a ct / mr compatible tandem - ovoid applicator that allows additional interstitial needle placement ( nucletron , an elekta company , elekta ab , stockholm , sweden ) .
for the first bt fraction , only the intracavitary part of the applicator was used . in six of the fifteen patients , the dose to 90% of the hr - ctv ( d90 hr - ctv ) of the first application
the number of needles and their position and depth of insertion were based on dosimetric and spatial shortcomings of the first application .
after bt , all patients received an additional 3d - conformal ebrt boost of 10 to 14 gy in 5 to 7 fractions to suspicious plnn , taking into account the dose contribution of bt .
twenty - seven patients with international federation of gynecology and obstetrics ( figo ) stage ib to iiib cervical cancer and enlarged plnn were treated between january 2007 and december 2010 with igabt high dose rate ( hdr ) applications .
the patients received ebrt to the pelvis or combined pelvic and para - aortic region to 45 gy in 25 fractions .
the dose to suspicious plnn was delivered with simultaneous integrated boosts ( 55 gy in 25 fractions ) .
brachytherapy was scheduled once a week during the fourth or fifth week of treatment , and twice a week after completion of the ebrt .
computed tomography ( ct)/magnetic resonance imaging ( mri ) compatible tandem - ring applicators ( nucletron , an elekta company , elekta ab , stockholm , sweden ) were used with mri - based planning for the first bt fraction followed by ct - based planning for subsequent treatments [ 6 , 12 ] .
the tandem length ranged from 40 to 60 mm , the ring- tandem angle was 45 to 60 , and ring diameter was 26 - 34 mm .
all patients were treated with 5 hdr fractions of 5.0 - 6.0 gy per fraction ( reference dose ) depending on the stage of disease and response to treatment .
planning aim was to cover 90% of the hr - ctv volume with the reference dose .
treatment planning optimization was performed using a combination of graphical and manual tools . for every patient , body mass index ( bmi )
fifteen patients with figo stage ibi to iva cervical cancer and enlarged plnn were treated between may 2009 and september 2011 .
treatment consisted of ebrt to the clinical target volume as described by van de bunt et al .
combined with 3d image - guided intracavitary or combined intracavitary / interstitial pulsed dose rate ( pdr ) bt applications .
brachytherapy consisted of two pdr applications of 32 pulses of 0.6 gy ( 2 19.2 gy planning aim ) with the first insertion after 22 ebrt fractions , and the second one after completion of ebrt .
brachytherapy was performed using the utrecht interstitial applicator , a ct / mr compatible tandem - ovoid applicator that allows additional interstitial needle placement ( nucletron , an elekta company , elekta ab , stockholm , sweden ) .
for the first bt fraction , only the intracavitary part of the applicator was used . in six of the fifteen patients , the dose to 90% of the hr - ctv ( d90 hr - ctv ) of the first application
the number of needles and their position and depth of insertion were based on dosimetric and spatial shortcomings of the first application .
after bt , all patients received an additional 3d - conformal ebrt boost of 10 to 14 gy in 5 to 7 fractions to suspicious plnn , taking into account the dose contribution of bt . also for these patients , bmi was calculated .
at upci , all 27 patients had mri examination ( ge signa hd excite 1.5 t , ge health care , milwaukee , wi , usa ) ; t2-weighted sequences with 3.5 mm slice thickness for the first bt fraction . for the following four fractions ,
a ct examination was performed ( ge lightspeed ultra-64 slice per rotation , ge health care , milwaukee , wi , usa ) with 2.5 mm slice thickness .
ct images were acquired without intravenous contrast , but with 60 ml diluted contrast injected into the bladder .
hr - ctv and organs at risk ( oar ) were contoured on an eclipse workstation for each fraction , according to the gec estro recommendations [ 3 , 4 ] .
thereafter , they were transferred to the nucletron plato brachytherapy planning system ( version 14.3 , nucletron , an elekta company , elekta ab , stockholm , sweden ) for three - dimensional planning . at umcu ,
all fifteen patients had mri examination for each application ( gyroscan nt intera 1.5 t , philips medical systems , best , the netherlands ) based on the institutional protocol , which includes t2-weighted turbo spin - echo images with 3 or 4.5 mm slice thickness , and a balanced steady - state free precession scan with a slice thickness of 1.5 mm for applicator reconstruction purposes .
hr - ctv and oar were contoured in volumetool according to gec estro recommendations [ 3 , 4 ] .
contours were transferred into the plato brachytherapy planning system , and applicator reconstruction and treatment planning was performed .
pre - treatment mri or pet - ct information was used to guide identification of residual plnn on bt scans . in case of complete response of the enlarged plnn ,
pelvic lymph nodes contours were generated by a specifically trained resident and verified by a radiation oncologist experienced in gynecological tumors in upci and umcu , respectively .
the dose contributions ( d90 ) to each individual plnn were calculated from dose - volume histograms ( dvh ) for each fraction ; both as absolute physical dose and as percentage of the reference dose .
the doses were expressed as biologically equivalent dose in 2 gy fractions ( eqd2 ) using lq - model ( with / = 10 for tumor , / = 3 for oar and half time of repair of 1.5 hours ) and were added for all fractions .
furthermore , the d90 for the hr - ctvs were determined in eqd2 . in order to get an estimate of the influence of radiobiology
, the pdr data were recalculated as if given by hdr in 4 fractions of 7 gy and scaled for oar constraints ( clinically used hdr schedule in our institute ) . for every patient
total reference air kerma divided by the reference dose was considered as an indicator of the volume encompassed by the 100% isodose .
the contoured plnn were grouped by three physicians into anatomical nodal regions , based on radiation therapy oncology group ( rtog ) definitions of pelvic nodal groups for intensity - modulated radiation therapy ( imrt ) and other published literature [ 1820 ] .
the common iliac region ( ci ) covers the area around the common iliac vessels from the aortic to iliac bifurcation . the internal iliac region ( ii )
is defined as an area of 7 mm around and along the internal iliac vessels , excluding plnn ventrally to external iliac vessels . from the point where the internal and external iliac vessels spread to a distance of more than 7 mm ,
the space around and along external iliac artery is called external iliac region ( ei ) .
the obturator region ( ob ) encompasses the space bordered by internal and external iliac regions .
at upci , all 27 patients had mri examination ( ge signa hd excite 1.5 t , ge health care , milwaukee , wi , usa ) ; t2-weighted sequences with 3.5 mm slice thickness for the first bt fraction . for the following four fractions ,
a ct examination was performed ( ge lightspeed ultra-64 slice per rotation , ge health care , milwaukee , wi , usa ) with 2.5 mm slice thickness .
ct images were acquired without intravenous contrast , but with 60 ml diluted contrast injected into the bladder .
hr - ctv and organs at risk ( oar ) were contoured on an eclipse workstation for each fraction , according to the gec estro recommendations [ 3 , 4 ] .
thereafter , they were transferred to the nucletron plato brachytherapy planning system ( version 14.3 , nucletron , an elekta company , elekta ab , stockholm , sweden ) for three - dimensional planning .
at umcu , all fifteen patients had mri examination for each application ( gyroscan nt intera 1.5 t , philips medical systems , best , the netherlands ) based on the institutional protocol , which includes t2-weighted turbo spin - echo images with 3 or 4.5 mm slice thickness , and a balanced steady - state free precession scan with a slice thickness of 1.5 mm for applicator reconstruction purposes .
hr - ctv and oar were contoured in volumetool according to gec estro recommendations [ 3 , 4 ] .
contours were transferred into the plato brachytherapy planning system , and applicator reconstruction and treatment planning was performed .
pre - treatment mri or pet - ct information was used to guide identification of residual plnn on bt scans . in case of complete response of the enlarged plnn ,
pelvic lymph nodes contours were generated by a specifically trained resident and verified by a radiation oncologist experienced in gynecological tumors in upci and umcu , respectively .
the dose contributions ( d90 ) to each individual plnn were calculated from dose - volume histograms ( dvh ) for each fraction ; both as absolute physical dose and as percentage of the reference dose .
the doses were expressed as biologically equivalent dose in 2 gy fractions ( eqd2 ) using lq - model ( with / = 10 for tumor , / = 3 for oar and half time of repair of 1.5 hours ) and were added for all fractions .
furthermore , the d90 for the hr - ctvs were determined in eqd2 . in order to get an estimate of the influence of radiobiology
, the pdr data were recalculated as if given by hdr in 4 fractions of 7 gy and scaled for oar constraints ( clinically used hdr schedule in our institute ) . for every patient
total reference air kerma divided by the reference dose was considered as an indicator of the volume encompassed by the 100% isodose .
the contoured plnn were grouped by three physicians into anatomical nodal regions , based on radiation therapy oncology group ( rtog ) definitions of pelvic nodal groups for intensity - modulated radiation therapy ( imrt ) and other published literature [ 1820 ] .
the common iliac region ( ci ) covers the area around the common iliac vessels from the aortic to iliac bifurcation .
the internal iliac region ( ii ) is defined as an area of 7 mm around and along the internal iliac vessels , excluding plnn ventrally to external iliac vessels . from the point where the internal and external iliac vessels spread to a distance of more than 7 mm ,
the space around and along external iliac artery is called external iliac region ( ei ) .
the obturator region ( ob ) encompasses the space bordered by internal and external iliac regions .
fifty - seven plnn were detected with a minimum of 1 and a maximum of 9 per patient .
the mean hr - ctv of these patients was 36.9 cm ( range 21.0 - 59.8 cm ) , and the mean bt d90 hr - ctv was 39.3 gy ( range 34.9 - 63.3 gy ) eqd2 .
the mean d90 received by the suspicious plnn was 10.8% ( range 5.7 - 25.1% ) of the reference dose , corresponding to 2.7 gy ( range 1.3 - 6.6 gy ) eqd2 .
forty plnn were detected with a minimum of 1 and a maximum of 5 per patient .
the mean hr - ctv was 39.4 cm ( range 15.9 - 115 cm ) , and the mean bt d90 hr - ctv was 47.1 gy ( range 0.3 - 52.4 gy ) eqd2 .
the mean d90 of the plnn was 20.5% ( range 6.8 - 93.3% ) resulting in 7.1 gy ( range 2.2 - 36.7 gy ) eqd2 ( table 2 ) .
figure 1 shows an illustration of the 3d eqd2 distribution of a bt pdr fraction displaying plnn with different locations and different dose contributions .
illustration of a transversal t2 weighted mr image ( a ) and corresponding 3d dose distribution in eqd2 of a brachytherapy pulsed dose rate fraction presented in three slices : ( b ) transversal , ( c ) coronal and ( d ) sagittal view .
blue = 0 and red > 20 gy eqd2 , yellow cross indicates common point for all images patient characteristics upci university of pittsburgh cancer institute , umcu university medical center utrecht , bmi body mass index upci
university of pittsburgh cancer institute , umcu university medical center utrecht , hdr high dose - rate , pdr pulsed dose - rate , plnn pathologically enlarged pelvic lymph - nodes , sd standard deviation , hr - ctv high risk - ctv , d90 dose to 90% of the volume , ref .
reference dose , eqd2 biologically equivalent dose in 2-gy fractions pdr data were recalculated as if given by hdr in 4 fractions of 7 gy and scaled for oar constraints
table 3 and figure 2 show the dose contributions of igabt to the different nodal regions . in upci , but not in umcu we saw a tendency that dose decreases from obturator to external , internal and common iliac nodes .
the dose contributions of igabt to individual plnn varied impressively in both centers ( range 5 to > 90% of the reference dose ) .
after having excluded the few parametrial , inguinal and peri - rectal nodes from the umcu data set ( leaving 35 plnn for the comparison ; see table 2 ) , the nodes received on average 16.7% ( range 6.8 - 34.7% ) of the reference dose , corresponding to 5.6 gy ( range 2.2 - 12.0 gy ) eqd2 . furthermore , recalculating the umcu pdr into hdr values , resulted in a reduced mean d90 of 3.9 gy ( range 1.5 - 8.2 gy ) eqd2 , and a smaller difference with upci values .
relative dose contributions of pdr and hdr treatments to individual lymph nodes in the different pelvic regions ; dose variations indicated between and within anatomical regions results for different nodal regions upci university of pittsburgh cancer institute ,
umcu university medical university utrecht , plnn pathologically enlarged pelvic lymph - nodes , d90 dose to 90% of the volume , eqd2 biologically equivalent dose in 2-gy fractions , sd standard deviation
figure 3 shows the d90 plnn as function of trak per reference dose ( as surrogate of the 100% isodose volume ) .
this overall results in higher dose contributions to the plnn in umcu , irrespective of pdr or hdr calculations .
the addition of interstitial needles for the second bt fractions in six of the umcu patients did not influence the d90 plnn systematically ; see wide range of values for those cases in figure 3 .
dose contributions to individual lymph nodes as function of trak / reference dose ; trak / reference dose is considered an indicator for the volume encompassed by the 100% isodose .
arrows indicate cases treated with a combined intracavitary / interstitial approach . diamonds upci ( hdr ) .
the differences between black and grey squares show that the bt dose contribution ( in eqd2 ) to plnn is influenced by dose rate body mass index was 25 ( range 19 - 32 ) in umcu patients and 30 ( range 18 - 41 ) in upci patients , respectively , without a relation between physical d90 dose to the nodes and bmi .
to our knowledge , this retrospective inter - institutional study represents the first dosimetric analysis of mri based igabt dose - contributions to pelvic lymph nodes in patients with advanced cervical cancer .
since the introduction of image guided bt , local control in cervical cancer has increased [ 5 , 7 ] , but regional control is still a matter of concern [ 6 , 9 ] .
additionally , modern imaging modalities allow a better visualization of plnn and help to consider them suspicious or not , which influences our intentions to treat nodal disease . for accurate dose reporting and for our understanding of nodal dose response relations
it is essential to learn how ebrt and bt contribute to the treatment of individual affected lymph nodes .
more detailed information on the dose administered per node will also help us to further optimize nodal boosting .
point b in this system was thought to give an estimate of the dose contribution to the lateral parametrium and the regional nodes .
it is located at the level of point a , 5 cm lateral from the center of the tandem and gets approximately 15% to 25% of the point a dose .
. already showed that the point b dose is not a good surrogate for the dose to pelvic nodes . in that study ,
the nodal regions defined on ct scans were overlaid with the bt dose distributions showing significantly lower dose to nodal groups than to point b ( on average 5 vs. 7 gy ) .
additionally , it was documented that there is some variation in dose delivered to the different nodal groups , defined as internal iliac , external iliac , and obturator nodes . in our study , we investigated the contribution of individualized igabt to the plnn .
we computed the bt dose to individual nodes located in the different groups by using the images at time of bt and the optimized bt treatment plans .
we found a variety of dose levels in individual nodes and patients , but no general dose level differences when comparing the earlier defined nodal groups .
this finding might be due to the fact that the spatial dose distributions of igabt in the central pelvis can be quite asymmetric as they follow the target and oar locations and shapes .
it can therefore be expected that individualized igabt also individually contributes to different plnn with the highest values to nodes in the vicinity of the hr - ctv and high dose regions .
based on the data presented here , we consider to calculate the igabt dose for the individual nodes , especially when ebrt boosting is part of the treatment schedule .
dose calculation for the different nodal regions is a rougher estimate and might still be used if individual node dose calculation is not possible .
the igabt dose contributions to plnn in upci were on average lower than the ones in umcu .
part of the difference was caused by the fact that in some umcu patients , different from upci , plnn were identified in the vicinity of the hr - ctv , especially in parametrium and perirectally .
after excluding these nodes and their corresponding high d90 values , the dose level difference was smaller .
the umcu pdr contributions to plnn into hdr values , the difference in dose again was smaller , but still remained .
this lasting difference is due to the fact that the prescribed and administered tumor dose in umcu is in principal higher , irrespective of the underlying radiobiology .
the on average higher d90 hr - ctv values in umcu are corresponding to higher totally administered doses and larger bt volumes irradiated to the reference dose .
the igabt dose contribution to individual plnn depends on patient and treatment related factors and varies considerably .
the difference is caused by the positions of the individual nodes in relation to the radiation source , by differences in radiobiology and by individual treatment planning , and spatial dose delivery to specific hr - ctv and oar locations and shapes .
based on the data presented here , calculation of the bt dose to the individual plnn for nodal dose reporting and individual node boost planning should be considered .
dose calculations for the different nodal regions is a rougher estimate and can be used as alternative if individual node dose calculation is not possible .
| purposethe goal of this study was to determine the dose contributions from image guided adaptive brachytherapy ( igabt ) to individual suspicious pelvic lymph nodes ( plnn ) in cervical cancer patients .
data were collected in two cancer centers , university of pittsburgh cancer institute ( upci ) and university medical center utrecht ( umcu).material and methods27 and 15 patients with node positive cervical cancer treated with hdr ( high dose rate ) or pdr ( pulsed dose rate)-igabt were analyzed .
hdr - igabt ( upci ) was delivered with ct / mri compatible tandem - ring applicators with 5.0 - 6.0 gy five fractions .
pdr - igabt ( umcu ) dose was delivered with utrecht tandem - ovoid applicators with 32 0.6 gy two fractions .
pelvic lymph nodes with short axis diameter of 5 mm on pre - treatment mri or pet - ct were contoured for all bt - plans .
dose contributions to individual plnn expressed as d90 ( dose to 90% of the volume ) were calculated from dose - volume histograms as absolute and relative physical dose ( % of the reference dose ) for each fraction . for each node ,
the total dose from all fractions was calculated , expressed in eqd2 ( equivalent total dose in 2 gy fractions).resultsfifty - seven ( upci ) and 40 ( umcu ) individual plnn were contoured .
the mean d90 plnn was 10.8% ( range 5.7 - 25.1% ) and 20.5% ( range 6.8 - 93.3% ) , respectively , and therefore different in the two centers .
these values translate into 2.7 gy ( 1.3 - 6.6 gy ) eqd2 and 7.1 gy ( 2.2 - 36.7 gy ) eqd2 , respectively .
differences are caused by the location of the individual nodes in relation to the spatial dose distribution of igabt , differences in total dose administered and radiobiology ( hdr versus pdr).conclusionsthe igabt dose contribution to individual pelvic nodes depends on patient and treatment related factors , and varies considerably . |
hypertensive encephalopathy ( he ) is heralded by headache , visual disturbance , seizures , and altered mental status , all associated with severe hypertension6 ) . without prompt
the most common feature of he by magnetic resonance imaging ( mri ) is hyperintensity of subcortical parietal and occipital white matter on t2-weighted views , suggestive of vasogenic edema . because such changes usually disappear upon bp stabilization , many authors refer to this state as reversible posterior leukoencephalopathy syndrome or posterior reversible encephalopathy syndrome ( pres)3,6,8,9 ) .
involvement of the brainstem and cerebellum in pres has rarely been described . in this report , however , we present a case of pres where the brainstem alone is affected , accompanied by intracranial hemorrhage .
a 36-year - old male with idiopathic hypertension ( duration > 6 yrs ) was admitted to our hospital for abrupt - onset headache , right facial palsy , dysarthria , and right hemiparesis that had developed three hours earlier .
laboratory studies were within normal range and ophthalmologic evaluation was unremarkable . specifically , no papilledema was evident .
routine chest x - ray and echocardiogram suggested severe concentric left ventricular hypertrophy , later confirmed by transthoracic echocardiography .
emergency computed tomographic ( ct ) scan of the brain revealed small amount ( 7 cc ) of intracranial hemorrhage involving the left basal ganglia , and there was relative hypodensity of the brainstem ( fig .
brain mri showed diffuse high - signal intensities on t2-weighted images and fluid - attenuated inversion recovery images without gadolinium enhancement in the pons and midbrain .
no high signal intensities in diffusion - weighted images ( dwi ) and high signal intensities in apparent diffusion coefficient ( adc ) images , implying that the changes were not due to cerebral infarction ( fig .
there were also no abnormal findings other than the small amount of hemorrhage in basal ganglia , which was confirmed already in ct ( fig .
follow - up mri ( including dwi ) on day 16 of hospitalization revealed complete resolution of prior imaging aberrations involving the brainstem ( fig .
as a distinct clinical entity , pres is characterized by progressive headache , visual changes , altered mental status , and seizures .
most commonly pres is secondary event , triggered by paroxysmal severe hypertension of various etiologies .
the spectrum of predisposing factors / conditions includes renal disease , immunosuppressive and cytotoxic drugs , collagen vascular disorders ( such as systemic lupus erythematosus ) , eclampsia , and hematologic conditions ( typically , thrombotic thrombocytopenic purpura and hemolytic - uremic syndrome)5 ) .
however , regardless of the underlying cause , the pathogenesis of the primary abnormality , cerebral vasogenic edema , is still under debate .
if the main cause is corrected , pres can be reversed . however , if patients with pres present with coma and/or status epilepticus , permanent neurological impairment or death may occur in some cases8 ) .
generally , subcortical white matter of the parietal and occipital lobes is affected , while involvement of brainstem is rare . in our patient ,
reversible brainstem hypertensive encephalopathy ( rbhe ) was first recognized by chang and keane2 ) in 1999 . when the brainstem alone is affected , most patients do not exhibit focal signs or symptoms .
intracerebral hemorrhage of the left basal ganglia , rather than the brainstem manifestations , was held responsible for the facial palsy , dysarthia and right - sided hemiparesis displayed in this instance .
because the neurologic effects of rbhe are reversible with prompt treatment , this syndrome must be differentiated from brainstem infarction , pontine glioma , central pontine myelinolysis , and infectious encephalitis .
severe hypertension , so - called " clinical - radiologic dissociation " ( signaling brainstem involvement ) , and rapid resolution of mri changes after antihypertensive treatment are pathognomonic of rbhe10 ) . the suggested etiology of rbhe is a failure of cerebrovascular autoregulation in the face of high bp , resulting in cerebral hyperperfusion , disruption of the blood - brain barrier ( bbb ) , and vasogenic edema1 ) .
while physiologic hypertension usually activates sympathetic pathways to maintain the bbb via cerebral vasoconstriction , experimentally induced hypertension in animal models has shown that transient reflex sympathetic hypertension can override the bbb . at breakthrough ,
constricted cerebral arterioles dilate under hypertensive force , allowing parenchymal extravasation of fluid , macromolecules , and red blood cells7 ) . the posterior circulatory network , in particular ,
is less endowed with sympathetic innervation than anterior vessels , and thus is less capable of a protective vasoconstrictive response to a sudden increase in arterial bp . a disruption in bbb
, this failure of autoregulation is marked by vasogenic ( as opposed to cytotoxic ) edema , confirmed by imaging studies .
consistent with other reports of rbhe , demonstrable hyperintensity on t2-weighted images and normal dwi with increased adc values were indicative of vasogenic edema in our patient .
what has yet to be explained is the predilection for brainstem ( versus cerebral ) involvement with rbhe . because the parietal and occipital lobes , as well as the brainstem , are within distribution of the vertebrobasilar artery and its branches , two tentative explanations have emerged4 ) . by allowing fluid to accumulate
, the brainstem may simply absorb much of the hypertensive " tidal wave " , dissipating pressure before other parts of the brain are reached .
in essence , the brainstem serves as a buffer to protecting cerebrum and the distal parieto - occipital region in the vertebrobasilar system , more distal in blood supply .
another explanation is that the parietal and occipital lobes may be resistant to relatively rich sympathetic innervation via the posterior communicating artery ( pcoa ) .
a well - developed pcoa , also known as fetal - type pcoa , is thought to possess the same degree of sympathetic innervation as the anterior circulation . in this manner
, individuals with a fetal - type pcoa may be protected from paroxysmal malignant hypertension .
consequently , it is clear that at least one of these theories is flawed and that more effort is needed to clarify the pathophysiologic mechanisms of rbhe .
thorough neurologic examination , in conjunction with mri with dwi , is essential for the correct diagnosis .
clinical - radiologic dissociation and rapid improvement of mri findings after antihypertensive treatment are features pathognomonic of rbhe . with prompt and proper management
however , physicians must cognizant of the potential for fatality and approach it as a medical emergency . | presented here is a 36-year - old male with arterial hypertension who developed brainstem edema and intracranial hemorrhage .
magnetic resonance scan revealed diffuse brainstem hyperintensity in t2-weighted and fluid - attenuated inversion - recovery images , with an increase in apparent diffusion coefficient values .
after a reduction in blood pressure , rapid resolution of the brainstem edema was observed on follow - up .
the patient 's condition was thus interpreted as hypertensive brainstem encephalopathy . while many consider this a vasogenic phenomenon , induced by sudden , severe hypertension , the precise mechanism remains unclear .
prompt recognition and aggressive antihypertensive treatment in such patients are essential to prevent permanent or life - threatening neurologic injury . |
hepatitis c virus ( hcv ) is the most common cause of chronic liver disease and also an important factor in liver transplantation ( cdc , www.cdc.gov/hepatitis/hcv/hcvfaq.htm ) .
the world health organization ( who ) has estimated that 3% of the world s population has been infected with hepatitis c virus ( hcv ) and currently more than 170 million carry chronic hcv ( who , www.who.int ) .
hcv is considered the cause of approximately one - third of hcc cases in the usa . according to various reports ,
it is estimated that the risk of development of hcc when liver cirrhosis has progressed is 0 to 3% per year .
the risk of hcc in chronic infections of hcv is highly associated with fibrosis stage .
the incidence of hcc is relatively high in patients with cirrhosis ( 1% - 7% per year ) ( 1 , 2 ) .
experimental observations have shown that hcv viral proteins interact with cell proliferation and survival pathways , which lead to the increased risk of hcc .
the over - expression of hcv protein enhances cell proliferation , transformation , and tumor development in mice , which suggests that viral proteins lead to the activation of oncogenic pathways ( 3 ) .
several studies have shown that core protein can participate in liver cancer caused by hcv .
it has been shown that the core protein expression can modulate cellular mrna transcription through the interaction with hnrnp k ( 4 ) .
it also has been shown that the core protein can affect p53 protein through various mechanisms such as physical connection ( 5 , 6 ) .
studies have suggested that non - structural hcv proteins may also play a role in carcinogenesis ( 7 ) . in parallel with this hypothesis ,
it has been reported that the expression of three hcv genes , including ns3 , ns5a and ns5b can affect cell proliferation .
ns3 and ns5a proteins can inhibit transcription of p21 ( 8 - 10 ) and ns5a protein impairs the cell cycle pathway by targeting cdk1/2-cyclin complex ( 11 ) .
ns5a protein also activates the wnt pathway and stimulates the -catenin - responsive transcription by activating the phosphoinositide 3-kinase ( 12 ) . finally ,
expression of non - structural ns5b protein , by targeting proteasomal degradation of prb , leads to up - regulation of e2f - responsive promoter and induction of cell entry into the s phase of the cell cycle ( 13 ) .
although the molecular mechanisms of hcv - induced hcc remain to be discovered , studies have suggested that hcv is the important factor in providing an appropriate microenvironment for progression and development of cancer ( 14 ) .
in addition , gene expression alterations of chronic liver disease and molecular mechanisms involved in the hcv - induced hcc are not well understood .
nowadays , the network - based analysis of high - throughput gene expression profiles is emerging as a reliable tool in the perception of biological processes . in the present study
, we studied the possible role of hcv in hcc initiation and invasion using topological analysis of ppi networks .
the gene expression profile gse38226 was obtained from the public functional genomics data repository gene expression omnibus ( geo ) database ( www.ncbi.nlm.nih.gov/geo/ ) .
there are two illumina platforms in this transcription profile , including gpl6947 and gpl8179 used as mrna and mirna expression beadchips , respectively . before the mrna and mirna expression profiling ,
the liver biopsies were collected from chronic hcv patients ( 24 samples ) , patients with hepatocellular carcinoma ( 12 samples ) and normal subjects ( 8 samples ) .
the samples were used to identify the differentially expressed genes ( degs ) and differentially expressed mirnas ( dems ) using geo2r ( 15 ) .
gene ontology ( go ) annotation of all degs was performed using g : profiler public web server ( http://biit.cs.ut.ee/gprofiler/ ) .
g : profiler is a powerful web tool for characterizing and manipulating the gene lists obtained from analysis of the high - throughput genomic data . the overrepresented go terms with significant p value filtered from the primary results . to determine functional categories of dems obtained from the previous step , the list of dems of both chronic hcv and hcc expression profiles were introduced to the tam tool ( 16 ) .
the list of dems was functionally annotated and summarized according to the integrated mirna categorical data .
there was a considerable overlap between the enrichment results of chronic hcv and hcc in the given dems lists .
based on the string ( a protein - protein interaction database ) ( 17 ) and genmania ( a web service used for prediction of gene function ) regulation databases , we constructed and visualized the corresponding ppi networks using cytoscape software ( 18 ) .
we considered directed interactions as well as important undirected interactions such as genetic interactions for building ppi networks .
the number of nodes - edges in the ppi networks constructed from chronic hcv and hcc gene expression profiles were 171 - 1090 and 175 - 1161 , respectively .
topological network analysis was performed for ppi networks and the most important hub genes were identified .
the network analyzer plugin of cytoscape was used to analyze topological properties of the networks . among nine evaluated methods , degree , betweenness centrality and closeness centrality had better outcomes and used for topological analysis of the ppi networks .
degree measure refers to the number of neighbors ( edges ) of a node and the nodes with higher degree centrality are considered biologically important hub genes within biological networks .
betweenness centrality is another centrality measure that represents the number of times each node is visited during traversing all shortest paths and is computed as follows : the s and t are nodes in the network different from n , st indicates the number of shortest paths from s to t and st ( n ) is the number of shortest paths from s to t that n lies on .
the closeness centrality of a node is a measure of how fast information spreads from a given node to other reachable nodes in the given network .
this measure is computed as follows : where l ( n , m ) is the length of the shortest path between two n and m nodes .
we selected the top 50 genes according to the ranked results of three mentioned methods .
furthermore , the putative mirnas regulating hub genes were predicted in silico using mirtarbase server ( 19 ) and a new mirna - hubgene network was reconstructed for both chronic hcv and hcc gene expression profiles .
topological analysis of the new mirna - hubgene networks was also performed using the degree measure to identify the hub mirnas in each network . interestingly
, most hub mirnas had been determined as dems after mirna expression analysis of chronic hcv and hcc samples with geo2r .
a network alignment allows us to find the conserved functional complexes between two or more given networks .
the alignment between chronic hcv and hcc networks was performed by netal tool ( 20 ) .
the netal is a new method for global alignment of ppi networks based on both biological and topological information of input networks .
the edge correctness ( ec ) measure was considered an important parameter to determine topological similarity between chronic hcv and hcc derived ppi networks .
after comparative analysis of chronic hcv and hcc networks , the most important common sub - network was determined and visualized .
the gene expression profile gse38226 was obtained from the public functional genomics data repository gene expression omnibus ( geo ) database ( www.ncbi.nlm.nih.gov/geo/ ) .
there are two illumina platforms in this transcription profile , including gpl6947 and gpl8179 used as mrna and mirna expression beadchips , respectively . before the mrna and mirna expression profiling ,
the liver biopsies were collected from chronic hcv patients ( 24 samples ) , patients with hepatocellular carcinoma ( 12 samples ) and normal subjects ( 8 samples ) .
the samples were used to identify the differentially expressed genes ( degs ) and differentially expressed mirnas ( dems ) using geo2r ( 15 ) .
gene ontology ( go ) annotation of all degs was performed using g : profiler public web server ( http://biit.cs.ut.ee/gprofiler/ ) .
g : profiler is a powerful web tool for characterizing and manipulating the gene lists obtained from analysis of the high - throughput genomic data . the overrepresented go terms with significant p value filtered from the primary results . to determine functional categories of dems obtained from the previous step , the list of dems of both chronic hcv and hcc expression profiles were introduced to the tam tool ( 16 ) .
the list of dems was functionally annotated and summarized according to the integrated mirna categorical data .
there was a considerable overlap between the enrichment results of chronic hcv and hcc in the given dems lists .
based on the string ( a protein - protein interaction database ) ( 17 ) and genmania ( a web service used for prediction of gene function ) regulation databases , we constructed and visualized the corresponding ppi networks using cytoscape software ( 18 ) .
we considered directed interactions as well as important undirected interactions such as genetic interactions for building ppi networks .
the number of nodes - edges in the ppi networks constructed from chronic hcv and hcc gene expression profiles were 171 - 1090 and 175 - 1161 , respectively .
topological network analysis was performed for ppi networks and the most important hub genes were identified .
the network analyzer plugin of cytoscape was used to analyze topological properties of the networks . among nine evaluated methods , degree , betweenness centrality and closeness centrality had better outcomes and used for topological analysis of the ppi networks .
degree measure refers to the number of neighbors ( edges ) of a node and the nodes with higher degree centrality are considered biologically important hub genes within biological networks .
betweenness centrality is another centrality measure that represents the number of times each node is visited during traversing all shortest paths and is computed as follows : the s and t are nodes in the network different from n , st indicates the number of shortest paths from s to t and st ( n ) is the number of shortest paths from s to t that n lies on .
the closeness centrality of a node is a measure of how fast information spreads from a given node to other reachable nodes in the given network .
this measure is computed as follows : where l ( n , m ) is the length of the shortest path between two n and m nodes .
we selected the top 50 genes according to the ranked results of three mentioned methods .
furthermore , the putative mirnas regulating hub genes were predicted in silico using mirtarbase server ( 19 ) and a new mirna - hubgene network was reconstructed for both chronic hcv and hcc gene expression profiles .
topological analysis of the new mirna - hubgene networks was also performed using the degree measure to identify the hub mirnas in each network .
interestingly , most hub mirnas had been determined as dems after mirna expression analysis of chronic hcv and hcc samples with geo2r .
a network alignment allows us to find the conserved functional complexes between two or more given networks .
the alignment between chronic hcv and hcc networks was performed by netal tool ( 20 ) .
the netal is a new method for global alignment of ppi networks based on both biological and topological information of input networks .
the edge correctness ( ec ) measure was considered an important parameter to determine topological similarity between chronic hcv and hcc derived ppi networks .
after comparative analysis of chronic hcv and hcc networks , the most important common sub - network was determined and visualized .
analysis of the functional annotation of chronic hcv and hcc related degs specifically indicated that most genes , in both chronic hcv and hcc related degs , are involved in important cellular processes such as cell signaling , cell proliferation , cell death , cell migration , etc .
furthermore , we enriched the list of dems obtained from mirna expression analysis of both hcc and chronic hcv samples .
interestingly , there was a considerable overlap between the functional categories of chronic hcv and hcc related dems ( figure 1 ) .
similarly to degs , a high number of mirnas predicted as regulators of several cancer - related pathways such as cell cycle , cell proliferation , cell death , apoptosis , epithelial - mesenchymal transition and angiogenesis .
we found that the four important onco - mirna ( hsa - mir-24 , hsa - mir-92a , hsa - mir-194 and hsa - mir-191 ) were up - regulated in both chronic hcv and hcc samples compared to the control liver cells .
furthermore , several mirna tumor suppressors , including hsa - mir-181b , hsa - mir-451 , hsa - mir-34a , hsa - mir-34b and hsa - mir-34c were significantly down - regulated in chronic hcv and hcc samples compared to the control samples .
the ppi networks for degs resulted from analysis of chronic hcv and hcc gene expression profiles were constructed with 171 - 1090 and 175 - 1161 nodes - edges , respectively .
we used the degree , betweenness centrality and closeness centrality measures to analyze topology of resulted ppi networks .
the top 50 hub genes of these three measures were selected for further analysis ( tables 2 and 3 ) . after filtering and removing duplicate values , we obtained 61 and 64 unique hub genes from the topological analysis of chronic hcv and hcc related ppis , respectively . there were 32 common hub genes between chronic hcv and hcc unique hub genes .
interestingly , the c - jun gene was identified as the most important hub gene in both chronic hcv and hcc related ppi networks . in parallel , validated corresponding mirnas for each hub gene were determined and a new mirnas - hubgenes network was reconstructed with 217 and 319 nodes and edges , respectively .
topological analysis of mirnas - hubgenes regulatory network was performed and the hub mirnas were identified and visualized along with their putative targets ( figure 2 ) .
we compared the hub mirnas with dems obtained from the mirna expression profile of chronic hcv and hcc samples .
two hub mirnas , including hsa - mir-34a and hsa - mir-155 were significantly down - regulated in both chronic hcv and hcc samples compared to normal liver samples .
moreover , three other hub mirnas ( hsa - mir-24 , hsa - mir-744 and hsa - mir-92a ) were up - regulated in chronic hcv and hcc samples compared to the control samples .
the common hub genes between chronic hcv and hcc are shown with dark cyan circles .
after networks alignment , several sub - networks were identified as common complexes between chronic hcv and hcc networks .
the ec measure was equal to 0.45 indicating approximately 45% of topological similarity between the given networks .
we selected a 22-node sub - network as the most important functional complex , which consists of 13 common genes with positive correlation ( figure 3 ) .
the functional annotation analysis demonstrated that this sub - network is actively involved in regulation of cell proliferation , cell migration and apoptosis .
the common genes between chronic hcv and hcc networks are shown in the green color boxes .
analysis of the functional annotation of chronic hcv and hcc related degs specifically indicated that most genes , in both chronic hcv and hcc related degs , are involved in important cellular processes such as cell signaling , cell proliferation , cell death , cell migration , etc .
furthermore , we enriched the list of dems obtained from mirna expression analysis of both hcc and chronic hcv samples .
interestingly , there was a considerable overlap between the functional categories of chronic hcv and hcc related dems ( figure 1 ) .
similarly to degs , a high number of mirnas predicted as regulators of several cancer - related pathways such as cell cycle , cell proliferation , cell death , apoptosis , epithelial - mesenchymal transition and angiogenesis .
we found that the four important onco - mirna ( hsa - mir-24 , hsa - mir-92a , hsa - mir-194 and hsa - mir-191 ) were up - regulated in both chronic hcv and hcc samples compared to the control liver cells .
furthermore , several mirna tumor suppressors , including hsa - mir-181b , hsa - mir-451 , hsa - mir-34a , hsa - mir-34b and hsa - mir-34c were significantly down - regulated in chronic hcv and hcc samples compared to the control samples .
the ppi networks for degs resulted from analysis of chronic hcv and hcc gene expression profiles were constructed with 171 - 1090 and 175 - 1161 nodes - edges , respectively .
we used the degree , betweenness centrality and closeness centrality measures to analyze topology of resulted ppi networks .
the top 50 hub genes of these three measures were selected for further analysis ( tables 2 and 3 ) . after filtering and removing duplicate values , we obtained 61 and 64 unique hub genes from the topological analysis of chronic hcv and hcc related ppis , respectively . there were 32 common hub genes between chronic hcv and hcc unique hub genes .
interestingly , the c - jun gene was identified as the most important hub gene in both chronic hcv and hcc related ppi networks . in parallel , validated corresponding mirnas for each hub gene were determined and a new mirnas - hubgenes network was reconstructed with 217 and 319 nodes and edges , respectively .
topological analysis of mirnas - hubgenes regulatory network was performed and the hub mirnas were identified and visualized along with their putative targets ( figure 2 ) .
we compared the hub mirnas with dems obtained from the mirna expression profile of chronic hcv and hcc samples .
two hub mirnas , including hsa - mir-34a and hsa - mir-155 were significantly down - regulated in both chronic hcv and hcc samples compared to normal liver samples .
moreover , three other hub mirnas ( hsa - mir-24 , hsa - mir-744 and hsa - mir-92a ) were up - regulated in chronic hcv and hcc samples compared to the control samples .
the common hub genes between chronic hcv and hcc are shown with dark cyan circles .
after networks alignment , several sub - networks were identified as common complexes between chronic hcv and hcc networks .
the ec measure was equal to 0.45 indicating approximately 45% of topological similarity between the given networks .
we selected a 22-node sub - network as the most important functional complex , which consists of 13 common genes with positive correlation ( figure 3 ) .
the functional annotation analysis demonstrated that this sub - network is actively involved in regulation of cell proliferation , cell migration and apoptosis .
the common genes between chronic hcv and hcc networks are shown in the green color boxes .
strong epidemiological evidence links hepatitis c infection with hepatocellular carcinoma ( 3 ) . liver cancer usually develops decades after hcv infection and liver cirrhosis .
hcv is classified as a positive - strand rna virus that replicates in the cytoplasm and does not integrate into the host dna ; however , its carcinogenic features are remarkable .
it is possible that indirect mechanisms such as chronic liver inflammation , oxidative damages , presence of cirrhosis and dna damage are involved in cancer development ( 21 , 22 ) .
cirrhosis as the result of fibrosis due to hepatitis c infection is the indication of preneoplastic stage in development of liver cancer , which can cause rapid proliferation of dysplastic liver cells ( 21 ) .
studies have shown that hcv proteins , especially core protein , are inherently oncogenic and their expression in the cell can be involved in the development of hcc directly and in the absence of immune responses or cirrhosis .
evidence has shown that replication of hcv replicon rnas is strongly dependent on cell proliferation ( 23 ) , while the hcv rna synthesis is stimulated in the s phase of the cell cycle ( 24 ) .
hepatocytes usually have low proliferation rate and it seems that some viral proteins expressed during hcv infection trigger cell division and are involved in carcinogenic effects of chronic hcv infection ( 22 , 25 ) .
however , the main mechanisms underlying pathogenesis of hcv - induced hcc has remained unclear .
we reported here molecular mechanisms regulated by hcv , which seem to be involved in hcc progression and invasion .
we found that the c - jun gene is the most important hub gene obtained from topological analysis of both hcv and hcc networks .
the c - jun has a key role in enhanced cell proliferation of cancer cells by regulating the expression of ccnd1 and cdc2 genes ( 26 ) .
erhadt and colleagues reported that hcv can protect various cells from tnf-induced apoptosis via activating nfkb signaling pathway ( 27 ) .
taken together , we suggest that the role of hcv in the inhibition of apoptosis is mediated by cjun function .
two proto - oncogene , bcl2 and erbb2 , were identified as the common hub genes of hcv and hcc - derived ppi networks .
the bcl2 overexpressing cells tend to have a prolonged survival and reduced apoptosis in a variety of cancer cells .
this gene is highly capable of suppressing apoptosis by either inhibiting the release of cytochrome c from the mitochondria or blocking the function of apaf1 ( 28 ) .
it is now well established that hcv triggers cancer cell apoptosis and survival through up - regulation of bcl2 expression , resulting in reduced casp3 activity .
the nonstructural protein 4b ( ns4b ) is the key gene responsible for stimulating bcl2 gene through inhibition of the function of socs3 gene ( 29 ) .
erbb2 is a protein tyrosine kinase that can bind to other growth factor receptors to form a heterodimer , stabilizing ligand - receptor complex and enhancing several downstream signaling pathways such as mitogen - activated protein kinase ( mapk ) and phosphatidylinositol-3 kinase ( pi3k / akt1 ) ( 10 , 30 ) .
this gene was identified as an important hub gene in both chronic hcv and hcc networks . the statistically significant correlation between ns3 ,
an hcv serine protease , and erbb2 has been reported in hcc cases ( 31 ) .
this hub gene was up - regulated in hcv and hcc samples compared to normal liver cells .
it is now widely accepted that hcv is of central importance for vascular development and angiogenesis through enhancing the expression of vegfc ( 32 ) .
extracellular matrix proteins play a pivotal role in tumor progression and their alterations are usually associated with an aggressive phenotype of various cancer types .
our results revealed that expression of col4a2 , col1a1 and col5a1 hub genes were significantly altered in the hcv - infected and hcc cells compared to the normal cell samples .
these genes are necessary for spreading hcc cells through remodeling of extracellular matrix ( 33 ) .
recently , it has been determined that hcv infection can lead to an increased invasive phenotype by regulating the matrix metalloproteinases ( mmps ) , a family of extracellular matrix components ( 34 ) .
however , the role of hcv in regulation of collagen genes is poorly understood . amongst non - coding rnas ,
varnholt and colleagues examined the mirna gene expression profile of hcv - infected hcc cells and identified several important mirnas that seem to be involved in the pathogenesis of hcv - associated hcc ( 26 ) .
we found that the mirna gene expression profile of hcv - infected cells is clearly similar to hcc cells .
the hsa - mir-155 and hsa - mir-34a were down - regulated in both hcv - infected and hcc cells compared to the control samples .
the hcv core and ns5a proteins have been implicated in pathogenesis of hcc through induction of wnt signaling pathway ( 28 ) .
hcv - associated activation of wnt signaling pathway may be mediated by expression alterations of hsa - mir-34a and has - mir-744 .
additionally , the hcv can actively induce cell proliferation and mapk signaling pathway in vitro ( 27 ) .
however , the distinct mechanism defining the effect of hcv on the cell proliferation has remained unknown .
here , we show that hcv can positively impair cell cycle progression through inhibition of hsa - mir-34a .
the expression of two cell proliferation - related genes , rrm2 and birc3 , is regulated by hsa - mir-34a ( figure 4 ) .
up - regulated and down - regulated hub mirnas with positive correlation in both hcv - infected and hcc cells are shown in red and green boxes , respectively .
however , activation of mapk , akt1 , nfkb1 and jak - stat signaling pathways is also mediated through down - regulation of hsa - mir-155 .
this hub mirna was significantly down - regulated in both hcv - infected and hcc cells compared to normal cells .
however , functional study of hsa - mir-155 targets revealed that down - regulation of this mirna can induce cell proliferation and angiogenesis and inhibit apoptosis through activating aforementioned signaling pathways ( figure 4 ) .
the hsa - mir-24 acts as an anti - apoptotic mirna by repressing the expression of bim gene ( 35 ) .
we found that this hub mirna was up - regulated in hcv - infected and hcc samples , suggesting its possible effect on the progression of hcc cells .
furthermore , the function of tumor suppressor brca1 is considerably modulated by hsa - mir-24 .
it has been shown that overexpression of hsa - mir-744 can moderately induce tube formation and reduce apoptosis in vitro ( 36 ) .
this study indicated that characteristics of invasive phenotype hcc cells are associated with differential expressions of several genes and mirnas .
the graph - based information may give us a new insight into molecular mechanisms underlying the pathogenesis of hcv - induced hcc .
this study indicated that characteristics of invasive phenotype hcc cells are associated with differential expressions of several genes and mirnas .
the graph - based information may give us a new insight into molecular mechanisms underlying the pathogenesis of hcv - induced hcc . | background : hepatitis c virus ( hcv ) has been known as a major cause of hepatocellular carcinoma ( hcc ) worldwide .
however , the distinct molecular mechanisms underlying the effects of hcv proteins on the hcc progression have remained unclear.objectives:in the present study , we studied the possible role of hcv in the hcc initiation and invasion using topological analysis of protein - protein interaction ( ppi ) networks.materials and methods : after analysis with geo2r , a ppi network of differentially expressed genes ( degs ) was constructed for both chronic hcv and hcc samples . the string and genemania databases
were used to determine the putative interactions between degs . in parallel ,
the functional annotation of degs was performed using g : profiler web tool .
the topological analysis and network visualization was carried outperformed using cytoscape software and the top hub genes were identified .
we determined the hub genes - related mirnas using mirtarbase server and reconstructed a mirna - hubgene network.results:based on the topological analysis of mirna - hubgene network , we identified the key hub mirnas . in order to identify the most important common sub - network , we aligned two ppi networks using netal tool .
the c - jun gene was identified as the most important hub gene in both hcv and hcc networks .
furthermore , the hsa - mir-34a , hsa - mir-155 , hsa - mir-24 , hsa - mir-744 and hsa - mir-92a were recognized as the most important hub mirnas with positive correlation in the chronic hcv and hcc samples .
functional annotation of differentially expressed mirnas ( dems ) using the tool for annotations of human mirnas ( tam ) revealed that there is a considerable overlap between mirna gene expression profiles of hcv - infected and hcc cells.conclusions:our results revealed the possible crucial genes and mirnas involved in the initiation and progression of hcc cells infected with hcv . |
the cnisn includes 554 sentinel hospitals conducting surveillance for influenza - like illness ( ili ; hereafter called sentinel hospitals ) and 408 network laboratories in all 31 provinces of china ( figure 1 ) . on a weekly basis ,
sentinel hospitals report the number of outpatient visits , by age group , for ili and the total number of outpatients . each week , 515 nasopharyngeal swab samples are collected from a convenience sample of patients who visit sentinel hospitals within 3 days of ili onset .
demographic and epidemiologic data , including age , sex , date of illness onset , and occupation , are also collected .
patient specimens are tested by real - time reverse transcription pcr or virus isolation in the affiliated laboratories .
geographic distribution of national influenza surveillance sentinel hospitals in beijing and shanghai municipalities and 8 provinces with confirmed human cases of avian influenza a(h7n9 ) virus infection , china , 2013 . on april 3 , 2013 ,
to enhance surveillance for influenza a(h7n9 ) virus , all network laboratories were required to increase the number of specimens to a minimum of 15/week and to test all specimens collected since march 4 , 2013 , for influenza a(h7n9 ) virus by real - time reverse transcription pcr as described ( 3,4 ) .
population data by age group were provided by the national bureau of statistics of china . during march 4april 28
samples included 20,739 specimens from patients with ili at 141 sentinel hospitals in 10 affected provinces / municipalities : anhui , jiangsu , zhejiang , shandong , henan , fujian , jiangxi , and hunan provinces and shanghai and beijing municipalities ( tables 1 , 2 ) .
the median number of specimens collected each week from affected provinces / municipalities was 244 ( range 72792 ) . of the 20,739 samples from patients with ili , 10,035 ( 48.4% ) were from persons 014 years of age , 9,319 ( 44.9% ) were from persons 1559 years of age , and 1,385 ( 6.7% ) were from persons > 60 years of age .
the age distribution of ili cases in the 10 affected provinces / municipalities was substantially different from that in the overall population ; persons 2559 years of age had a lower proportion of ili than would be expected had ili distribution mirrored the age distribution of the population .
( technical appendix , figure 1 ) . in the affected provinces / municipalities , the number of specimens tested increased from a mean of 2,643 during the week starting april 1 to a peak of 3,259 during the week starting april 9
; the increase was highest among persons 1524 and 2559 years of age ( technical appendix
figure 2 ) .
areas include beijing and shanghai municipalities and anhui , jiangsu , zhejiang , shandong , henan , fujian , jiangxi , and hunan provinces .
all areas are provinces , except beijing municipality . positive patient , a resident of shanghai , stayed for 23 d in hunan , where he visited the sentinel hospital and then returned to shanghai .
percentage of hospital visits attributed to influenza - like illness , china , april 2 , 2012may 6 , 2013 .
hospital visits were made to sentinel surveillance hospitals in 7 southern provinces ( sp ) and 3 northern provinces / municipalities ( nm , np ) with confirmed human cases of avian influenza a(h7n9 ) virus infection .
arrows indicate march 31 , 2013 , the date the first human case of influenza a(h7n9 ) virus infection was reported . during april 128 ,
the percentage of visits for ili increased in 5 of the 7 affected southern provinces and 2 of 3 affected northern provinces / municipalities ( figure 2 ) .
however , during the same period , the proportion of specimens positive for influenza decreased in the affected provinces / municipalities . of the 10 affected provinces / municipalities , 5 reported > 1 ili patient with test results positive for influenza a(h7n9 ) virus .
the percentage of specimens positive for influenza a(h7n9 ) virus , by province / municipality , ranged from 0 to 0.06% ( table 2 ) .
we detected influenza a(h7n9 ) virus in samples from 6 ( 0.03% ) of the 20,739 patients with ili ; these cases were then reported as confirmed to the local cdcs and china cdc .
no unsubtypeable influenza samples were reported in the affected provinces / municipalities during the study period ( technical appendix table ) .
epidemiologic investigations found that 2 of the 6 patients with influenza a(h7n9 ) infection had not been hospitalized , and the other 4 had been hospitalized for pneumonia complications .
of the 4 hospitalized patients , 3 were 2559 years of age , and 1 was 69 years of age .
four of the patients had a history of contact with live chickens or visiting a live poultry market .
after the avian influenza a(h7n9 ) virus outbreak was identified in china , cnisn increased sampling and testing of ili case - patients .
cnisn has tested > 46,807 specimens from all provinces , including 20,739 specimens from affected provinces / municipalities . as a result of this testing , cnisn identified 6 influenza a(h7n9 ) virus positive specimens in 5 provinces that were already known to have cases .
these data demonstrate that avian influenza a(h7n9 ) virus is an uncommon cause of ili in any age group and in the areas reporting confirmed cases of influenza a(h7n9 ) infection . the confirmed case - patients included 2 children who did not require hospitalization and 4 adults with more severe disease , possibly indicating that influenza a(h7n9 ) virus causes milder disease in younger persons .
although the proportion of all outpatient visits for ili increased in affected provinces / municipalities , virologic surveillance data showed that the proportion of ili patient specimens positive for influenza decreased , and there was no increase in unsubtypeable influenza viruses during the study period .
this suggests that any increase in the percentage of consultations for ili might be a result of increased healthcare seeking behavior after media reports of the avian influenza a(h7n9 ) virus outbreak or the circulation of non - influenza respiratory viruses .
the spectrum of illness caused by other avian influenza viruses varies tremendously and can also vary by age group .
previous human infections with avian influenza a(h7 ) viruses ( i.e. , subtypes h7n3 , h7n2 , and h7n7 ) have been generally mild , causing conjunctivitis , with the exception of very occasional cases of pneumonia and a single fatal case in the netherlands in a highly exposed veterinarian ( 510 ) .
in contrast , avian influenza a(h5n1 ) virus has an overall case fatality rate of 60% , and persons with confirmed cases are usually severely ill ( 11 ) .
recent reviews of avian influenza a(h5n1 ) virus seroprevalence studies found little evidence that large numbers of human infections are going undetected ( 1214 ) . among the 82 human influenza a(h7n9 )
virus infections reported as of april 17 , 2013 , a total of 38 ( 46% ) were in persons > 65 years of age ( 2 ) .
we did not find evidence of widespread mild disease , suggesting that the reported cases reflect the true distribution of infection and not a surveillance artifact .
the 554 cnisn sentinel hospitals are located in urban areas , so the surveillance system may not detect influenza a(h7n9 ) virus infections in rural areas .
in addition , most sentinel hospitals are tertiary care hospitals , and their patient populations are not representative of the general population with ili .
the distribution of those patients who had specimens tested is not necessarily random and may not reflect the population of those with ili .
last , our system lacks a straightforward way to calculate rates of disease because it lacks denominators .
the emergence of a reassortant between avian influenza a(h7n9 ) virus and seasonal influenza subtype viruses , with possible increased human transmissibility , is possible during the upcoming summer influenza season in southern china .
careful monitoring and rapid characterization of influenza a(h7n9 ) viruses and unsubtypeable viruses from infected humans will be critical .
enhanced surveillance studies of mild and severe respiratory disease and seroprevalence studies in focal areas are necessary to further characterize the epidemiology and clinical spectrum of this emerging virus .
number of influenza virus positive respiratory specimens , by virus type / subtype , in provinces with confirmed human influenza a(h7n9 ) virus infections ; age distribution of patients seen for influenza - like illness in areas with confirmed human influenza a(h7n9 ) virus infections ; and number of specimens tested for avian influenza a(h7n9 ) virus in 10 provinces / municipalities with confirmed human cases , china , march 4april 28 , 2013 . | in china during march 4april 28 , 2013 , avian influenza a(h7n9 ) virus testing was performed on 20,739 specimens from patients with influenza - like illness in 10 provinces with confirmed human cases : 6 ( 0.03% ) were positive , and increased numbers of unsubtypeable influenza - positive specimens were not seen .
careful monitoring and rapid characterization of influenza a(h7n9 ) and other influenza viruses remain critical . |
histoplasmosis is an infective condition caused by a dimorphic , saprophytic fungus , histoplasma capsulatum and is acquired by inhalation of its spores .
soil rich in bird and bat dropping is its natural habitat , and it exists as a mycelium in the atmosphere . the disease is endemic in the united states , africa and asia .
although most infections are asymptomatic , self - limiting acute pneumonitis and hilar lymphadenopathy may occur with inhalation of large aerosol .
it may occur in disseminated form in some patients with impaired host defence mechanism and frequently involves liver , spleen , lymph node , bone marrow and adrenal.[24 ] adrenal involvement is usually sequelae of previous infection .
we encountered two patients presenting with nonspecific features of systemic disease and imaging revealed bilateral adrenomegaly .
transabdominal ultrasound guided fine - needle aspiration cytology ( fnac ) of the adrenal showed histoplasmosis . a brief review of imaging of adrenal histoplasmosis is discussed along with the cases .
a 60-year - old diabetic male presented with low grade , intermittent fever , loss of weight and appetite for duration of 4 months .
he had been on oral hypoglycemics for noninsulin - dependent diabetes mellitus for 7 years .
routine blood examination , urine examination , renal function test and chest x - ray were normal .
contrast - enhanced ct of the abdomen showed hepatosplenomegaly with enlarged , hypoattenuated bilateral adrenals showing peripheral rim enhancement and enhancing internal septations [ figure 2 ] .
transabdominal ultrasound - guided fnac [ figure 3 ] revealed numerous organism ranging in size from 2 to 4 m in a necrotic background which were positive on periodic acid schiff staining consistent with histoplasmosis .
the patient was treated with itraconazole 200 mg twice daily and clinical improvement was seen within 4 weeks .
abdominal ultrasonography showing enlarged , hypoechoic right ( a ) and left ( b ) adrenal glands ( arrows ) contrast - enhanced axial ct section of the upper abdomen showing enlarged and hypodense bilateral adrenals ( white arrows ) with peripheral rim enhancement and enhancing internal septations ( black arrows ) fine - needle aspiration smear showing numerous degenerating histoplasmosis capsulatum organisms in the necrotic background ( periodic acid schiff stain , 1000 ) a 56-year old male presented with 6 month history of on and off fever , loss of weight and appetite , and hyperpigmentation all over the body .
nmol / l ( 171 - 536 nmol / l ) and post stimulation was 115
he had decreased calcium 8.3 gm / dl ( 8.6 - 10.2 gm / dl ) and potassium 3.04 meq / l ( 3.5 - 5 meq / l ) levels .
rest of the laboratory investigations including routine blood examination and blood sugar levels were normal .
ultrasonography of the abdomen revealed bilateral adrenomegaly [ figure 4 ] and contrast - enhanced ct showed peripherally enhancing hypodense bilateral adrenals with peripheral rim and septate enhancement [ figure 5 ] .
the fnac showed a similar picture as in the case 1 confirming histoplasmosis which was also positive by fungal culture studies .
the patient was treated with hydrocortisone , fludrocortisone and amphotericin b. the patient showed clinical improvement within one week .
ultrasonography of the abdomen revealing right ( a ) and left ( b ) adrenomegaly ( arrows ) contrast - enhanced axial ct showing enlarged adrenals glands ( white arrows ) with rim enhancement ( black arrows ) of periphery and internal septations and central hypodensity
a 60-year - old diabetic male presented with low grade , intermittent fever , loss of weight and appetite for duration of 4 months .
he had been on oral hypoglycemics for noninsulin - dependent diabetes mellitus for 7 years .
routine blood examination , urine examination , renal function test and chest x - ray were normal .
contrast - enhanced ct of the abdomen showed hepatosplenomegaly with enlarged , hypoattenuated bilateral adrenals showing peripheral rim enhancement and enhancing internal septations [ figure 2 ] .
transabdominal ultrasound - guided fnac [ figure 3 ] revealed numerous organism ranging in size from 2 to 4 m in a necrotic background which were positive on periodic acid schiff staining consistent with histoplasmosis .
the patient was treated with itraconazole 200 mg twice daily and clinical improvement was seen within 4 weeks .
abdominal ultrasonography showing enlarged , hypoechoic right ( a ) and left ( b ) adrenal glands ( arrows ) contrast - enhanced axial ct section of the upper abdomen showing enlarged and hypodense bilateral adrenals ( white arrows ) with peripheral rim enhancement and enhancing internal septations ( black arrows ) fine - needle aspiration smear showing numerous degenerating histoplasmosis capsulatum organisms in the necrotic background ( periodic acid schiff stain , 1000 )
a 56-year old male presented with 6 month history of on and off fever , loss of weight and appetite , and hyperpigmentation all over the body .
nmol / l ( 171 - 536 nmol / l ) and post stimulation was 115
he had decreased calcium 8.3 gm / dl ( 8.6 - 10.2 gm / dl ) and potassium 3.04 meq / l ( 3.5 - 5 meq / l ) levels .
rest of the laboratory investigations including routine blood examination and blood sugar levels were normal .
ultrasonography of the abdomen revealed bilateral adrenomegaly [ figure 4 ] and contrast - enhanced ct showed peripherally enhancing hypodense bilateral adrenals with peripheral rim and septate enhancement [ figure 5 ] .
the fnac showed a similar picture as in the case 1 confirming histoplasmosis which was also positive by fungal culture studies .
the patient was treated with hydrocortisone , fludrocortisone and amphotericin b. the patient showed clinical improvement within one week .
ultrasonography of the abdomen revealing right ( a ) and left ( b ) adrenomegaly ( arrows ) contrast - enhanced axial ct showing enlarged adrenals glands ( white arrows ) with rim enhancement ( black arrows ) of periphery and internal septations and central hypodensity
histoplasmosis is usually a self - limiting pulmonary infection , which is usually asymptomatic and can be associated with mild influenza like symptoms .
there are three major clinical presentations : pulmonary , progressive disseminated and primary cutaneous histoplasmosis .
progressive disseminated form of the disease is rare and occurs in the immunocompromised patients in the endemic areas .
progressive disseminated disease may manifest as chronic disease in immunocompetent host or acute progressive disease in immunosuppressed hosts . in the patients with disseminated histoplasmosis
the patients of adrenal histoplasmosis usually reveal bilateral adrenal masses of varied imaging features . on ultrasonography
bilateral symmetrical adrenomegaly with preservation of the contour of the gland , central hypodensity with peripheral enhancement and presence of calcification has been described in ct . in both cases ,
the differential diagnoses of bilateral adrenomegaly are metastasis , lymphoma , adrenal haemorrhage , sarcoidosis and infections which include histoplasmosis , tuberculosis , cryptococosis , coccidioidomycosis and blastomycosis .
but , central hypodensity and peripheral rim enhancement of the adrenals narrow down the differentials only to tuberculosis and histoplasmosis . as the adrenal gland is frequently the only demonstrable site of active disease
, adrenal fnac can suggest the diagnosis which is further confirmed by culture , polymerase chain reaction , and urine antigen .
h. capsulatum is an intracellular dimorphic fungus which is commonly seen within the cytoplasm of the macrophages and exhibit narrow based budding.[1012 ] in fine - needle aspiration smears , these organism can also be seen extracellularly as the fragile cytoplasm of the macrophages may get disrupted at the time of making smears . in our two cases , there was necrosis in the smears which may have resulted in their presence extracellularly scattered throughout the necrotic material .
these fungi can also be easily identified at the time of onsite adequacy evaluation in contrary to previous data which suggest that it is difficult to appreciate on routine cytological stains.[1012 ] rapid onsite cytopathological evaluation can suggest immediate diagnosis of the infection and hence the treatment can be started at the earliest .
although this was not performed in our cases , the diagnosis was obtained the next day permitting prompt institution of treatment . in the regions where endemic , possibility of
histoplasmosis should always kept in a patient with enlarged , hypoattenuating bilateral adrenal glands with peripheral rim enhancement and enhancing internal septations .
image guided fnac will demonstrate the disease and should be performed without delay as a great number of patients with adrenal histoplasmosis may develop life - threatening adrenal insufficiency if untreated . | histoplasmosis is a geographically restricted form of fungal infection .
adrenal involvement is seen in disseminated disease but sometimes it may be the only site of demonstrable disease .
early diagnosis and treatment may save the patient from catastrophic adrenal insufficiency .
we present two patients showing bilateral adrenomegaly on ultrasonography and contrast - enhanced ct , and was diagnosed to have histoplasmosis on fine - needle aspiration cytology . |
concern regarding facial esthetics is one of the most probable reasons to seek orthodontic and/or surgical orthodontic treatment .
the dentition and the occlusal relationship play a vital role in determining the overall perception of facial esthetics .
the degree of anteroposterior ( ap ) discrepancy , either dental or skeletal , often serves as an indicator of malocclusion , and by inference the need for the treatment . the treatment plan , orthodontics and/or orthognathic surgery , for patients with more severe problems is frequently dictated in part by esthetic concerns .
orthodontic patients and their parents believe that well - aligned teeth are important for an overall pleasing appearance .
the decision to undergo orthodontic treatment seems to be motivated by social norms and the beauty culture in their reference group .
therefore , the opinion of the laymen is an important parameter in determining the success of orthodontic treatment .
orthodontists , however , prefer to use objective parameters instead of opinions for their diagnosis , treatment plan and evaluation of the outcome of their clinical intervention .
the objective parameters used by the orthodontists usually include quantitative descriptions of ap , vertical discrepancies , and dental irregularities .
studies carried out to relate the laypersons perception of facial esthetics to the orthodontist 's objective parameters of facial and dental value are scarce .
it is postulated that laypeople will vary in their subjective viewpoints , and this would be dependent upon the environment that they are exposed to .
hence , this study was done to determine the objective parameters used in daily orthodontic practice related to facial esthetics , as perceived by the laypeople in the selected district .
the objective parameters used represented ap characteristics ( overjet and anb angle ) , vertical characteristics ( sn - gogn angle ) , and dental esthetics esthetic component of the index of orthodontic treatment need ( ac / iotn ) .
this study examined and correlated the contribution of the objective measures representing ap and vertical characteristics , dental esthetics , or their combination that are used in daily orthodontic practice with subjective measures in the assessment of facial esthetics for the test group population .
inclusion criteria for subjects for the collection of data are as follows :
the age is between 10 and 25 yearshad no dental or facial traumahad no history of orthodontic treatmenthad no congenital defectsnot been wearing glassesthe panel of laypeople should be from different professional backgrounds .
the age is between 10 and 25 years had no dental or facial trauma had no history of orthodontic treatment had no congenital defects not been wearing glasses the panel of laypeople should be from different professional backgrounds .
about 64 patients were randomly selected after stratification , from the records of the department of orthodontics of a dental hospital .
eight boys and eight girls each , in angles class i ; class ii division i ; class ii division ii ; class iii malocclusions , were shortlisted .
the objective parameters were obtained from the pretreatment records ( dental casts , cephalograms , extraoral and intraoral color photographs ) [ figures 13 ] of these patients .
subjective parameters : 64 laypeople ( 32 males and 32 females ) were chosen from professional colleges in the area .
each subject was given a performa having 64 patients ' names and a visual analog scale ( vas ) ( a vas , operationally , a measurement instrument with horizontal line , which is 100 mm in length , anchored by 0 in beginning and 100 at the end representing least attractiveness and most attractiveness parameters marked , respectively .
the subject marks on the line , the point that they feel represents their perception of the variable under consideration .
the vas score is then determined by measuring the length of the line marked , in millimeters , from the left - hand end of the line to the point that the subject had marked against each patient .
a slide show presenting the digital images in the ( a ) frontal view , ( b ) three - quarter smiling view , and ( c ) profile view of each patient was prepared .
overjet measurement using ruler anb and sn - gogn sella - nasion - gonion gnathionmeasurement from lateral cephalogram a sample of extraoral photographs of a patient sample of intraoral photographs of a patient the following objective parameters were obtained from the records :
overjet was measured on the dental casts as the ap distance between the maxillary and mandibular central incisors at the most labial point of the most prominent incisor [ figure 1]anb angle and sn - gogn angle were measured on tracing of lateral head film [ figure 2]the horizontal sum obtained as the summation of the overjet in millimeters and anb in degreesac / iotn was determined on intraoral pictures [ figure 5 ] by mutual agreement between two independent observers ( as per the guidelines of shaw wc ) .
overjet was measured on the dental casts as the ap distance between the maxillary and mandibular central incisors at the most labial point of the most prominent incisor [ figure 1 ] anb angle and sn - gogn angle were measured on tracing of lateral head film [ figure 2 ] the horizontal sum obtained as the summation of the overjet in millimeters and anb in degrees ac / iotn was determined on intraoral pictures [ figure 5 ] by mutual agreement between two independent observers ( as per the guidelines of shaw wc ) .
microsoft power point demonstration to laypersons after the collection of data on the varied objective and subjective criteria , the data were subjected to statistical analysis .
then , the data were analyzed by one - way analysis of variance ( anova ) test to find out the significant difference between more than two groups followed by tukeys multiple post hoc procedures for pairwise comparison .
further , the multiple linear regression was performed to see the influence of independent variables on the dependent variable .
the following objective parameters were obtained from the records :
overjet was measured on the dental casts as the ap distance between the maxillary and mandibular central incisors at the most labial point of the most prominent incisor [ figure 1]anb angle and sn - gogn angle were measured on tracing of lateral head film [ figure 2]the horizontal sum obtained as the summation of the overjet in millimeters and anb in degreesac / iotn was determined on intraoral pictures [ figure 5 ] by mutual agreement between two independent observers ( as per the guidelines of shaw wc ) .
overjet was measured on the dental casts as the ap distance between the maxillary and mandibular central incisors at the most labial point of the most prominent incisor [ figure 1 ] anb angle and sn - gogn angle were measured on tracing of lateral head film [ figure 2 ] the horizontal sum obtained as the summation of the overjet in millimeters and anb in degrees ac / iotn was determined on intraoral pictures [ figure 5 ] by mutual agreement between two independent observers ( as per the guidelines of shaw wc ) .
microsoft power point demonstration to laypersons after the collection of data on the varied objective and subjective criteria , the data were subjected to statistical analysis .
then , the data were analyzed by one - way analysis of variance ( anova ) test to find out the significant difference between more than two groups followed by tukeys multiple post hoc procedures for pairwise comparison .
further , the multiple linear regression was performed to see the influence of independent variables on the dependent variable .
the statistical significance for one way anova was set at 5% level of significance ( p < 0.05 ) .
the values and standard deviations of the various objective parameters obtained and segregated into angle 's classes [ table 1 ] .
one - way anova test done to compare various classes with respect to the different objective parameters indicated that all the parameters , except the vertical parameter ( sn - gogn ; p = 0.1758 , ( p > 0.05 ) not significant ) , showed a statistically significant variation between different classes .
overjet , anb , iotn , and horizontal sum were significantly correlated with p value of 0.0000 , 0.0001 , 0.00400 , and 0.0008 , respectively [ table 2 ] .
it was deduced that the value of each parameter was significantly different from the same parameter in other class .
the esthetic scores ( vas scores ) given by male and female reviewing laypeople were compared [ table 3 ] .
it was observed that the comparison of different classes with respect to vas scores given by male and female laypeople was found to be nonsignificant .
multiple regression analysis of the subjective parameter , the vas scores ( dependent variable ) with the objective parameters taken , indicated that though the overjet , horizontal sum , and ac ( iotn ) negatively correlated with the objective parameters , only the ac ( iotn ) showed a statistically significant negative correlation [ table 4 ] .
a backward stepwise multiple regression analysis of vas scores according to the total sample of laypersons showed a significant correlation between subjective vas score with the ac ( iotn ) [ table 5 ] .
the mean and standard deviation of different objective parameters obtained grouped according to angle 's classification comparison of classes with respect to different objective measurements by one - way anova comparison of the different angle 's classes ( i , ii division i , ii division ii , iii ) with respect to visual analog scale scores given by male and female laypersons multiple regression analysis of visual analog scale scores according to the total laypersons ( male and female laypersons ) ( dependent variable ) with objective measurements ( independent variables ) backward stepwise multiple regression analysis of visual analog scale scores according to the total sample of laypersons ( male and female laypersons ) ( dependent variable ) with index of orthodontic treatment need
the most commonly used parameter for ap characteristics is angle classification , which is a rough estimate having four discrete classes . for proper orientation of the jaw , combination of anb and overjet has been indicated .
the effect of the vertical on facial attractiveness has been studied on constructed profiles or manipulated photographs .
most orthodontists use sn - gogn angle for the evaluation of vertical dimensions in daily clinical practice , but the relation of this parameter to facial attractiveness has been debatable .
both orthodontists and laymen are well able to use vas scores to judge facial esthetics from photographs in a more or less intuitive way although facial esthetics seem to be subjective and not a well - defined variable . when the various classes of angle 's classification were compared to different objective parameters , all the parameters , except the vertical parameter sn - gogn , showed significant variation between the different classes .
these parameters were distinctly different despite overlapping ranges for different classes as indicated by the one - way anova test done between the classes .
however , no difference was found between two sexes of patients , when their objective parameters were compared .
observed that the objective parameters used in his study showed wide overlapping ranges for different classes and none of them was decisive for angle classification . in our study , however , the objective parameters were distinctly different between the classes but did not differ between the male and female subjects
. the comparison of different classes with respect to vas scores given by male and female laypeople did not show significant differences , so it could be deduced that the laypeople were not appreciative of facial beauty with respect to different angle classes . in this study ,
class i and class ii / division i malocclusions were rated lower than the class ii / division ii and class iii malocclusions as against the previous studies which rated class ii and class iii lower than class i malocclusions .
this could be interpreted that the laypeople of the area under study are more appreciative of a protrusive profile as against a straight or retrusive profile .
the male laypeople gave higher esthetic scores than female laypeople showing that females were more critical in evaluating both male and female subjects than the males .
however , this observation should be subjected to further research . when the esthetic scores given by male and female laypersons were compared with the objective measurements , the ac ( iotn ) and the horizontal sum showed significant negative correlation with the subjective parameters .
the horizontal sum , introduced by kiekens et al . appeared to be a useful parameter to measure the horizontal discrepancy .
it is related to the dentition ( overjet ) measured from the dental cast and to the skeletal parameter
however , when the esthetic values given by males and females separately were compared with the objective parameters , only the ac ( iotn ) significantly correlated with the objective parameters , negatively . a multiple regression analysis of the subjective parameter , the vas scores ( dependent variable ) with the objective parameters taken , indicated that only the ac ( iotn ) was significant and negatively correlated and individually only female laypersons were negatively correlated and significant .
the laypeople gave the highest vas score on the factors with a horizontal sum of 7.25 for male and 8.3 for female laypersons .
the fact that for the variable horizontal sum , degrees , and millimeters are summed , might be surprising , but it appears to be reliable parameter ( cronbach 's alpha = 0.7391 ) .
, matoula and pancherz found that the ap discrepancies , as measured by soft tissue anb , showed minimal correlation with facial attractiveness .
however , a trend emerged that would suggest that in faces where the anb varies widely from 5 , the face is considered less attractive .
sn - gogn angle , however , is a measure for mandibular rotation or growth direction and not for facial height .
found that both orthodontists and layperson are sensitive to small horizontal changes and orthodontists are relatively less sensitive to vertical changes compared to the horizontal changes .
studies showed that orthodontists are significantly more likely to choose class i profile as most attractive and vast majority of the orthodontists and laymen considered class ii profile as least attractive .
michiels and sather found profiles with increase vertical features or convex or class ii tendency profiles were judged as being most unattractive . in this study , however , class ii division ii and class iii malocclusions were judged as being more attractive than the class ii or class i angle malocclusion .
showed that the iotn ( ac ) index appeared robust in its reflection of the perception of malocclusion by children and parents , respectively .
assessments were little affected by gender or ethnicity . however , the scores of children and parents were much lower than those of an orthodontist trained in the use of iotn .
johansson and follin found that photographs matching higher grades on the ac scale were perceived as the most unattractive , indicating what could be a layperson 's priority when considering an orthodontic treatment policy in tanzania .
synonymously , in this study , the ac / iotn appeared to be a valuable indicator of facial esthetics and the higher grades on the scale were considered to be the most unattractive , whereas the lower scales were more attractive .
the ac / iotn , a measure of dental esthetics , appears to be the most vital contributor of facial esthetics and addition of horizontal sum increased the explained variance to 35 .
this increases the prognostic value of the ac / iotn and horizontal sum when used in combination in evaluating facial esthetics , but the remaining 65% of the variance is left unaccounted for by these parameters and could probably be attributed to other facial features such as the eyes , skin , and hair .
the abovementioned study revealed that objective measurement showed overlapping between different angle classes , but the differences were statistically significant for each angle classes .
correlation between esthetic scores done by male and female laypersons together with objective measurements showed that only ac / iotn was negatively correlated and a statistically significant parameter .
the prognostic value of the ac / iotn increased when used with the horizontal sum in evaluating facial esthetics .
thus , horizontal sum was found to be a reliable variable for ap characteristics of the patient .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
| background : the study mentioned was aimed to examine the contribution of the objective measures representing anterior - posterior ( ap ) and vertical characteristics , dental esthetics , or their combination that are used in daily orthodontic practice in the assessment of the facial esthetics.materials and methods : a panel of 64 laypersons evaluated the facial esthetics of 32 boys and 32 girls , stratified over four different angle classes , on a visual analog scale .
the relationship between the objective parameters and facial esthetics was evaluated by the backward multiple regression analysis.results:dental esthetics , expressed by the esthetic component of the index of orthodontic treatment need ( ac / iotn ) , appeared to be the most vital indicator for facial esthetics .
the horizontal sum , a variable for ap characteristics of the patient , could be a better variable when compared with the overjet.conclusion:addition of this newly defined parameter to the ac / iotn improved the prognostic value from 25% to 35% . |
sexual dysfunction is a common complication in diabetic patients which accompanies with some complications of diabetes and should be considered especially in patients with nephropathy or retinopathy .
nowadays , 246 million people in the world are affected by diabetes and it is expected to affect 380 million by 2025 . moreover , the largest increases in prevalence of diabetes will occur in developing countries by 2025 . among diabetic patients
sexual health is an important , but often neglected . according to the world health organization , term sexual dysfunction
is defined as various ways in which an individual is unable to participate in a sexual relationship as he / she would wish .
it was first mentioned in the 10 century ad by the persian physician and philosopher avicenna describing the collapse of sexual functions in diabetic males .
previous reports have shown that diabetic men are at increased risk for sexual dysfunction with an incidence ranging from 20 - 85% .
in contrast to male sexual dysfunction , sexual dysfunction has not been well studied among diabetic women and its prevalence in diabetic women seems to be slightly lower ( 20 - 80% ) .
sexual problems in diabetic women mostly include sexual satisfaction and desire , orgasmic and arousal disorder and lubrication .
several neurological , vascular , endocrine , and psychological factors have been suggested as the etiology of sexual dysfunction among diabetics .
it is stated that infections , hyperglycemia , and vascular , neural , neurovascular and psychosocial disorders are pathogenetic factors of diabetic female sexual dysfunction . in this study
, we aimed to evaluate the prevalence of sexual dysfunction in diabetic women in addition to evaluate the association of diabetic parameters including diabetes duration , glycemic control and other parameters with chronic complications of diabetes .
this cross - sectional study was conducted among 110 diabetic women referred to shahid beheshti hospital of qom .
the sample size was estimated on the basis of fatemi and taghavi 's research .
the inclusion criteria were identified as registered for two year or longer ( these patients had done all their two to three recent hba1c tests in a year and the mean of their hba1c was used as hba1c level ) , aged 30 years or older and history of diabetes for five years or longer and being married .
exclusion criteria included record of mastectomy , total hysterectomy , pregnancy , existence of sexual disorders in patients ' spouse , anemia , presence of sexual disorders before getting diabetes and known history of psychiatric illness .
body weight of study participants was measured in light clothing to the nearest 0.1 kg on a bathroom scale ( beurer gmbh and co , made in germany ) .
also , height was measured to the nearest 0.5 cm and the participants were standing upright with bared foot , the heels put together and the head in the horizontal plane against a wall - mounted ruler .
body mass index ( bmi ) was calculated by dividing weight ( kg ) by the height squared ( m ) .
systolic and diastolic blood pressures were measured by a richter manometer , made in germany .
patients were considered hypertensive if they had a diastolic blood pressure of 90 mmhg , or systolic blood pressure of 140 mmhg .
people who exercised at least 150 min aerobic exercise per week in the last two months were considered as exercise positive .
the female sexual function index ( fsfi ) is a known instrument that assesses sexual function in women with six domains : desire , arousal , lubrication , orgasm , satisfaction and pain during sexual intercourse . in women ,
this cut - off point was the same figure validated by other researchers we used available data from shahid beheshti hospital where all patients received routine clinical examination that included recording of diabetes duration , body mass index ( bmi ) , fasting blood sugar , insulin resistance ( based on homa - ir ) , metabolic syndrome criteria , blood pressure , cholesterol and triglyceride levels and diabetes - related complications .
serum total cholesterol , triglyceride , and hdl - cholesterol were measured using enzymatic techniques .
fasting plasma glucose was measured and glycosylated hemoglobin ( hba1c ) percentage was determined by ion - exchange method .
insulin level was determined by using kits of dia metra ( made in italy ) , insulin resistance was calculated by homeostasis model of assessment . insulin resistance ( homa - ir )
was calculated using the following us formula : ( fasting glucose ( mg / dl)fasting insulin ( 405/u / ml ) .
all measurements were done under standard conditions by one technician and with the same device .
the micro - vascular complications included recording of retinopathy ( ophthalmology visit ) , nephropathy , ( 24 h urine analysis ) and neuropathy ( peripheral , autonomic ) , monofilament and the diapason therapeutic examination .
data was analyzed using software package used for statistical analysis ( spss ) version 16.0 in a descriptive and analytical fashion . to investigate the associations of demographic and clinical characteristics of the patients with sd , the test was used . in all statistical tests , a p < 0.05 was considered being significant .
diabetic women were assured regarding the confidentiality of the information and the questionnaire was completed by a person with the same gender .
this cross - sectional study was conducted among 110 diabetic women referred to shahid beheshti hospital of qom .
the sample size was estimated on the basis of fatemi and taghavi 's research .
the inclusion criteria were identified as registered for two year or longer ( these patients had done all their two to three recent hba1c tests in a year and the mean of their hba1c was used as hba1c level ) , aged 30 years or older and history of diabetes for five years or longer and being married .
exclusion criteria included record of mastectomy , total hysterectomy , pregnancy , existence of sexual disorders in patients ' spouse , anemia , presence of sexual disorders before getting diabetes and known history of psychiatric illness .
body weight of study participants was measured in light clothing to the nearest 0.1 kg on a bathroom scale ( beurer gmbh and co , made in germany ) .
also , height was measured to the nearest 0.5 cm and the participants were standing upright with bared foot , the heels put together and the head in the horizontal plane against a wall - mounted ruler .
body mass index ( bmi ) was calculated by dividing weight ( kg ) by the height squared ( m ) .
systolic and diastolic blood pressures were measured by a richter manometer , made in germany .
patients were considered hypertensive if they had a diastolic blood pressure of 90 mmhg , or systolic blood pressure of 140 mmhg .
people who exercised at least 150 min aerobic exercise per week in the last two months were considered as exercise positive .
sexual dysfunctions were measured in women using a standard questionnaire . the female sexual function index ( fsfi ) is a known instrument that assesses sexual function in women with six domains : desire , arousal , lubrication , orgasm , satisfaction and pain during sexual intercourse . in women ,
this cut - off point was the same figure validated by other researchers we used available data from shahid beheshti hospital where all patients received routine clinical examination that included recording of diabetes duration , body mass index ( bmi ) , fasting blood sugar , insulin resistance ( based on homa - ir ) , metabolic syndrome criteria , blood pressure , cholesterol and triglyceride levels and diabetes - related complications .
serum total cholesterol , triglyceride , and hdl - cholesterol were measured using enzymatic techniques .
fasting plasma glucose was measured and glycosylated hemoglobin ( hba1c ) percentage was determined by ion - exchange method .
insulin level was determined by using kits of dia metra ( made in italy ) , insulin resistance was calculated by homeostasis model of assessment .
insulin resistance ( homa - ir ) was calculated using the following us formula : ( fasting glucose ( mg / dl)fasting insulin ( 405/u / ml ) .
all measurements were done under standard conditions by one technician and with the same device .
the micro - vascular complications included recording of retinopathy ( ophthalmology visit ) , nephropathy , ( 24 h urine analysis ) and neuropathy ( peripheral , autonomic ) , monofilament and the diapason therapeutic examination .
sexual dysfunctions were measured in women using a standard questionnaire . the female sexual function index ( fsfi ) is a known instrument that assesses sexual function in women with six domains : desire , arousal , lubrication , orgasm , satisfaction and pain during sexual intercourse . in women ,
we used available data from shahid beheshti hospital where all patients received routine clinical examination that included recording of diabetes duration , body mass index ( bmi ) , fasting blood sugar , insulin resistance ( based on homa - ir ) , metabolic syndrome criteria , blood pressure , cholesterol and triglyceride levels and diabetes - related complications .
serum total cholesterol , triglyceride , and hdl - cholesterol were measured using enzymatic techniques .
fasting plasma glucose was measured and glycosylated hemoglobin ( hba1c ) percentage was determined by ion - exchange method .
insulin level was determined by using kits of dia metra ( made in italy ) , insulin resistance was calculated by homeostasis model of assessment . insulin resistance ( homa - ir )
was calculated using the following us formula : ( fasting glucose ( mg / dl)fasting insulin ( 405/u / ml ) .
all measurements were done under standard conditions by one technician and with the same device .
the micro - vascular complications included recording of retinopathy ( ophthalmology visit ) , nephropathy , ( 24 h urine analysis ) and neuropathy ( peripheral , autonomic ) , monofilament and the diapason therapeutic examination .
data was analyzed using software package used for statistical analysis ( spss ) version 16.0 in a descriptive and analytical fashion . to investigate the associations of demographic and clinical characteristics of the patients with sd , the test was used . in all statistical tests , a p < 0.05 was considered being significant .
diabetic women were assured regarding the confidentiality of the information and the questionnaire was completed by a person with the same gender .
in this study , 110 diabetic women with age range of 30 - 55 years and the mean age of 48.18 6.04 years were evaluated .
the minimum and maximum duration of diabetes was 5 and 25 years respectively with the mean duration of 8.56 4.38 years .
the mean age of women was 48.22 6.61 in sexual dysfunction group and 48.14 5.37 years in healthy patients and it was not statistically significant in both groups ( p = 0.94 ) .
the mean diabetes duration , fbs , hba1c and insulin resistance was not statistically significant in both groups [ table 1 ] . also , abdominal circumference and bmi were evaluated and were not statistically significant in both groups [ table 1 ] .
other characteristics of both groups including blood lipid and serum insulin level are shown in [ table 1 ] .
characteristics and laboratory findings of both groups of the studied patients , 33 ones ( 33% ) had microalbuminuria and 11 ones ( 10% ) had macroalbuminuria and sexual dysfunction was detected in 23 ( 39% ) of microalbuminuria and 10 ( 16.9% ) of macroalbuminuria patients .
a significance association was found between albuminuria and sexual dysfunction ( p = 0.001 ) [ table 2 ] . also , retinopathy and sexual dysfunction were statistically significant ( p = 0.007 ) [ table 2 ] but diabetic neuropathy and sexual dysfunction were not associated ( p = 0.79 ) .
history of hypertension , coronary artery disease and exercise were not associated with sexual dysfunction [ table 2 ] .
female sexual dysfunction has several causes including interpersonal , contextual ( social ) , psychological and biological factors .
in fact , every factor contributing normal female sexual function can be a potential cause of sexual dysfunction and there is a strong association with psychological health issues such as anxiety , depression , low self - esteem , body image perception disorders , sexual performance anxiety , fear of rejection , traumatic sexual experience in the past , and abuse history .
biological risk factors include several medical conditions ( urogenital , neurological and endocrine disorders , pelvic floor disorders , menopause , pregnancy , obesity ) in addition to pharmacological and other therapies ( antineoplastic agents , antipsychotic and antidepressant medications , antihypertensive agents , major surgical operations , radiation therapies ) . finally , sociocultural risk factors of sexual dysfunction include limited social relations , financial difficulties , employment status , religious beliefs , educational background and lack of exercise .
generally , sexual dysfunction appears to be quite complex in diabetic women , involving neurological , vascular , hormonal , and psychosocial aspects .
it is difficult to identify the specific risk factors , including those related to diabetes .
it has been hypothesized that hyperglycemia results in poor vaginal lubrication and dyspareunia , by reducing hydration of mucus membrane in the vaginal tissue . also , hyperglycemia may potentially lead to dyspareunia because of its association with an increased incidence of genitourinary infections .
diabetes causes vascular and nerve dysfunction which can cause structural and functional changes in female genitalia and may impair sexual response .
moreover , a reduced vibration sense in the genitalia has been observed in diabetic women , but its relationship with sexual problems should be clarified .
vascular changes or diabetic damage may change the local blood flow and inhibition of the clitoris engorgement and vaginal lubrication during arousal , resulting in dyspareunia or decreased arousal during sexual activity .
disorders of arousal , orgasm and sexual pain are the main consequences of the diabetic neuropathy . hormonal imbalance
accompanying diabetes has been hypothesized to play a potential role in the pathogenesis of the female sexual dysfunction .
additionally , several other endocrinopathies that may accompany diabetes , such as thyroid and hypothalamic - pituitary disorders , and polycystic ovarian syndrome , can contribute to sexual problems in these women .
it seems that depression is the most established risk factor for sexual dysfunction in diabetic women .
the prevalence of sexual dysfunction in diabetic women is estimated to be 20 - 80% . in our study , it was 53.6% .
one factor contributing this prevalence is age and some studies consider it as an effective factor in this disorder . on the contrary
, some studies have not confirmed that association . in our study , no association was found between age and risk of sexual dysfunction ( p = 0.94 ) .
another finding of this research was considering the impact of diabetes duration on sexual dysfunction .
some studies confirmed it while others did not confirm it . in our study , the association between duration of diabetes and sexual dysfunction was observed ( p = 0.71 ) .
some studies have shown that glycemic control has no effect on the incidence of sexual dysfunction in women with diabetes .
, we found no significant association between blood sugar and sexual dysfunction ( p = 0.29 ) .
of course , our measure of glycemic control was the mean hba1c within last year but due to chronic nature of sexual dysfunction , prolonged evaluation of glycemic control , may provide more reliable results .
some believe that overweight is another factor , but we found no association between bmi and abdominal circumference with sexual dysfunction ( p = 0.86 and p = 0.053 respectively ) and it was similar to fatemi and taghavi 's study . of course , as it can be seen , the very close relationship between the abdominal circumference and sexual dysfunction is close to the significant amount .
one of the discussed issues is the effect of other chronic diabetic complications like retinopathy and nephropathy on sexual dysfunction and considering them as risk factors for this complication .
some studies considered some diabetic complications as risk factor for sexual dysfunction and some others consider them independent to the other diabetic complications .
this relationship has not been observed in some studies , but some studies have shown this association . in our study , no significant association was found between sexual dysfunction and diabetic neuropathy ( p = 0.79 ) .
the criterion for the diagnosis of diabetic neuropathy was therapist 's examination and the patients have not undergone nerve conduction velocity ( ncv ) .
one of the significant correlations was detected between sexual dysfunction and diabetic nephropathy ( p = 0.001 ) and it appears that there is a significant relationship between these two disorders , it is notable that due to this statistically significant association , albuminuria can be considered as an important risk factor in the development of sexual dysfunction .
it was evaluated in a study on men and albuminuria was considered as an important risk factor in the development of sexual dysfunction ( 14 ) and it seems that further prospective studies are needed for this evaluation .
another finding of this study was the association between sexual dysfunction and retinopathy ( p = 0.007 ) , which was confirmed in abu ali et al 's study .
also no significant association was found between hypertension and sexual dysfunction in our study , ( p = 0.13 ) which was the same as abu ali et al 's study .
sexual dysfunction is a common complication in patients with diabetes that should be considered by clinicians .
the very meaningful relationship of this disorder with diabetic nephropathy reminds that in patients with diabetes , sexual dysfunction should be considered . | background : sexual dysfunction is a common complication of diabetes that adversely affects their quality of life . its prevalence is known to be higher in diabetic men with and it is estimated to affect 20 - 85% of patients but the problem is probably less common in diabetic women .
this study investigated the prevalence of sexual dysfunction and its risk factors among women with diabetes.materials and methods : this descriptive - analytic study was performed during may 2012 to feb 2013 at diabetes clinic of shahid beheshti hospital of qom and the female sexual function index ( fsfi ) was used for evaluation of sexual dysfunction.conclusion:in this study , 59 ( 53.6% ) women had sexual dysfunction .
the mean age of patients with sexual dysfunction and healthy people was 48.22 6.61 and 48.14 5.37 years respectively and it was not statistically different in both groups ( p = 0.94 ) .
also , there was no significant difference between two groups in average duration of diabetes , fasting blood sugar ( fbs ) , glycosylated hemoglobin ( hba1c ) level , insulin resistance , abdominal circumference and body mass index bmi . although the history of hypertension , coronary artery disease and exercise levels were not significantly associated with sexual dysfunction , but there was a significant association between albuminuria and sexual dysfunction ( p = 0.001 ) .
retinopathy and sexual dysfunction had statistically significant relationship ( p = 0.007 ) while no association was found between diabetic neuropathy and sexual dysfunction ( p = 0.79).results : sexual dysfunction is a common complication in diabetic patients which accompanies with some complications of diabetes and should be considered especially in patients with nephropathy or retinopathy . |
sct is a carrier state of sickle cell disease with one copy of normal beta globulin gene and one copy of sickle variant gene - producing heterozygous hbas ( 1 ) .
sct is estimated to have prevalence of 300 million worldwide , mostly concentrated in africa , middle east , and mediterranean region ( 2 ) . in the united states ,
although sct is considered a benign condition with no increased mortality in individuals ( 4 ) , it has been associated with several complications ( 2 ) .
a number of abnormalities have been reported in sct , including renal medullary carcinoma , papillary necrosis , thromboembolic events , and hyposthenuria ( 5 , 6 ) .
renal complications of sickle cell trait renal papillary necrosis is frequently associated with sickle cell disease , but it is not uncommon in sct as well .
it has been usually associated with diabetes , analgesic nephropathy , chronic pyelonephritis , and renal tuberculosis .
renal papillary necrosis is usually precipitated by some factors such as intense exercise , dehydration , and taking nsaids . in our patient ,
to date no comparative data exist with regard to the distribution and frequency of occurrence of renal papillary necrosis among persons with sct , hemoglobin sc , and thalassemias ( 5 , 9 ) ; however , there have been case reports on hemoglobin sc ( 9 , 10 ) but none to date on thalassemia unless associated with a sickling hemoglobin ( s ) ( 11 ) .
papillary necrosis results from obstruction of the vasa recta producing ischemia and micro infarction of medullary region of the kidneys ( 12 ) .
polymerization of the rbcs in the vasa recta of the renal medulla is the main trigger for renal papillary necrosis .
numerous factors are involved in initiating the sickling of rbcs including hypoxia , hypertonicity , and acidosis ( 13 ) .
once sickling starts it leads to increased blood viscosity and decreased blood flow to medulla , which ultimately results in micro infarction of vasa recta causing papillary necrosis .
the extent of polymerization of rbcs depends upon the concentration of hbs ( 11 ) .
the clinical presentation varies from painless gross hematuria to acute condition with pain , fever , and obstructive urinary symptoms ( 14 , 15 ) .
it usually manifests in the age group between 3040 years and there seems to be no sex predominance ( 16 ) .
any patient with sct , who presents with hematuria , needs a thorough clinical investigation especially in young patients because renal medullary carcinoma can also presents in a similar fashion .
renal medullary carcinoma is a rare , aggressive fatal tumor , which presents in young patients with sct ( 6 ) . because renal medullary cancer is very rare
, there is no epidemiological data available , but according to one report in 2003 , there are 55 cases of renal medullary cancer that were reported in literature ( 6 ) .
renal papillary necrosis should be in the differential diagnosis in any sct patient who comes in with hematuria .
intravenous pyelogram has been traditionally used for establishing the diagnosis but it is now largely replaced with ct urography because of its high sensitivity and specificity ( 17 , 18 ) .
ct urography is also superior to other imaging studies because of its ability to diagnose papillary necrosis in early stages ( 19 ) but more studies are required for comparison between ivp , ultrasound , and ct urography . in all reported cases , there was no biopsy performed and renal angiogram is not required for diagnosis . in sct ,
the most common findings on urinalysis of patients with sickle cell disease is proteinuria as this proteinuria occurs in 2030% of patients with sickle cell disease .
additionally , there is decreased concentrating ability with urine osmolarity of 400450 mosm / kg under water deprivation ( 12 ) .
although there are no large studies done to standardize the treatment of papillary necrosis secondary to sct , the underlying pathophysiology guides the treatment modalities . as mentioned earlier , papillary necrosis starts with sickling of rbcs , which is itself affected by factors like acidosis , hypertonicity , and hypoxia .
treatment options that have been utilized are listed in the table 2 , ( 7 , 15 , 16 ) .
initial management involves conservative measures , as most of the cases of hematuria are self - limiting .
patient should be started on hypotonic fluids along with diuretics to increase the urine flow and reduce the tonicity in the medullary interstitial area .
sodium bicarbonate is also used to alkalinize the urine , which reduces the acidosis , but whether it prevents further necrosis is uncertain . in some reports desmopressin (
increases clotting by dose - dependent increase in plasma factor viii , and vwf ) and epsilon - amino caproic acid ( inhibits fibrinolysis by inhibiting plasmin activity ) can be used to treat hematuria in resistant patients ( 7 , 2022 ) .
if all the aforementioned measures fail , then invasive measures can be taken which involve renal artery embolization and nephrectomy .
it should be considered in the differential diagnosis of gross hematuria , especially in sickle cell patients , because if it is not caught earlier it can lead to devastating complications .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | we describe a case of renal papillary necrosis in a middle - aged female with sickle cell trait who presented with gross hematuria .
we wish to highlight this case for several reasons .
sickle cell trait is often viewed as a benign condition despite the fact that it is associated with significant morbidity such as renal papillary necrosis and renal medullary carcinoma .
appropriate evaluation needs to be undertaken to promptly diagnose renal papillary necrosis and differentiate it from renal medullary carcinoma as this can result in deadly consequences for patients .
ct urography has emerged as a diagnostic study to evaluate hematuria in such patients .
we review the pathophysiology , diagnosis , and management of renal papillary necrosis in patients with sickle cell trait . |
a 14-year - old siamese neutered male cat was evaluated for anorexia and a left periorbital mass .
skull radiographic findings showed a well - defined lesion resembling new compact bone formation without destruction .
the tumor was removed intact with a normal tissue margin of at least 1 cm . there were no postsurgical complications .
periorbital tumors are infrequently diagnosed in companion animals and most are malignant . in this case , the diagnosis was orbital osteoma .
the most commonly affected bone for osteoma in cats is the mandibular bone ; few cases have been identified in orbital bones .
orbital surgery has the potential to be challenging owing to complex anatomy , difficult exposure and the tendency to bleed .
surgical complications are common . in this case , although the disease was advanced , subtotal orbitectomy was successfully performed .
periorbital tumors are infrequently diagnosed in companion animals and most are malignant . diagnosis is based on clinical signs , radiographic features and histological diagnosis .
orbital surgery has the potential to be challenging owing to complex anatomy , difficult exposure and a tendency to bleed .
the aim of this study is to report a successful subtotal orbitectomy in a cat with orbital osteoma .
a 14-year - old siamese neutered male cat was evaluated for anorexia and a left periorbital mass .
the left eye could not be visualized . a complete blood count ( cbc ) and
the results from the cbc did not reveal any abnormality , and the only abnormal value on the biochemical panel was a mildly elevated creatinine level ( 2.5 mg / dl ; reference interval 0.91.8 mg / dl ) .
skull radiographic findings showed a well - defined lesion resembling new compact bone formation without destruction ( figure 2 ) . in thoracic
the cat was premedicated intramuscularly with acepromazine ( 0.03 mg / kg ) and methadone ( 0.2 mg / kg ) by subcutaneous injection 15 mins prior to induction .
anesthesia was induced with propofol ( 4 mg / kg ) by intravenous injection and maintained with isoflurane .
methadone was given intramuscularly q6h for 24 h. the cat was positioned in ventral recumbency . the tumor was removed intact and surrounded by at least a 1 cm margin of normal tissue .
it was a circular , well - defined mineral mass measuring 3.2 cm in diameter at the rostral aspect of the left orbit .
an osteotomy of frontal bone combined with an osteotomy of maxilla and zygomatic bone were performed using an osteotome and mallet .
bony margins of the orbit were removed en bloc with the eye and structures within the orbit .
the surgical defect was closed by two single - pedicle advancement flaps ( dorsum of head and cheek ) and apposition of subcutaneous tissues and skin .
microscopically , the lump was ovoid and well - delineated from surrounding dermal connective tissue .
the lesion was composed of numerous trabeculae consisting of both woven and lamellar bone , osteocytes of normal appearance being present in lacunae within the osseous matrix .
amoxicillin ( amoxicilin ; medley ) 15 mg / kg orally q12h for 10 days ; dipyrone 25 mg / kg orally q12h for 3 days ; and tramadol ( dorless v ; agener ) 2 mg / kg orally q12h for 7 days , were prescribed , and an elizabethan collar was recommended .
six percent of all primary bone tumors commonly affect the skull of dogs and cats .
few cases of osteoma have been described in cats and they are rarely identified in the orbit . the most commonly affected bone is the mandible .
diagnosis for orbital osteoma is based on clinical signs ( usually not painful on palpation ) , radiographic features ( well - circumscribed , dense bony projections ) and histological diagnosis ( similar to reactive bone ) .
it is a compact bone formation without destruction and with continuous growth at a slow rate .
orbitectomy is a painful surgical procedure.effective pain management generally involves a balanced or multimodal strategy using several classes of pain - modifying medications .
a fentanyl patch was the better option for this case ; however , it was not available to be used in this cat .
fentanyl patches provide long - lasting analgesia and can be placed prior to surgery in order to be effective postoperatively .
non - steroidal anti - inflammatory drugs ( nsaids ) are a mainstay for the management of chronic pain , as well as for perioperative use .
nsaids are avoided in cats with renal dysfunction because an important side effect associated with nsaids is nephrotoxicity .
the cat had a mildly elevated creatinine level , so nsaids were avoided . because animals are non - verbal and can not self - report the presence of pain , appropriate pain management is required and includes several classes of pain - modifying medications .
if the tumor remains static and asymptomatic it may be possible to consider no treatment but monitor it closely .
orbital surgery has the potential to be challenging owing to the complex anatomy , difficult exposure and a tendency to bleed . in this case , the osteoma was removed without difficulty and complication .
surgical complications like blindness in the contralateral eye , neurological signs , cardiopulmonary arrest , facial swelling , hemorrhage and dehiscence may develop after orbitectomy . in this case , no postsurgical signs were observed .
the osteoma was completely removed without complications and the cat s quality of life was subjectively better than before surgery .
early intervention is recommended for cats with osteoma , as surgical excision of the mass may be curative .
subtotal orbitectomy was a successful treatment in this case , even though the mass was advanced ( 3.2 cm in diameter ) . | case summarya 14-year - old siamese neutered male cat was evaluated for anorexia and a left periorbital mass .
skull radiographic findings showed a well - defined lesion resembling new compact bone formation without destruction .
a subtotal orbitectomy was indicated .
the tumor was removed intact with a normal tissue margin of at least 1 cm . there were no postsurgical complications .
histopathologic examination revealed an osteoma .
the cat returned to normal appetite and activity 15 days after surgery .
six months after surgery , there were no gross signs of recurrence.relevance and novel informationperiorbital tumors are infrequently diagnosed in companion animals and most are malignant . in this case , the diagnosis was orbital osteoma .
the most commonly affected bone for osteoma in cats is the mandibular bone ; few cases have been identified in orbital bones .
orbital surgery has the potential to be challenging owing to complex anatomy , difficult exposure and the tendency to bleed .
surgical complications are common . in this case , although the disease was advanced , subtotal orbitectomy was successfully performed . |
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