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rheumatoid arthritis ( ra ) is an inflammatory polyarthritis with a variable course and prognosis . the prevalence is approximately 1% and affects females more commonly than males ( 3:1 ) . upwards of 80% of patients report foot problems during the course of the disease . acquired pes planovalgus ( ppv ) with associated involvement of the tibialis posterior ( tp ) tendon is common . pes planovalgus is a complex multi - planar deformity characterised by valgus alignment of the rearfoot with corresponding midfoot collapse and forefoot abduction . the cause is unknown but inflammatory damage in the peri - talar complex , tibialis posterior ( tp ) tendinopathy and altered foot mechanics frequently co - exist in published cohorts [ 36 ] . currently only one study has investigated electromyographic ( emg ) activity of tp in ra . here the magnitude of activity of tp in stance was increased in patients with valgus rearfoot alignment compared to those without . these findings have been replicated in non - ra cohorts with tp tendon dysfunction and flat foot . despite preliminary evidence regarding differences in emg activity of tp with varying foot posture ; there is minimal evidence in terms of how these features respond to intervention or how they behave over time in a chronic inflammatory joint disease such as ra . the consequence of tp dysfunction can also be indirectly assessed through structural and functional changes , for example by 3d kinematics of the peri - talar complex via multi - segmented foot models . we aim to develop and understand the reliability characteristics of an extended multi - segmented foot model which includes a midfoot segment combined with fine - wire emg to tp and surface emg to selected lower limb muscles applied in patients with ra and ppv . in particular , the motivation to investigate between - day reliability is a prerequisite for accurately quantifying progressive change in muscle and joint function associated with chronic inflammatory disease , and as a technique to assess , monitor and evaluate function and outcome for non - pharmacological , pharmacological and surgical interventions . patients were recruited from outpatient clinics at glasgow royal infirmary and gartnavel general hospital , glasgow uk . patients were eligible for inclusion if they had a confirmed diagnosis of ra based on the 1987acr criteria and passively correctable ppv . patients were excluded if they had a history of a significant neurological or musculoskeletal condition affecting the lower limb in terms of gait or muscle function or if they were taking anti - coagulant medication . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . participant age , gender and disease duration were recorded . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , inc . , rockville , md , usa ) was used to build the multi segmented foot model . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . the midfoot segment could not be recorded in shoe so the model was reduced to four segments in the shod condition . five trials were recorded and the ensemble average created for each condition on two occasions 1 week apart . the limb selected for analysis was based on clinical severity of foot posture . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . a shoe ( flextop diabetic shoe , reed medical ltd . , uk ) was modified for the shod trials . windows were cut to allow the tracking of markers for the rear and forefoot segments and to record vertical height of the navicular . the shoe was selected for the soft leather upper and flexible vamp in order to accommodate forefoot deformity . velcro straps were stitched to the superior aspect of the heel counter in order to maintain stability during walking ( supplementary file ) . in order to avoid undertaking an invasive procedure on subjects potentially at risk of infection , intramuscular emg tibialis anterior , soleus , peroneus longus and medial gastrocnemius emg signals were recorded using trigno wireless surface electrodes ( delsys inc . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . intramuscular emg of tp was undertaken using bi - polar stainless steel nylon coated fine wire electrodes ( 0.051 mm diameter ) inserted via 50 mm length 15 gauge needles ( motion lab systems inc . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . electrodes were inserted under ultrasound guidance ( esaote mylab 70 ) using a 134 mhz linear array transducer via the posterior - medial approach at 50% of the distance between the medial malleolus and the medial joint line of the knee . placement of the electrode was verified by checking the signal while applying manual resistance in the direction of dorsiflexion and eversion while participants plantarflexed and inverted ; the signal was also checked when participants flexed their toes to ensure the electrode was not placed inadvertently in the flexor digitorum longus muscle . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . all emg data ( including mvics ) were subject to a root mean squared ( rms ) moving average of 25 ms in order to create a wave envelope . emg data was normalised to maximum voluntary isometric contractions ( mvic ) ; three mvics were recorded for each muscle following completion of walking trials . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . and murley et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . kinematic data were subject to a fourth order butterworth low pass filter with a cut off of 6 hz . demographic and group characteristics were summarised with the mean , and either standard deviation ( sd ) or range . biomechanical and emg data were normalised to 100% of stance and analysed using the absolute coefficient of multiple correlation ( cmc ) . within subject variation was assessed using two way mixed model intra class correlation coefficients ( icc ) with 95% confidence intervals ( ci ) . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . the standard error of measurement ( sem ) with 95% ci was calculated to express the level of error in original and clinically meaningful units . patients were recruited from outpatient clinics at glasgow royal infirmary and gartnavel general hospital , glasgow uk . patients were eligible for inclusion if they had a confirmed diagnosis of ra based on the 1987acr criteria and passively correctable ppv . patients were excluded if they had a history of a significant neurological or musculoskeletal condition affecting the lower limb in terms of gait or muscle function or if they were taking anti - coagulant medication . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . participant age , gender and disease duration were recorded . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , rockville , md , usa ) was used to build the multi segmented foot model . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . the midfoot segment could not be recorded in shoe so the model was reduced to four segments in the shod condition . five trials were recorded and the ensemble average created for each condition on two occasions 1 week apart . the limb selected for analysis was based on clinical severity of foot posture . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . a shoe ( flextop diabetic shoe , reed medical ltd . , uk ) was modified for the shod trials . windows were cut to allow the tracking of markers for the rear and forefoot segments and to record vertical height of the navicular . the shoe was selected for the soft leather upper and flexible vamp in order to accommodate forefoot deformity . velcro straps were stitched to the superior aspect of the heel counter in order to maintain stability during walking ( supplementary file ) . in order to avoid undertaking an invasive procedure on subjects potentially at risk of infection , intramuscular emg was restricted to the tp muscle because of its inaccessibility via surface electrodes . tibialis anterior , soleus , peroneus longus and medial gastrocnemius emg signals were recorded using trigno wireless surface electrodes ( delsys inc . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . intramuscular emg of tp was undertaken using bi - polar stainless steel nylon coated fine wire electrodes ( 0.051 mm diameter ) inserted via 50 mm length 15 gauge needles ( motion lab systems inc . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . electrodes were inserted under ultrasound guidance ( esaote mylab 70 ) using a 134 mhz linear array transducer via the posterior - medial approach at 50% of the distance between the medial malleolus and the medial joint line of the knee . placement of the electrode was verified by checking the signal while applying manual resistance in the direction of dorsiflexion and eversion while participants plantarflexed and inverted ; the signal was also checked when participants flexed their toes to ensure the electrode was not placed inadvertently in the flexor digitorum longus muscle . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . all emg data ( including mvics ) were subject to a root mean squared ( rms ) moving average of 25 ms in order to create a wave envelope . emg data was normalised to maximum voluntary isometric contractions ( mvic ) ; three mvics were recorded for each muscle following completion of walking trials . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . and murley et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . kinematic data were subject to a fourth order butterworth low pass filter with a cut off of 6 hz . statistical analyses were performed using spss 17.0 ( spss inc . , chicago , il , usa ) . demographic and group characteristics were summarised with the mean , and either standard deviation ( sd ) or range . biomechanical and emg data were normalised to 100% of stance and analysed using the absolute coefficient of multiple correlation ( cmc ) . within subject variation was assessed using two way mixed model intra class correlation coefficients ( icc ) with 95% confidence intervals ( ci ) . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . the standard error of measurement ( sem ) with 95% ci was calculated to express the level of error in original and clinically meaningful units . five patients with ra ( 3f:2 m ) and ppv with a mean ( sd ) age of 53 ( 9 ) years and mean disease duration of 7 ( 5 ) years were recruited . numerical differences were recorded between time points in global and foot related measures of disability ( table 1 ) . analyses were restricted to the stance phase of gait as preliminary analyses indicated that cmcs can be falsely inflated due to muscle inactivity during the swing phase . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . the lowest mean value was 0.68 for peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent ; values for peroneus longus and tp again fell below 0.75 . discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . a number of icc values fell into the poor category ( < 0.40 ) and negative values were found in tp and peroneus longus . a negative icc value is suggestive of greater levels of intra - session variation than inter session variation ; where negative values occurred these are represented by 0 . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . the sem varied across both the temporal and the amplitude characteristics in all muscle groups ; however there was a trend towards reduced levels of error in the temporal domain . the sem for magnitude of tp contraction ranged from 8% to 27% of mvic across both phases of stance . for timing of peak tp activity the sem ranged from 1% to 4% of the stance phase . despite the smaller levels of error in the temporal domain , in almost all cases the 95% ci crossed zero across both domains . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . five patients with ra ( 3f:2 m ) and ppv with a mean ( sd ) age of 53 ( 9 ) years and mean disease duration of 7 ( 5 ) years were recruited . numerical differences were recorded between time points in global and foot related measures of disability ( table 1 ) . analyses were restricted to the stance phase of gait as preliminary analyses indicated that cmcs can be falsely inflated due to muscle inactivity during the swing phase . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . the lowest mean value was 0.68 for peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . a number of icc values fell into the poor category ( < 0.40 ) and negative values were found in tp and peroneus longus . a negative icc value is suggestive of greater levels of intra - session variation than inter session variation ; where negative values occurred these are represented by 0 . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . the sem varied across both the temporal and the amplitude characteristics in all muscle groups ; however there was a trend towards reduced levels of error in the temporal domain . the sem for magnitude of tp contraction ranged from 8% to 27% of mvic across both phases of stance . for timing of peak tp activity the sem ranged from 1% to 4% of the stance phase . despite the smaller levels of error in the temporal domain , in almost all cases the 95% ci crossed zero across both domains . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane . there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . this study set out to assess the within- and between - day reliability of emg activity patterns for tp and other selected lower limb muscles as well as 3d multi - segmented foot kinematics in an ra cohort with ppv . the emg and kinematic results demonstrated superior within - day reliability to between - day reliability in this small cohort . however , in this patient cohort discrete variables for muscle timing and amplitude demonstrated unacceptably poor levels of reliability between - days . tibialis posterior is the most powerful supinator of the rearfoot , it also adducts the forefoot and contributes to ankle plantarflexion ; in addition it acts as a dynamic stabiliser of the medial longitudinal arch . increased emg signal amplitude and activity during the stance phase of gait has been consistently demonstrated in ra and non - ra ppv cohorts [ 79 ] this suggests the muscle is working to counteract the motion tendencies of eversion and dorsiflexion in the ankle / subtalar complex and forefoot abduction which characterise ppv . we recorded tp emg patterns in ra patients with ppv and demonstrated that this could be reliably measured using a fine - wire , indwelling electrode approach in a single session both barefoot and in - shoe . however , while the overall emg muscle pattern was similar 1 week later , discrete timing and magnitude variables were not reliable . altered muscle activity has also been described for other lower leg muscles including gastrocnemius , peroneus longus , peroneus brevis and flexors digitorum and hallucis longus [ 79 ] . this evidence suggests these muscles must be studied if the complexity of the primary impairment and compensatory mechanisms are to be fully understood . however , we observed the same trend for poor reliability of other lower limb muscles as for tp : this confirms and extends the observations of murley et al . to include an inflammatory joint disease . factors related to emg technique , the effects of ra on emg measurement , and the effects of ra directly on muscle structure and function should be considered when interpreting these results . potential sources of error with emg are well described and include subcutaneous soft - tissue volume , cross talk , motion artefact , intramuscular bleeding , accuracy of electrode placement , skin preparation and retraction of electrodes during dynamic tasks . in an attempt to minimise their effect however , there are specific issues related to ra which may compound these potential sources of error . moreover , muscle activation capacity can be reduced depending on disease activity state and joint pain and effusion . structural muscle changes such as decreased volume may have influenced the precision of surface and fine - wire electrode placement and signal detection . effusions or pain in the ankle , subtalar or midtarsal joints where the lower leg muscle tendons cross to insert into the foot may have influenced muscle activation patterns . changes in ra disease activity state with up or down regulation of inflammatory cytokines may have altered muscle physiology and emg signal detection . we did not measure muscle cross - sectional areas or volume so can not account for the influence of structural changes . however , we attempted to limit error by guiding the electrode to the tp muscle belly using ultrasound ; a technique we have shown to be highly accurate . ra is a disease with a variable course , characterised by flares and remissions with associated fluctuations in patient symptoms . we found evidence that self - reported disease state and pain changed between testing periods so this may have changed muscle physiology , muscle activity , and subsequently emg signal detection . muscle - specific force and muscle activation patterns have been found to be normal in ra patients with stable disease , even with significant muscle loss . in our cohort disease activity may have fluctuated but a firmer conclusion can only be reached on this if objective measures were employed . finally we detected difference in joint tenderness between time points and this may have adversely influenced reproducibility of muscle activity capacity during mvic tests , as well as muscle function during gait . ra patients seldom walk barefoot and this may explain the superior reliability in the shod condition . combining emg muscle activation patterns with multi - segmented foot kinematics presents an opportunity to understand the relationship between muscle function and joint motion . the reliability of the kinematic data in this study was consistent with that reported for other inflammatory joint conditions . we confirmed previous observations where larger foot segments have greater reliability both within- and between - days in the sagittal plane in comparison with smaller segments with smaller ranges of motion , particularly in the frontal and transverse planes . inflammatory joint diseases present challenges for identification of surface landmarks , in particular when joints are swollen , tender and deformed . nevertheless , the model presented here showed excellent reliability for discrete variables both barefoot and shod . firstly the complexity and time burden of the protocol restricted the sample size , particularly in a heterogeneous disease such as ra . future work should attempt to recruit a larger sample in order to draw more meaningful conclusions . secondly , normalising the emg signals to mvics has inherent limitations in a patient population with fluctuating symptoms , particularly joint pain and effusions . thirdly , while the use of the intensity and duration of muscle activation are appealing variables to study for clinical and research applications , other forms of analysis may show greater reliability . in conclusion , patterns of muscle activation and kinematic motion appeared more consistent than discrete variables and absolute measures of error . the findings demonstrate that , in this cohort of ra patients using these emg variables , the use of discrete emg variables between time points is not supported either barefoot or shod . within session reliability is greater than between session and this should be considered when planning intervention or longitudinal studies ; a single session evaluation or alternative analysis may be more appropriate due to the lack of measurement precision . kinematic reliability has been established in the presence of pathology in this cohort but should be interpreted within the error limits .
biomarker expression data are usually characterized by a small number of sample vectors of high dimension , which makes it very difficult to be treated with many kinds of single classifiers . up to now , a possible approach to reduce the dimensionality consists in applying straightforward statistical feature selection operation . ensemble methods based on re - sampling technique are addressed to solve problems arising from small samples and biological variability of the data ( 1 ) . ensembles consisting of a certain number of single classifiers outperform a single classifier greatly in terms of classification accuracy 1 . , the generation performance of an ensemble classifier mainly depends on its base learners classification accuracy and relativity . therefore , how to choose a group of single classifiers to compose a high - powered ensemble is a hot topic in this field . in the present study , we propose a constructive ensemble algorithm based on double - layer hierarchical fusion strategy , that is , the outputs of all base learners are combined together by a specific single classifier . upon the fusion strategy , the support vector machine recursive feature elimination ( svm - rfe ) method ( 6 ) is adopted to produce a rank of base learners by their contributions to the upper - layer decision machine . the first several base learners in this rank are selected and the ensemble size ( or the number of selected base learners ) is given by 10-fold cross - validation . ensembles constructed by our method not only have relatively simpler structures but also have better performance than those constructed by bagging . this algorithm has been tested on two ovarian cancer datasets previously obtained by proteomic mass spectrometry ( ms ) . the training set of biomarker expression is represented as gtr = { ( xi ; yi)\| i = 1 , 2 , , l } , where xi r is a d - dimensional vector , in which every dimension corresponds to the expression data from a specific biomarker , and yi { 1 , + 1 } represents the class label , that is , which class the sample belongs to . each replicate training set is used to train a certain svm ; the base learners used to constitute the lower layer of ensemble will be selected from these k svms . a new svm is trained to fuse the output of these k svms , and these ( k+1 ) svms form a hierarchical structure . using fk to be the decision function of the k svm in the lower layer and f to be the decision function of the svm in the upper layer , the final decision value of a given sample xi is determined as:(1)d(xi)=f(f1(xi),f1(xi), ,fk(xi ) ) the experimental results from previous studies 3 . , 4 . indicate that most gain of ensemble s performance comes from an optimal combination of several specific base learners . in our method , the optimal combination is realized by the svm in the upper layer , where the focus is how to select these specific base learners . according to equation ( 1 ) , f1(xi ) , f1(xi ) , , fk(xi ) are regarded as the k features or inputs of the upper - layer decision machine . therefore , to choose a group of base learners for constructing ensembles means to choose a group of most discriminative features for the upper - layer decision machine . ( 6 ) has shown sound performance on bio - feature selection in bioinformatics 7 . , 8 . and key variable identification in chemical industrial process ( 9 ) . in the present study , this method is adopted to rank the lower - layer decision machines according to their importance to the upper - layer decision machine . in brief , rfe is a circulation procedure for eliminating features by a criterion . it consists of three steps : ( 1 ) train the classifier ; ( 2 ) compute the ranking criterion ; ( 3 ) remove the features with the smallest ranking scores . the ranking criterion is relative to the realization of classifier , that is to say , rfe is a wrapper algorithm . when the linear kernel svm f(x)=w , x+b=i=1laiyi < xi , x>+b is used as a classifier in rfe , the contribution of each feature to the discriminative function , j(i ) , lies on its weight value , namely j(i ) = ( wi ) , where w=(wi)=i=1laiyixi , and the decision coefficients a = ( ai ) and b are obtained by training of svm ( 10 ) . svm is retrained after each elimination operation , because a feature of medium - low importance may be promoted by removing a correlated feature . finally , 10-fold cross - validation is used to determine the number of classifiers in ensemble . the linear kernel svm was used throughout our experiments . to avoid the noise resulted from the oversize number of features , the fisher criterion score f(j)=(j+j)2/((j+)2+(j)2 ) was used to preselect 100 biomarkers , where j+ and j denote the mean value of the j biomarker for classes 1 and 2 , respectively , while j+ and j denote the standard deviation of the j biomarker for classes 1 and 2 , respectively . in bootstrapping , the size of each resampled dataset was set as 50% of the original training set . the initialized number of base learners in the lower layer of ensemble was set as 100 . two ovarian cancer datasets from the surface - enhanced laser desorption ionization time - of - flight ( seldi - tof ) experiments by ms ( 11 ) were analyzed in this study . dataset 1 was prepared manually , consisting of 200 samples that were separated into a training set and a test set . each set has 100 samples , including 50 ovarian cancer samples and 50 control ( normal ) samples . dataset 2 was prepared with a robotic instrument , consisting of 162 ovarian cancer samples and 91 control samples . its training set was made up of 60 cancer samples and 40 control samples drawn out statistically , and the test set consisted of the remaining samples . each feature corresponded to the relative intensity of a certain kind of ionized proteomic molecule with specific m / z value . to prove the effectiveness of classifier selection , majority voting and double - layer fusion strategy were used as the decision method respectively on the classifier ranking results to test the ensemble accuracy . the classification accuracy analysis on dataset 1 is shown in figure 1 . the ensemble size indicated by 10-fold cross - validation is 7 , and these seven base learners are selected by svm - rfe . the positive predictive values are 100% on the training set and 96% on the test set , with the ensemble constructed by these seven classifiers . the ensemble constructed by all base learners can not achieve a lower error rate whether it is fused by majority voting or double - layer fusion strategy . the similar result achieved on dataset 2 is shown in figure 2 . the ensemble size indicated by 10-fold the positive predictive values are 100% on both the training set and the test set , with the ensemble constructed by these three classifiers . it is also concluded that the ensemble constructed by all base learners can not achieve a lower error rate whether it is fused by majority voting or double - layer fusion strategy . by all the above analysis , it can be seen that majority voting and double - layer fusion strategy can depress the error rate of cancer diagnosis resulted from single svm . in most cases , ensembles constructed by double - layer fusion strategy with classifier selection we have presented a constructive algorithm for training cooperative support vector machine ensembles ( csvmes ) . csvme combines ensemble architecture design with cooperative training for individual svms in ensembles and puts emphasis on both base learner s accuracy and collaboration among individual svms . this kind of svme has been tested on two ovarian cancer datasets previously obtained by proteomic ms . by combining several optimally selected individual svms , the proposed method achieves better performance and simpler structure than the svme of all base svms . in addition , csvme performs better than a single svm trained on the whole training set .
microglia are the resident innate immune cells of the central nervous system ( cns ) and play an important role in immune surveillance and in the pathogenesis and progression of a number of cns disorders . microglia are constantly mobile , spending time scanning the extracellular space of the cns . in response to brain injury or immunological stimuli , these cells become activated and undergo dramatic morphological and functional changes , which are highly dependent on the context of their activation . activated microglia phagocytose debris and peptides , present antigens , and produce a number of soluble factors . these factors may be inflammatory , regulatory , or cytotoxic in nature and include reactive oxygen species ( ros ) , nitric oxide ( no ) , proinflammatory and anti - inflammatory cytokines , prostaglandins , and growth factors [ 4 , 5 ] . microglia can be both neuroprotective or neurotoxic when activated , depending on the factors they produce and the quantity and context in which they are released , with prolonged or excessive activation of these cells being associated with neuroinflammation and the progression of a number of cns disorders . the mechanisms behind enhanced microglial activation in these disorders and the features determining the balance between neuroprotection and neurotoxicity are not fully understood . the p2x7 receptor is a trimeric ligand - gated cation channel belonging to the p2x family of purinergic receptors . p2x7 is predominately expressed on mononuclear leukocytes including macrophages and microglia and plays a role in inflammation and immunity . in particular activation of p2x7 by extracellular adenosine-5-triphosphate ( atp ) , or the most potent p2x7 agonist , 2(3)-o-(4-benzoylbenzoyl ) atp ( bzatp ) , causes the passage of small cations including ca , na , and k across the plasma membrane , as well as organic cations , such as the fluorescent dyes ethidium and yo - pro-1 . compared to other p2x receptors , p2x7 requires relatively high atp concentrations for activation , with a half maximal effective concentration ( ec50 ) of 100300 m . activation of p2x7 leads to a number of cell - specific downstream signalling events , including the formation of ros and reactive nitrogen species , and either cell proliferation or death [ 11 , 12 ] . using molecular , immunochemical , and pharmacological techniques , we demonstrate in the current study that the murine microglial eoc13 cell line expresses functional p2x7 . activation of p2x7 by atp in this cell line induces the uptake of organic cations , ros formation , and cell death . rpmi-1640 and dmem / f12 media , glutamax , normal horse serum ( nhs ) , 0.05% trypsin , yo - pro-1 , 2,7-dichlorodihydrofluorescein diacetate ( h2dcfda ) , and 2,7-difluorofluorescein diacetate ( daf - fm da ) were from invitrogen ( grand island , ny ) . fetal bovine serum ( fbs ) ( heat - inactivated before use ) was from bovogen biologicals ( east keilor , australia ) . atp , bzatp , ethidium bromide , dimethyl sulfoxide ( dmso ) , glycerol gelatin , and the p2x7 antagonist brilliant blue g ( bbg ) were from sigma - aldrich ( st . louis , mo ) . the p2x7 antagonists a438079 , az10606120 , and az11645373 were from tocris bioscience ( ellisville , mo ) . protease inhibitor cocktail tablets ( complete , mini , edta - free ) and annexin - v - fluorescein were from roche diagnostics ( penzberg , germany ) . the viability dye 7-aminoactinomycin d ( 7aad ) and the ros scavenger n - acetyl - l - cysteine ( nac ) were from enzo life sciences ( plymouth meeting , pa ) . the broad - spectrum ros inhibitor diphenyleneiodonium ( dpi ) was from cayman chemical ( ann arbor , mi ) . phenyl - methyl - sulfonyl - fluoride ( pmsf ) , n - dodecyl -d - maltoside , and ethylene glycol tetraacetic acid ( egta ) were from amresco ( solon , oh ) . cells preincubated with h2o soluble compounds ( bbg , a438079 , and az10606120 ) were compared to cells preincubated in the absence of each compound . cells preincubated with dmso soluble compounds ( az11645373 and dpi ) were compared to cells preincubated with dmso alone . solutions containing 40 mm nac were prepared in nacl medium ( 140 mm nacl , 5 mm naoh , 5 mm kcl , 10 mm hepes , and 5 mm glucose , ph 7.4 ) and adjusted to ph 7.4 ; cells were then preincubated in nacl medium with or without nac . rabbit anti - mouse p2x7 ( extracellular epitope ) polyclonal antibody ( ab ) and rabbit anti - rat p2x7 ( c - termini epitope ) ab ( and corresponding blocking peptide ) were from alomone labs ( jerusalem , israel ) . peroxidase - conjugated goat anti - rabbit igg ab was from rockland immunochemicals ( gilbertsville , pa ) . cy3-conjugated donkey anti - rabbit igg ab was from jackson immunoresearch ( west grove , pa ) . rat anti - mouse p2x7 monoclonal antibody ( mab ) ( clone hano43 ) was from enzo life sciences . rat igg2b isotype control mab and allophycocyanin- ( apc- ) conjugated donkey anti - rat igg ab were from ebioscience ( san diego , ca ) . the murine macrophage j774 cell line , the murine microglial eoc13 cell line , and the murine lymphoblast ladmac cell line , all originally obtained from the american type culture collection ( manassas , va ) , were kindly provided by jasmyn dunn ( university of queensland , brisbane , australia ) ( j774 ) and iain campbell ( university of sydney , sydney , australia ) ( eoc13 and ladmac ) . j774 cells were maintained in rpmi-1640 medium containing 10% fbs and 2 mm glutamax ( complete rpmi medium ) . eoc13 cells were maintained in dmem / f12 supplemented with 10% fbs , 2 mm glutamax , and 20% ladmac conditioned medium ( complete dmem medium ) . cell lines were maintained at 37c and 95% air/5% co2 and passaged every 3 - 4 days . quarterly mycoplasma testing was carried out using the mycoalert mycoplasma detection kit ( lonza , rockland , me ) , as per the manufacturer 's instructions . for experiments , cells were washed in nacl medium ( 300 g for 5 min ) , resuspended in nacl medium , and equilibrated at 37c for 5 min ( 1 10 cells/1 ml / tube ) . cells were then incubated with 25 m ethidium ( or 1 m yo - pro-1 where indicated ) in the absence or presence of the p2x7 agonists atp or bzatp ( as indicated ) for 5 min . in some experiments , atp - induced cation uptake was assessed with cells suspended in kcl medium ( 150 mm kcl , 5 mm glucose , and 10 mm hepes , ph 7.4 ) or in nacl medium containing 1 mm cacl2 or 100 m egta . in other experiments , cells were preincubated in the absence or presence of p2x7 antagonists or the ros scavenger nac ( as indicated ) for 15 and 30 min , respectively , prior to cation and atp addition . incubations with nucleotides were stopped by the addition of an equal volume of ice - cold nacl medium containing 20 mm mgcl2 ( mgcl2 medium ) followed by centrifugation ( 300 g for 5 min ) . cells were washed once with nacl medium and events collected using a lsr ii flow cytometer ( bd biosciences , san diego , ca ) ( excitation 488 nm , emission collected with 575/26 and 515/20 band - pass filters for ethidium and yo - pro-1 , resp . ) . the mean fluorescence intensity ( mfi ) of relative cation uptake total rna isolation from cells was performed using the rneasy mini kit ( qiagen , hilden , germany ) as per the manufacturer 's instructions . pcr amplification was performed as described previously using superscript iii one - step rt - pcr system platinum taq dna polymerase ( invitrogen ) with 500 ng of rna , and p2x7 forward ( 5-atatccacttccccggccac-3 ) and reverse ( 5-tcggcagtgatgggaccag-3 ) primers for 42 cycles ( 94c , 1 min ; 68c , 1 min ; 72c , 1 min ) . pcr products were separated on a 2% agarose gel in tris - acetate edta buffer and visualised with ethidium bromide staining . images of gels were collected using a gel logic 212 pro imaging system ( carestream health , rochester , ny ) . cells were washed three times with phosphate - buffered saline ( pbs ) ( 300 g for 5 min ) and lysed ( 1 10 cells / ml ) over 60 min in ice - cold lysis buffer ( 50 mm bistris , 750 mm 6-aminohexanoic acid , 1% n - dodecyl -d - maltoside , 1 mm pmsf , and protease inhibitor cocktail , ph 7.0 ) . cells were sheared by passing ten times through a 21 g needle and stored at 20c until needed . cells were then thawed and cleared ( 16,000 g at 4c for 10 min ) . supernatants ( 25 g protein / lane ) were separated under reducing conditions ( 5% -mercaptoethanol ) using a discontinuous sds - page system with a 4% stacking gel and 10% separating gel . proteins were then transferred to nitrocellulose membranes ( bio - rad , hercules , ca ) and blocked at 4c overnight with tris - buffered saline ( 250 mm nacl and 50 mm tris , ph 7.5 ) containing 0.2% tween-20 and 5% milk powder . the following day , nitrocellulose membranes were incubated at room temperature for 2 h with anti - mouse p2x7 ab ( 1 : 500 ) in tris - buffered saline containing 0.2% tween-20 and 5% milk powder . membranes were washed three times over 30 min with tris - buffered saline containing 0.2% tween-20 and then incubated at room temperature for 1 h with peroxidise - conjugated anti - igg ab ( 1 : 1000 ) in tris - buffered saline containing 0.2% tween-20 and 5% milk powder . membranes were washed as above and visualised using chemiluminescent substrate and amersham hyperfilm ecl ( ge healthcare , little chalfont , buckinghamshire , uk ) . images of films were collected using a gs-800 calibrated densitometer ( bio - rad ) . cells in nacl medium containing 10% nhs and 0.02% nan3 ( 1 10 cells/200 l / tube ) were incubated with anti - p2x7 or rat igg2b isotype control mab ( 5 g / ml ) at room temperature for 30 min . cells were then washed twice with nacl medium ( 300 g for 5 min ) and incubated with apc - conjugated anti - rat igg ab ( 1.3 g / ml ) and 7aad ( to exclude dead cells ) for 30 min protected from light . events were then collected using a lsr ii flow cytometer ( excitation 633 nm , emission collected with 660/20 band - pass filter for apc ; excitation 488 nm , emission collected with 695/40 band - pass filter for 7aad ) . relative cell - surface p2x7 was determined using flowjo software and is expressed as the difference in the mfi of specific mab labelling and isotype control labelling . eoc13 or j774 cells in their respective complete culture medium were plated into 24-well plates with 13 mm glass coverslips ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight to allow time to adhere . the following day , cells were fixed with 4% paraformaldehyde in pbs at room temperature for 15 min and then washed three times with pbs over 10 min . cells were incubated with permeabilisation solution ( pbs containing 0.1% dmso , 2% nhs , and 0.1% triton x-100 ) at room temperature for 10 min and washed three times with pbs . cells were then blocked with 20% nhs in pbs at room temperature for 20 min . cells were incubated at 4c overnight with anti - rat p2x7 ab ( 5 g / ml ; preincubated for 1 h in the absence or presence of blocking peptide as per the manufacturer 's instruction ) in pbs containing 1% bsa , 0.2% nhs , and 0.05% nan3 . cells were then washed as above and incubated at room temperature for 1 h with cy3-conjugated anti - rabbit igg ab ( 15 g / ml ) in pbs containing 0.2% nhs . cells were washed as above and then the coverslips mounted onto slides with 50% ( v / v ) glycerol gelatin in pbs . cells were visualised using a dm ibre inverted microscope and tcs sp confocal imaging system ( leica , mannheim , germany ) ( excitation 488 nm , emission collected at 560600 nm ) . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , cells were then incubated with nacl medium containing 10 m h2dcfda ( 0.5 ml / well ) at 37c , 95% air/5% co2 , protected from light for 30 min . the medium was removed , and cells were further incubated in nacl medium ( containing 1 mm cacl2 ) in the absence or presence of 2 mm atp at 37c , 95% air/5% co2 for 15 min . incubations were stopped by the addition of an equal volume of ice - cold mgcl2 medium . cells were harvested using 0.05% trypsin ( 5 min , 37c ) and were washed once with nacl medium . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected at 515/20 nm ) and the mfi of relative dichlorofluorescein ( dcf ) determined using flowjo software . in some experiments , atp - induced ros formation was assessed in kcl medium , in nacl medium in the absence of 1 mm cacl2 or presence of 100 m egta , or in complete dmem medium in the absence or presence of 10 m az10606120 ( 15 min preincubation , prior to atp addition ) . as free ca lowers the concentration of atp , cells incubated in the absence of 1 mm ca were incubated with 1.4 mm atp to provide equimolar atp concentrations ( 575 m ) , as calculated using the bound and determined program . in other experiments , cells were preincubated in the absence or presence of az10606120 , nac , or dpi ( as indicated ) for 15 , 30 , and 30 min , respectively , prior to atp addition . cells prior to harvesting were also visualised by differential interference contrast ( dic ) imaging using an eclipse te2000 inverted microscope ( nikon , tokyo , japan ) to examine cell morphology , and dic images were captured using image - pro ams ( version 6.1 ) ( media cybernetics , rockville , md ) . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight . cells were then incubated with nacl medium containing 10 m daf - fm da ( 0.5 ml / well ) at 37c , 95% air/5% co2 , protected from light for 30 min . cells were then preincubated with nacl medium in the absence or presence of 10 m az10606120 at 37c , 95% air/5% co2 for 15 min . following this , cells were further incubated in the absence or presence of 1.4 mm atp for 15 min . incubations were stopped by the addition of an equal volume of ice - cold mgcl2 medium . cells were harvested using 0.05% trypsin ( 5 min , 37c ) and were washed once with nacl medium . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected at 515/20 nm ) and the mfi of relative benzotriazole derivative determined using flowjo software . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight . cells were then incubated with filter sterile atp ( as indicated ) at 37c , 5% co2 for 24 h. in some experiments , cells were preincubated in the absence or presence of 10 m az10606120 or 40 mm nac for 15 or 30 min , respectively , prior to atp addition . in other experiments , cells were preincubated in the absence or presence of 40 mm nac for 90 min , with 2 mm atp added in the final 1560 min , and then the medium replaced and cells incubated at 37c , 95% air/5% co2 for a further 24 h. following the 24 h incubations , cells were harvested from wells using 0.05% trypsin ( 5 min , 37c ) and washed once with annexin - v binding medium ( nacl medium containing 5 mm cacl2 ) . cells were then incubated with annexin - v binding medium containing annexin - v - fluorescein and 7aad at room temperature protected from light for 15 min . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected with 515/20 and 695/40 band - pass filters for annexin - v - fluorescein and 7aad , resp . ) and the mfi of annexin - v - fluorescein and 7aad determined using flowjo software . quadrant markers were used to determine the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells . in some experiments , cells prior to harvesting were visualised by dic imaging to examine cell morphology , and dic images captured as outlined in section 2.8 . differences between multiple treatments were compared by anova paired with tukey 's hsd posttest using prism 5 for windows ( version 5.04 ) ( graphpad software , san diego , ca ) , with differences considered significant for p < 0.05 . concentration response and inhibition curves were fitted using prism 5 and assuming a variable slope , with normalised and nonnormalised response curves , respectively , selected to obtain the best fit . estimates of ec50 values and half maximal inhibitory concentrations ( ic50 ) were obtained from individual fits of these plots . the murine macrophage j774 cell line is well known to express functional p2x7 . moreover , our group has demonstrated the presence of functional p2x7 in various cell types using a fixed - time fluorescent cation uptake assay ( e.g. , [ 14 , 18 ] ) . therefore , this technique was used to confirm the presence of p2x7 in j774 cells and to validate the use of this cell line as a positive control . incubation of j774 cells with the p2x7 agonist atp and the most potent p2x7 agonist bzatp induced significant ethidium uptake into these cells compared to cells incubated in the absence of nucleotide ( figure 1(a ) ) . in addition , incubation of j774 cells with atp induced significant yo - pro-1 uptake compared to cells incubated in the absence of atp ( figure 1(b ) ) . however , atp - induced yo - pro-1 uptake was significantly lower than atp - induced ethidium uptake ( figure 1(b ) ) . a number of highly specific p2x7 antagonists , including a438079 , az10606120 , and az11645373 , have recently become available . in addition , bbg is commonly used as a p2x7 antagonist in vitro and in vivo ( e.g. , [ 22 , 23 ] ) . therefore , to determine the optimum concentrations of these antagonists required to inhibit murine p2x7 , j774 cells were preincubated in the absence or presence of varying concentrations of bbg , a438079 , az10606120 , and az11645373 and the atp - induced ethidium uptake assessed . each antagonist impaired 1 mm atp - induced ethidium uptake in a concentration - dependent manner , with ic50 values of 1.8 0.2 , 7.9 0.4 , 1.0 0.1 , and 1.5 0.1 m , respectively ( figure 1(c ) ) . az10606120 and a438079 completely inhibited ethidium uptake at respective concentrations of 10 and 100 m . in contrast , bbg and az11645373 were partial antagonists at the atp concentration used ( 1 mm ) . to determine whether eoc13 microglial cells express p2x7 , a series of experiments using j774 cells as a positive control were performed . firstly , rna was isolated from eoc13 and j774 cells and amplified by rt - pcr using primers for p2x7 . similar to j774 cells , eoc13 cells were found to express p2x7 mrna , as evident from the 230 base pair band corresponding to the size of the predicted product ( figure 2(a ) ) . the presence of total p2x7 protein was determined by probing separated whole lysates of both cell lines with an anti - p2x7 ab . immunoblotting revealed one major protein band of 75 kda for both eoc13 and j774 cells ( figure 2(b ) ) , corresponding to the predicted size of glycosylated p2x7 . moreover , both cell lines were incubated with an anti - p2x7 mab and the presence of cell - surface p2x7 determined by flow cytometry . immunolabelling demonstrated cell - surface p2x7 on both eoc13 and j774 cells , with mfis of 13 2 and 14 4 , respectively ( n = 3 ) ( figure 2(c ) ) . finally , both cell lines were stained with an anti - p2x7 ab and analysed by confocal microscopy . this similarly demonstrated the presence of cell - surface p2x7 , as well as intracellular p2x7 , with bright staining observed on all cells ( figure 2(d ) ) . preincubation of the anti - p2x7 ab with blocking peptide completely abrogated the detection of p2x7 in both cell lines ( data not shown ) . together , these results indicate that p2x7 is expressed in eoc13 cells . to determine whether p2x7 was functional in eoc13 cells , the fixed - time ethidium uptake assay was performed . both atp and bzatp were found to induce significant ethidium uptake into eoc13 cells compared to cells incubated in the absence of nucleotide ( figure 3(a ) ) . atp induced ethidium uptake in a concentration - dependent manner , with maximal uptake obtained at an atp concentration of 1 mm and with an ec50 of 130 30 m ( figure 3(b ) ) . subsequent characterisations of p2x7 in eoc13 microglia were performed using this maximal concentration of atp ( 1 mm ) . to determine if the observed atp - induced ethidium uptake into eoc13 cells was mediated by p2x7 , cells were preincubated in the absence or presence of p2x7 antagonists at inhibitory concentrations optimal for 1 mm atp - induced ethidium uptake in j774 cells , as demonstrated above ( figure 1(c ) ) . preincubation of eoc13 cells with 30 m bbg , 100 m a438079 , 10 m az10606120 , and 30 m az11645373 resulted in significant impairment of atp - induced ethidium uptake by 75 2 , 90 1 , 100 0 , and 99 1% , respectively ( figure 3(c ) ) . none of the p2x7 antagonists except az11645373 significantly altered the basal ethidium uptake into eoc13 cells . again with the exception of az11645373 , which reduced the amount of gated viable cells by ~30% , cell viability ( as assessed by forward and side scatter ) was similar between treatments ( data not shown ) . to determine if p2x7 activation could induce the uptake of a second organic cation into eoc13 cells , cells were preincubated in the absence or presence of az10606120 , and atp - induced yo - pro-1 uptake examined . similar to ethidium uptake , 1 mm atp induced significant yo - pro-1 uptake into eoc13 cells compared to cells incubated in the absence of atp ( figure 3(d ) ) . moreover , preincubation of cells with 10 m az10606120 resulted in complete inhibition of atp - induced yo - pro-1 uptake ( figure 3(d ) ) . incubation with az10606120 did not significantly alter the basal yo - pro-1 uptake ( figure 3(d ) ) . furthermore , cell viability ( as assessed by forward and side scatter ) was similar between treatments ( data not shown ) . p2x7 activation has been reported to induce ros formation in a number of cell types , including primary microglia [ 24 , 25 ] . thus , atp - induced ros formation in the eoc13 cell line was investigated using the ros sensitive probe dcf . cells loaded with h2dcfda ( which is converted to dcf inside cells ) were incubated in the absence or presence of atp , and the subsequent ros formation analysed by flow cytometry . as extracellular ca has been reported to be important for p2x7-induced ros formation in a number of cell types [ 24 , 26 , 27 ] , assays were initially conducted in the presence of 1 mm ca . however , due to the inhibitory action of ca on p2x7 , assays were initially conducted with 2 mm atp . incubation with 2 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( mfi of ros formation 16.3 0.6 and 5.18 0.06 , resp . furthermore , preincubation of cells with 10 m az10606120 resulted in complete inhibition of atp - induced ros formation ( figure 4(a ) ) , indicating that this process is mediated by p2x7 activation . as for cation uptake ( figure 3 ) , az10606120 did not significantly alter the basal ros formation ( figure 4(a ) ) or cell viability ( as assessed by forward and side scatter ) ( data not shown ) . p2x7 is a ligand - gated cation channel ; therefore the possible roles for cation fluxes in p2x7-induced ros formation were next investigated . p2x7-induced ros formation has been reported to be partially dependent on ca influx in human promyelocytes and rat submandibular gland cells . thus , to determine if ca influx is required for p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation in the absence and presence of ca was investigated . for this comparison , equivalent amounts of atp ( 575 m ) were used by adding 1.4 or 2 mm atp to nacl medium nominally free of ca or containing 1 mm ca , respectively . atp induced significant ros formation in both the absence and presence of 1 mm ca compared to similarly treated cells in the absence of atp ( figure 4(b ) ) . cells incubated in the absence of ca had significantly higher atp - induced ros formation compared to those incubated in the presence of ca . in contrast , atp - induced ethidium uptake ( p2x7 function ) was similar in cells incubated in the absence or presence of ca ( figure 4(b ) ) , indicating that the differences in atp - induced ros formation were not due to altered p2x7 function . thus , to further exclude a role for ca in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was investigated in the absence and presence of the ca chelator egta . incubation with 1.4 mm atp induced significant but similar amounts of ros formation in both the absence and presence of 100 m egta compared to similarly treated cells in the absence of atp ( figure 4(c ) ) . again , atp - induced ethidium uptake was similar in cells incubated in the absence or presence of egta ( figure 4(c ) ) . finally , the role of k in p2x7-induced ros formation in eoc13 cells was investigated . both ros and k efflux have been reported to be involved in interleukin-1 ( il-1 ) release from monocytes , although whether these downstream processes are linked is yet to be established . thus , to determine if k efflux is involved in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was compared with cells in nacl medium and kcl medium , which prevents the loss of intracellular k. incubation with 1.4 mm atp induced significant ros formation in both nacl and kcl media , with similar levels of ros formation in both media ( figure 4(d ) ) . likewise , atp - induced ethidium uptake was similar in nacl and kcl media ( figure 4(d ) ) , indicating that the inability of high extracellular k to impair atp - induced ros formation was not due to altered p2x7 function . to confirm that p2x7 activation induced ros formation in eoc13 microglia , dcf - loaded cells in nacl medium were preincubated in the absence or presence of the ros scavenger nac , before incubation in the absence or presence of atp . as above ( figure 4 ) , 1.4 mm atp induced significant ros formation ( figure 5(a ) ) . preincubation with 40 mm nac inhibited atp - induced ros formation by 73.7 0.3% ( figure 5(a ) ) . basal ros formation ( figure 5(a ) ) and cell viability ( as assessed by forward and side scatter ) ( data not shown ) were similar between treatments . preincubation of cells with 40 mm nac inhibited atp - induced ethidium uptake by 30 2% ( figure 5(b ) ) . thus , the inhibitory effect of nac on p2x7-induced ros formation may be partially attributed to inhibition of p2x7 itself . however , incubation with nac and atp , but not either compound alone , reduced the amount of gated viable cells by ~40% in the ethidium uptake assay ( as assessed by forward and side scatter ) ( data not shown ) . this suggests that the inhibitory action of nac on atp - induced ethidium uptake may be a result of cytotoxicity in this assay . to confirm that nac did not induce morphological changes or cause cytotoxicity under the conditions used for the ros assay , dic images of adherent cells were acquired following incubation in the absence or presence of atp . cells incubated in the absence or presence of nac ( without atp ) displayed discrete cell bodies with long , spindled shaped processes ( figure 5(c ) ) , as previously observed . cells incubated in the absence or presence of nac ( with atp ) also displayed discrete cell bodies , but with retracted and branched processes ( figure 5(c ) ) , typical of atp causing membrane changes . therefore , in the ros assay , eoc13 cell morphology was not altered by nac when compared to the corresponding treatment . dcf - loaded cells were also preincubated in the absence or presence of the broad - spectrum ros inhibitor dpi and the atp - induced ros formation investigated . however , a 30 min preincubation with dpi at various concentrations ( 540 m ) led to high amounts of cell death ( data not shown ) , and thus this compound was not examined further . to further verify that p2x7 activation induces the formation of reactive species in eoc13 cells , atp - induced no formation was investigated using the no sensitive probe daf - fm da . cells loaded with daf - fm da ( which reacts with no to form a fluorescent benzotriazole ) were preincubated in the absence or presence of az10606120 , followed by incubation in the absence or presence of atp , and the subsequent no formation analysed by flow cytometry . incubation with 1.4 mm atp induced significant no formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 6 ) . furthermore , preincubation of cells with 10 m az10606120 inhibited atp - induced no formation by 82 11% ( figure 6 ) , indicating that this process is mediated by p2x7 activation . again , az10606120 did not significantly alter the basal no formation ( figure 6 ) or cell viability ( as assessed by forward and side scatter ) ( data not shown ) . p2x7 activation results in the death of various cell types [ 11 , 12 ] . to determine whether atp induces the death of eoc13 microglia , cells in complete dmem medium were incubated in the absence or presence of atp for 24 h , and then the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells examined by flow cytometry ( figure 7(a ) ) . cell death is expressed as the total of dying ( annexin - v/7aad ) and dead ( annexin - v/7aad and annexin - v/7aad ) cells . incubation with either 2 or 3 mm atp but not 1 mm atp resulted in significantly higher percentages of total cell death compared to cells incubated in the absence of atp ( figure 7(a ) ) . next , to determine if the atp - induced eoc13 death was mediated by p2x7 activation , cells were preincubated in the absence or presence of az10606120 and then incubated in the absence or presence of atp for 24 h. as above ( figure 7(a ) ) , 2 mm atp induced significant cell death , with higher percentages of total cell death compared to cells incubated in the absence of atp ( figure 7(b ) ) . preincubation with 10 m az10606120 completely inhibited atp - induced cell death ( figure 7(b ) ) , indicating that this process is mediated by p2x7 activation . p2x7-induced death of murine raw264.7 macrophages is mediated by ros formation . therefore , a role for ros in p2x7-induced death of eoc13 microglia was investigated . to confirm that p2x7 induced ros formation under conditions used to induce cell death , dcf - loaded eoc13 cells in complete dmem medium were incubated in the absence or presence of atp , and then subsequent ros formation determined by flow cytometry . similar to atp - induced cell death ( figure 7(a ) ) , incubation with 2 or 3 but not 1 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 7(c ) ) . the requirement for higher atp concentrations to induce cell death ( figure 7(a ) ) or ros formation ( figure 7(c ) ) compared to pore formation ( figure 3(b ) ) is in line with the inhibitory action of divalent cations , which are present in the culture medium but not in the nacl medium used . to confirm that this ros formation was mediated by p2x7 , cells were preincubated with az10606120 and the amounts of atp - induced ros formation determined . again , atp induced significant ros formation compared to cells incubated in the absence of atp ( figure 7(d ) ) . preincubation with 10 m az10606120 completely inhibited this atp - induced ros formation ( figure 7(d ) ) . eoc13 cells were preincubated in the absence or presence of nac followed by atp for 24 h. however , 24 h incubation with 40 mm nac in the absence of atp led to significant amounts of eoc13 cell death ( data not shown ) . therefore , to reduce the total exposure to 40 mm nac , cells were incubated in the absence or presence of nac for 90 min , with atp added in the final 1560 min . the medium was then replaced with fresh complete dmem medium and the cells incubated for 24 h. incubation with 2 mm atp for 30 or 60 min but not 15 min resulted in significant cell death compared to cells incubated in the absence of atp ( figure 7(e ) ) . in contrast to the 24 h incubation with nac , 90 min incubation with 40 mm nac ( without atp ) did not induce significant cell death compared to cells incubated for the same length of time in the absence of both nac and atp ( figure 7(e ) ) . a 60 min preincubation with nac inhibited cell death induced by 30 min incubation with atp by 99 6% ( figure 7(e ) ) . in contrast , a 75 and 30 min preincubation with nac , followed by 15 and 60 min atp treatment , respectively , had no effect on the percentage of cell death compared to cells incubated for the same time length with atp in the absence of nac ( figure 7(e ) ) . to further confirm that nac did not induce morphological changes or cause cytotoxicity under the conditions used for the cell death assay , dic images of adherent cells were acquired following the 24 h incubation . as above ( figure 5(c ) ) , cells incubated in the absence or presence of nac ( without atp ) displayed discrete cell bodies with long , spindled shaped processes ( figure 7(f ) ) . in addition , cells incubated in the presence of nac and atp displayed a similar morphology to that of cells incubated in the absence of atp ( figure 7(f ) ) . in contrast , cells preincubated with atp alone displayed rounded and granular cell bodies with no or few processes ( figure 7(f ) ) , characteristic of cell death . furthermore , wells preincubated with atp alone had a high amount of nonadherent cells compared to the other treatments ( data not shown ) . thus , preincubation with nac prevented the morphological changes associated with atp incubation , but nac alone had no effect on cell morphology . the current study demonstrates for the first time that the murine microglial eoc13 cell line expresses functional p2x7 . firstly , the presence of p2x7 mrna and protein was established using rt - pcr and immunoblotting techniques . in addition , the presence of cell - surface p2x7 was demonstrated using immunofluorescence staining . moreover , p2x7 on eoc13 microglia was functional , as the p2x7 agonists atp and bzatp induced significant ethidium or yo - pro-1 uptake into these cells . in these experiments , atp induced ethidium uptake with an ec50 value which falls within the typical range for atp - induced cation fluxes mediated by recombinant murine p2x7 . furthermore , pretreatment of cells with p2x7 antagonists inhibited atp - induced organic cation uptake . lastly , atp could induce ros formation in and the death of eoc13 cells , and both of these events , which are often associated with p2x7 activation [ 1012 ] , could be inhibited by the p2x7 antagonist az10606120 . the presence of functional p2x7 on eoc13 microglia is consistent with the presence of this receptor on primary microglia and microglial cell lines ( including n9 , n13 , bv-2 , and ntw8 cells ) [ 3235 ] . ros formation has been reported in primary microglia [ 24 , 25 ] , and a role for this process in microglia has been highlighted by several studies . p2x7 activation induces the production of the ros , superoxide , in primary rat microglia , while this receptor is upregulated in a transgenic mouse model of alzheimer 's disease . although a direct link between p2x7 , superoxide , and alzheimer 's disease was not established , the authors proposed a link between these molecules and this disease . this link is supported by subsequent observations by others , where fibrillar -amyloid peptide , which is associated with alzheimer 's disease , caused atp release and autocrine activation of p2x7 leading to ros formation in primary rat microglia . in addition , another group demonstrated that p2x7-induced superoxide release from primary rat microglia induced injury of rat cortical neurons . collectively , this data indicates that p2x7-induced ros formation from microglia may be involved in various neuroinflammatory and neurodegenerative disorders . this may be of particular importance in diseases where microglial p2x7 is reported to be upregulated such as in alzheimer 's disease , multiple sclerosis , and amyotrophic lateral sclerosis [ 25 , 37 ] . it should be noted , however , that dcf , as employed in the current study and as widely used by others to detect ros , can also propagate ros formation . nevertheless , our observation that p2x7 activation also induces the formation of no in eoc13 cells supports a role for this receptor in the formation of reactive species . the current study excluded an essential role for ca influx in p2x7-induced ros formation in eoc13 microglia . this finding is similar to other observations with other murine cell types , including submandibular glands and erythroid cells . in contrast , p2x7-induced ros formation in primary rat microglia and rat submandibular glands is dependent on an influx of ca . the reason for this difference between these two species remains unknown but may reflect differences in experimental protocols or differences in signalling molecules between mice and rats . the current study also excluded an essential role for k efflux in p2x7-induced ros formation in eoc13 microglia . both ros formation and k efflux are involved in il-1 release from monocytes , although whether these downstream processes are linked has not been established . thus , our results indicate that p2x7-induced ros formation does not require k efflux and that ros formation and k efflux may be independent events in il-1 release from myeloid cells . use of an annexin - v/7aad assay suggested that this process was mediated by apoptosis . however , in the absence of other markers of apoptosis and necrosis , this remains to be established , especially since p2x7 activation induces both apoptosis and necrosis in the microglial n13 cell line . nevertheless , our observations support previous studies in which p2x7 activation induced death in primary microglia and other microglial cell lines [ 41 , 42 ] . the physiological role of p2x7-induced microglia death is unclear . further obscuring this is the known role of p2x7 activation in inducing the proliferation of microglia . this paradoxical role of p2x7 is thought to be related to the relative atp concentration , with high concentrations promoting cell death and low concentrations promoting cell proliferation . in support of this , our study observed that atp only induced eoc13 cell death at 2 or 3 but not 1 mm atp . moreover , our data also showed that a transient incubation with atp of 3060 but not 15 min induced cell death in eoc13 microglia . this suggests that transient atp release and subsequent p2x7 activation may be sufficient to kill microglia in vivo . the current study examined a potential link between p2x7-induced ros formation and death in eoc13 microglia . a previous study demonstrated that the atp - induced death of murine raw264.7 macrophages was mediated by ros derived from nadph oxidase downstream of p2x7 activation . this contrasts with another study , which found that p2x7-induced ros formation , but not death , was attenuated in primary macrophages from nadph oxidase deficient mice . our data using the ros scavenger nac supports a role for ros formation in the p2x7-induced death of eoc13 microglia . the capacity of nac to prevent p2x7-induced eoc13 microglia death was dependent on the preincubation time with nac , as well as the total incubation time with atp , with only 4560 min preincubations with nac preventing cell death induced by transient 3045 min exposures to atp . in contrast , 24 h incubation with nac induced significant amounts of eoc13 microglia death , equivalent to that induced by atp alone . this cytotoxicity of nac may have occurred due to increased toxic metabolic byproducts such as reduced glutathione . alternatively , scavenging of ros by nac may indicate that low amounts of ros are important for eoc13 cell homeostasis . of note , the ros inhibitor dpi also induced the death of eoc13 microglia , albeit over a much faster time course . finally , it should be noted that nac inhibition of p2x7-induced death and ros formation in eoc13 microglia may have been partly due to direct inhibition of p2x7 . nac inhibited atp - induced pore formation by 30% compared to a 74 and 99% inhibition of atp - induced ros formation and cell death , respectively . this direct inhibition of p2x7 by nac was not due to an acidic ph , which is known to impair p2x7 function , as the nac - containing solutions were adjusted to ph 7.4 before each assay . thus , our results indicate that either cellular signalling involving ros may modulate p2x7 activation in eoc13 microglia or that nac may directly impair p2x7 at 40 mm . the concentration of nac used in these experiments ( 40 mm ) is 48-fold higher than that used in a number of similar studies ( e.g. , ) . the requirement for this high concentration of nac remains unknown but may reflect a reduced ability of nac to cross the plasma membrane or to be converted to glutathione in eoc13 cells . the presence of functional p2x7 on j774 macrophage cells was confirmed in the current study . p2x7 is present in this cell line , and activation of p2x7 leads to the release of mature il-1 , the formation of macrophage - derived multinucleated giant cells [ 5052 ] , and cell death . in this study , the potency of four p2x7 antagonists against 1 mm atp , the atp concentration most commonly used to study p2x7 , was determined . the ic50 values for bbg , a438079 , and az11645373 ( 1.8 , 7.9 , and 1.5 m , resp . ) were within one log range of those published for recombinant murine p2x7 [ 54 , 55 ] . in contrast , the ic50 value for az10606120 has not been reported for murine p2x7 , although this compound has been shown to specifically bind to and inhibit rat and human p2x7 . in the current study , this highly specific p2x7 antagonist also completely impaired atp - induced ethidium uptake , ros formation , and death of murine eoc13 cells . thus , az10606120 will be useful for future studies of murine p2x7 . in the cns , atp acts as a neurotransmitter and is released from neurons during synaptic transmission and from dying cells . under normal physiological conditions , extracellular atp concentrations in the cns are estimated to be in the nanomolar to micromolar range , depending on the balance between atp release and degradation , while intracellular microglial atp concentrations are in the millimolar range . after cns injury , however , extracellular atp concentrations increase and can reach as high as the millimolar range . furthermore , it is hypothesised that atp may act on microglial p2x7 at very close range where the concentration of atp may be quite high . activation of p2x7 on primary microglia and microglial cell lines leads to the release of proinflammatory il-1 and tumour necrosis factor- [ 33 , 58 ] and ros formation . although proinflammatory factors are important for immunity , prolonged or inappropriate release of such factors from chronically activated microglial can be highly toxic to neurons and can promote neuroinflammation and neurodegeneration . there are a number of diseases in the cns characterised by the presence of activated microglia , including alzheimer 's disease , prion infection , cerebral ischemia , multiple sclerosis , and amyotrophic lateral sclerosis . in such diseases , p2x7 has also been reported to be upregulated [ 25 , 37 , 59 , 60 ] . this raises questions of possible roles for p2x7 in mediating inappropriate microglial responses in cns disorders . activation of this receptor by atp resulted in organic cation uptake , ros formation , and death in these cells . moreover , the eoc13 cell line may be useful for investigating p2x7-mediated events in microglia and the role of this receptor in microglia - mediated inflammatory disorders .
autoimmune diseases arise from an overactive immune response of the body against tissues normally present in the body . organ - specific autoimmune disease can develop through a combination of hereditary and environmental factors that regulate adaptive immune responses to self antigens ( mills , 2011 ) . several studies have implicated microorganisms in the environmental etiology of autoimmune disorders based on observations such as , infection with chlamydia pneumoniae , human herpesvirus 6 , and epstein barr virus ( ebv ) are triggered or exacerbated by multiple sclerosis ( mills , 2011 ) . the regression of autoimmune thrombocytopenia is seen after the eradication of helicobacter pylori ( gasbarrini et al . , 1998 ) . in guillain barr syndrome ( gbs ) , amino acid similarities exist between the gangliosides of the nerve system and the lipopolysaccharides ( lpss ) of campylobacter jejuni , suggesting that sensitization by microbes may be based on autoimmunity from molecular mimicry between bacteria and the targeted system of the host ( yuki et al . , 2004 ; houliston et al . , the link has been attributed to either molecular mimicry between pathogen - derived antigens and self antigens or non - specific activation of innate immunity leading to a breakdown in immunological tolerance and the development of self antigen - specific t cell and antibody response ( mills , 2011 ) . a common recent theory of the cause of autoimmune diseases is that an infectious agent triggers a cycle of events , which leads to the upregulation of the host immune response to self antigens ( aoki , 1999 ; tlaskalov - hogenova et al . , 2004 ) . autoimmune pancreatitis ( aip ) is a putative autoimmune disease and is a chronically progressing inflammatory disease of the pancreas ( park et al . , 2009 ; okazaki et al . , 2011a ) the morphological characteristics of aip include diffuse or localized enlargement of the pancreas and irregular narrowing of the main pancreatic duct . histologically , the disease is also associated with progressive lymphoplasmacytic infiltration , predominantly localized to the ductal structures , and varying degrees of parenchymal and acinar destruction ( okazaki et al . , 2008 ) . there are two types of aip that differ in their clinical features , such as the gender ratio , mean age , and associated immune - related diseases . type 1 aip is associated with the histological finding of lymphoplasmacytic sclerosing pancreatitis ( lpsp ) . its serological hallmark is an elevation in the serum levels of the igg4 subclass of igg ( okazaki et al . , 2011a ) . type 1 aip appears to be the pancreatic manifestation of a systemic disease called igg4-associated systemic disease ( isd ) or igg4-related sclerosing disease , affecting not only the pancreas , but also other organs including the bile duct , retroperitoneum , kidney , lymph nodes , and salivary glands ( kamisaw and okamoto , 2006 ; okazaki et al . , type 2 aip is a form of idiopathic chronic pancreatitis ( icp ) , histologically associated with granulocyte - epithelial lesions ( shimosegawa and kanno , 2009 ) . genetic associations between susceptibility to the disease and the human leukocyte antigen ( hla ) drb1 * 0405-dqb1 * 0401 haplotype ( kawa et al . , 2002 ; muraki et al . , 2006 ; ota et al . , 2007 ) , fc receptor - like gene 3 ( fcrl3 ; kojima et al . , 2007 ) , and an outstanding finding in type 1 aip is hypergammaglobulinemia and the existence of a high serum concentration for igg4 , which has been documented in 90% of patients ( hamano et al . , this occurs in parallel to an abundant igg4 positive plasma cell infiltration in the pancreatic tissue ( aoki et al . , the fibroinflammatory process characterizing aip occurs at the pancreatic basement membranes where igg4/igg / complement immune complexes are deposited ( detlefsen et al . , 2010 ) . igg1 is believed to be an opsonizing antibody and activates the complement classical pathway , namely opsonization of a bacterium by activated complement and antibodies . combined opsonization by both complement and antibodies considerably enhances the uptake of the bacterium by the phagocyte ( wilson et al . , 2011 ) . aip is occasionally associated with elevated circulating immune complex levels , which are significantly linked to increased serum igg1 and complement activation via the classical pathway ( muraki et al . , 2006 ) . igg4 is unable to activate the classical pathway of complements , but binds igg1 , 2 , and 3 and forms an immune complex by fc although the role of igg4 in the immune response and autoimmunity has not yet been fully elucidated , it may be hypothesized that igg4 blocks the fc - mediated effector functions of igg1 and dampens the inflammatory response to an as yet unidentified primary trigger of the inflammatory process in aip ( ito et al . , 2010 ) . to which antigens the immunoglobulins in the deposits along the basal membranes of the pancreatic ducts and acini react remains unclear and definitive disease - specific antibodies have not been identified . however , several candidates for aip - specific autoantibodies have been reported , such as anti - lactoferrin ( lf ; okazaki et al . , 2000 ) , anti - carbonic anhydrase ( ca)-ii ( okazaki et al . , 2000 ) , anti - ca iv ( nishimori et al . , 2005 ) , anti - pancreatic secretory trypsin inhibitor ( psti ; asada et al . , 2006 ) , anti - amylase - alpha ( endo et al . , 2009 ) , anti - heat shock protein ( hsp ) 10 ( takizawa et al . , 2009 ) , and anti - plasminogen - binding protein ( pbp ) peptide autoantibodies ( frulloni et al . , 2009 ) . these putative aip - specific autoantibodies give us some hints toward considering the possibility that microorganisms are involved in the pathogenesis of aip . in order to survive in a host , a pathogenic microbe has to follow these steps ; ( i ) attach to the host cells for colonization , ( ii ) evade the host s innate and adaptive immune defense and persist in the host , ( iii ) obtain iron and other nutrients , ( iv ) disseminate or spread within a host , and ( v ) produce symptoms of disease in the host ( although production of symptoms is not necessary ; wilson et al . , 2011 ) . following the above mentioned processes , it might be valid to hypothesize that microbes are the pathogenic factors of aip , based upon the possible roles of aip - specific antibodies . lf : considering the fact that lf is distributed in the ductal cells of several exocrine organs , including the pancreas , salivary gland , biliary duct , lungs , and renal tubules ( okazaki et al . , 2011b ) , an anti - lf antibody might be induced as a consequence of the tissue damage caused by microbial infection and anti - lf might exert direct effects on these ductal cells of exocrine organs . most pathogenic bacteria require iron and thus there is a strong correlation between iron availability and virulence ( wilson et al . , 2011 ) . however bacteria have to cope with the fact that iron concentrations in nature are quite low . the concentration of free iron is particularly low in the host body due to the actions of host proteins , such as lactoferrin , transferring , ferritin , and heme , that bind most of the available iron . to survive in the body , bacteria must have some mechanism for acquiring this sequestered iron ( wilson et al . , 2011 ) . lf is a glycoprotein , and a member of a transferrin family , thus belonging to those proteins capable of binding and transferring fe ions ( wilson et al . , 2011 ) . lf binds fe with an affinity and stability much higher than that of transferrin in the serum ( valenti and antonini , 2005 ) . lf is secreted from the exocrine glands and in specific granules of neutrophils , which are the main source of lactoferrin in blood plasma after degranulation . neutrophils are at the front line of the innate immunity process ( wilson et al . , 2011 ) . lf binds iron released from transferrin , which prevents its further usage for bacterial proliferation . besides iron , lf is capable of binding a large amount of other compounds and substances such as lps , heparin , glycosaminoglycans , or other metal ions and certain dna and rna viruses . the concentration of lf in the blood increases during infection and inflammation ( adlerova et al . , 2008 ) . since many bacteria synthesize and secrete small iron - chelating molecules ( siderophores ) that can compete with lf for insoluble fe ions , the growth - inhibiting activity of the protein is thus reversed . iron sequestration by apo - lf , which is an iron - free form of lf , can effectively inhibit the growth of many bacterial species ( valenti and antonini , 2005 ) . lf combines with the bacterial surface through electrostatic and hydrophobic interactions , resulting in a perturbation of bacterial membranes . the molecular mechanisms of this bactericidal activity of lf , which is not related to iron withholding , appears to be similar for either gram - negative or gram - positive bacteria . taking these characteristics of lf into consideration , blocking the lf function by an anti - lf antibody might be beneficial for microbes to get the essential growth element iron to survive and prolong persistent infection in the body of aip patients . assessing whether or not the anti - lf antibody obtained from aip patients actually possesses the blocking function of the binding of fe3 ion to lf would be of interest . hsp : mammalian hsp10 and hsp60 , also known as chaperonins 10 and 60 , are mitochondrial proteins involved in protein folding . hsps exported to the plasma membrane or released from dying cells are believed to be a source of danger signals , informing the innate and adaptive immune systems of tissue damage induced by various insults including infection , toxins , and cellular stress ( johnson et al . it has not been clarified whether anti - hsp10 production is the cause or the consequence of aip progression , but hsp10 consistently inhibits lps - induced tlr4 signaling by interacting with hsp60 in the extracellular milieu and inhibits lps - induced secretion of the pro - inflammatory cytokines , tnf- , il-6 , and the pro - inflammatory chemokine regulated upon activation , normal t cell expressed and secreted ( rantes ) and anti - inflammatory cytokine il-10 ( johnson et al . , 2005 ) . from this point of view , it might be possible that the production of anti - hsp10 antibodies indicates the implication of bacteria in the pathogenesis of aip . molecular mimicry : homology between alpha - ca of h. pylori and human ca - ii has been also reported . moreover , the homologous segments contained the binding motif of drb1 * 0405 ( guarneri et al . , 2005 ) . notably , the possession of the hla - drb1 * 0405-dqb1 * 0401 genotype confers a risk for aip development ( ota et al . , 2007 ) . ( 2009 ) reported that 94% of aip patients , but only 5% of pancreatic cancer patients , exhibit igg antibodies to a pbp that is homologous to the human protein ubiquitin - protein ligase e3 component n - recognin 2 ( ubr2 ) , which is expressed in pancreatic acinar cells and is also homologous to the pbp of h. pylori . these data suggest that h. pylori infection may trigger aip in genetically predisposed subjects through autoimmune responses triggered by molecular mimicry . cas : immunization with ca - ii or lf has been reported to induce systemic lesions such as pancreatitis , sialadenitis , cholangitis , and interstitial nephritis ( nishimori et al . , 1995 ; ueno et al . , 1998 the distribution of ca - ii and iv are both in the ductal cells of several exocrine organs , including the pancreas , salivary gland , biliary duct , lungs and renal tubules ( okazaki et al . , 2011b ) . ( 2005 ) reported that serum antibodies to cas i and ii might be detected in patients with sjgren s syndrome and icp perhaps as a consequence of the cross - reactivity of an antibody against another unknown antigen that mimics cas i and ii . expression of ca iv is more restricted in tissues and cell types in the inflamed lesions that have been observed in patients with aip and related diseases . it has been suggested that ca iv is more likely to function as the target antigen ( nishimori et al . , 2005 ) . cas on the luminal surface of the ductal cells would eliminate excess acid from the ductal lumen , inhibiting spontaneous autoactivation of trypsin and other proteases ( nishimori et al . , 1999 ) , and therefore may contribute to attrition of bacterial killing by these enzymes in the pancreas . psti : psti , a 56-amino - acid peptide , is synthesized in pancreatic acinar cells , distributed in the ductal cells of several exocrine organs , including the pancreas , salivary gland , biliary duct , lungs , and renal tubules , and colocalizes with trypsinogen in zymogen granules . in addition to its protective role in acinar cells , psti inhibits activation of trypsinogen in the pancreatic duct ( asada et al . , 2006 ) . it might be beneficial for preventing degradation of bacterial pathogen(s ) . additionally , it would be of interest to study whether or not the anti - psti antibody inhibits the function of psti . besides putative aip - specific autoantibodies , other factors such as channel(s ) , transporters , and some of aip associated genes also give us hints to consider the possible involvement of bacteria in the pathogenesis of aip . aqp1 : aquaporin ( aqp ) water channels are intrinsic membrane proteins expressed most of the cell types which have high osmotic water permeability . among them aqp1 ( aquaporin 1 ) is a predominant water channel expressed in the plasma membranes of human pancreatic ducts ( ko et al . , 2009 ) . altered tissue water homeostasis may contribute to edema formation during various stress including bacterial infection ( schweitzer et al . aqp1 indeed contributes to maintain water homeostasis even in cells with plasma membrane damage following insult with bacterial infection ( schweitzer et al . , 2007 ) . interestingly , aqp1 expression was significantly increased in plasma membranes of pancreatic ducts in aip ( ko et al . , 2009 ) . upregulation of aqp1 expression seen in pancreatic ducts of patients with aip is speculated to be caused by the reduced fluid secretion from the pancreas as compensation ( ko et al . , 2009 ) . membranes serve as a barrier to prevent pathogenic bacteria from entering the nutrient - rich host cytosol ( radtke and oriordan , 2008 ) . aquaporins may be an integral part of infection and pathology caused by some microbial pathogens , so regulation of aquaporin expression and function is a key aspect of host pathogen interaction ( radtke and oriordan , 2008 ) . in addition , aqp1 regulates swelling of secretory vesicles , associated with pathogen - containing vacuoles ( pcv ) . aqp1-dependent modulation of pcv was triggered by bacterial induced membrane damage and ion flux ( radtke and oriordan , 2008 ) . as described above , in the pancreas of aip patients , expression of aqp1 was increased markedly , not only to on the plasma membrane but also in the cytoplasm of pancreatic duct cells . to clarify the association between the upregulation of aqp1 in the cytoplasm and pcv cftr : it was reported that aberrant cystic fibrosis transmembrane conductance regulator ( ctfr ) localization in the pancreatic ducts was observed in aip patients . the ctfr regulates overall pancreatic ductal fluid / overall ion transport across most epithelia and plays a central role in pancreatic ductal hco3- secretion ( ko et al . , 2010 ) . altered acidity of the pancreatic fluid may affect growth of the normal bacterial inhabitant and immunity of the upper intestinal environment . abcf1 : the abcf1 ( atp - binding cassette , sub - family f ) gene proximal to c3 - 2 - 11 microsatellite in hla class i regions , is thought to be one of the hla - linked susceptibility regions for aip ( ota et al . biological function of the putative abcf1 protein is mostly speculative , however it is thought to be regulated by tnf- , a prime cytokine in inflammatory reactions ( ota et al . , 2007 ) . from this point , it could be possible to suspect that bacteria would lie in the pathogenesis of aip . several experimental models of aip have been described ( table 1 ) . as for specific induction models , ca - ii , lf , and amylase - specific t cells could be listed ( nishimori et al . , 1995 ; uchida et al . , 2002 ; davidson et al . , 2005 ) as for the microorganisms related induction group , virus - induced aip models , such as c57bl/6 mice infected with the murine leukemia retrovirus lp - bm5 , developed histological findings similar to human aip ( suzuki et al . , 1993 ; watanabe et al . , 2003 ) . the spontaneous development of pancreatitis via an autoimmune mechanism in mrl / mp mice is accelerated by the administration of polyinosinic : polycytidylic acid ( poly i : c ) , a synthetic double - stranded rna and tlr 3 ligand ( qu et al . , 2002 ; soga et al . , 2009 ; asada et al . , 2010 ; sensitization occurs with not only viral components , such as double - stranded rna poly i : c , but also bacterial lps in interleukin ( il)-10-deficient mice ( nishio et al . , tlrs play important roles in innate immunity by initiating intracellular signaling to macrophages and dendritic cells after stimulation with various antigens . the majority of known tlrs mediate the development of th1 cell - inducing dendritic cells ( li et al . thus , pattern - recognition receptors ( prrs ) that bind pathogen - associated molecular patterns ( pamps ) may trigger an autoimmune response . experimental animal models of autoimmune pancreatitis . ca - ii , carbonic anhydrase ii , lf , lactoferrin ; psti , pancreatic secretary trypsin inhibitor ; poly i : c , polyinosinic : polycytidylic acid ; ntx , neonatal thymectomy . modified from references ( 2011 ) . as for specific induction , thymectomized mice immunized with ca - ii or lf develop a pathology that closely resembles aip under a regulatory t ( treg ) cell - depleted background ( uchida et al . , 2002 ) . however , t cells specific for ca - ii and lf were unable to induce pancreatitis in the adoptive transfer of an amylase - specific rat model ( davidson et al . , 2005 ) , suggesting that autoantibodies against these enzymes in aip represent a late consequence of tissue destruction . as for the spontaneous induction group , recently , the spontaneous development of aip in cd4 t cell - competent hla - dr*0405 transgenic ab0 nod mice ( freitag et al . , 2010 ) , treg - deficient backgrounds in neonatally thymectomized mice ( uchida et al . , 2002 ) , nod.cd28 knockout mice ( meagher et al . , 2008 ) , wbn / kob rats ( sakaguchi et al . , 2008 ) , and tgfbr2 knockout mice ( boomershine et al . , 2009 ) have demonstrated genetic polymorphisms of the effector cells in the etiologies of aip . however , because these mice are genetically engineered they may not completely reflect the onset of human diseases . instances of where microbial antigens might underlie the pathogenesis of igg4-related disease have been reported ( akitake et al . , 2010 ; watanabe et al . , 2012 ) . we previously reported that when c57bl/6 mice were inoculated intraperitoneally ( i.p . ) with heat - killed e. coli weekly for 8 weeks , marked cellular infiltration with fibrosis was observed in the exocrine pancreas accompanied by a high serum gamma globulin level and the production of autoantibodies against ca - ii and lf . bacterial infection apparently triggered aip - like pathological alterations in mice that strikingly resembled aip in humans ( haruta et al . c57bl/6 mice inoculated weekly with e. coli for 8 weeks were utilized as donors , and the spleens were intravenously transferred to rag2 mice . the pancreas in the recipient rag2 mice showed cellular infiltration in the exocrine pancreas , especially around the pancreatic ducts , indicating that the e. coli - inoculated mouse spleen cells possess the ability to reproduce pathological alterations in the pancreas of nave mice . similarly , when the spleen cells of donor s. intermedius - inoculated mice were transferred to rag2 mice , primary biliary cirrhosis ( pbc)-like cholangitis in the liver was induced , similar to that seen in the donor ( haruta et al . , 2010b ) . the aip - like inflammatory region in the pancreases of recipient mice with spleen cells transferred from e. coli - inoculated mice ( haruta et al . , 2010a ) , showed that most of the cellular infiltrates in the target organs were cd3-positive , indicating that these cells in both models originated from the donor mice . the criteria for determining whether a condition may be considered to be autoimmune , according to witebsky s postulates with modern revision by rose and bona ( 1993 ) , include ( i ) indirect evidence based on the reproduction of the autoimmune disease in experimental animals , ( ii ) direct evidence of the transfer of pathogenic antibodies , or ( iii ) pathogenic t cells and indirect evidence of the isolation of autoantibodies or autoreactive t cells . several lines of evidence have suggested that repeated e. coli - inoculated aip - like inflammation in the c57bl/6 mice pancreas is possibly of autoimmune origin . it is worth mentioning that long before the concept of aip was proposed as a definite disease entity , yamaki et al . . repeated injection of pancreatic extract from syngeneic mice mixed with capsular polysaccharide ( cps ) of the klebsiella pneumoniae type 1 kasuya strain ( cps - k ) , as an adjuvant , caused pathological alteration in the pancreas , consistent with infiltration by lymphocytes , plasma cells and mononuclear cells , degradation and lysis of the acinar cells , and destruction of the lobular architecture , replacement of fatty tissue and fibrous connective tissue and anti - pancreas antibody production . no histological changes were observed in tissues other than the pancreas and either cps - k or the pancreatic extract alone were insufficient to induce inflammation in the pancreas . k. pneumoniae is one of the commonly found bacteria in enterobacteriaceae ( yamaki et al . , 1980 ) , the natural habitat of the intestinal tract , and it is an opportunistic bacterium . the cps is a voluminous outer layer and is involved in protection against complement deposition that occurs mainly in the inhibition of macrophage phagocytosis ( evrard et al . , 2010 ) . it is of great interest that inflammation had been induced only when the syngeneic pancreatic antigen and bacterial component both existed , which suggest the possibility that the bacterial component may exhibit an adjuvant effect to the antigen . it is currently believed that the microbiota is essential for the development of a functional immune system and the gut microbiota has a far - reaching influence on the immune system , beyond the gastrointestinal tract , i.e. , within and outside the gut . in other words , gut microbiota has a greater influence on the systemic immune system than previously anticipated ( kranich et al . , 2011 ) . from the aspect of gut microbiota and autoimmunity , several animal models have been reported . for instance , it was reported that the relationship between certain harmful species of gut bacteria affect t cell populations in the periphery and thereby control the development of autoimmune arthritis . segmented filamentous bacteria could promote experimental autoimmune encephalomyelitis ( kranich et al . , 2011 ) . . however , additional factor(s ) such as genetic factors including the haplotype of class ii antigen of the major histocompatibility complex ( mhc ) and so on would be synchronized with the existence of microorganisms , and the progression switch to generate the status of aip would be turned on . during the initiation phase , infection with microorganisms possessing pamps and/or non - pathogenic microorganisms associated molecular patterns ( mamps ) trigger and inflammatory t cells are induced by molecules derived from pathogens or commensal microorganisms , as well as by endogenous stress - induced self molecules . these respective mamps and damage - associated molecular patterns ( damps ) induce inflammatory t cells either indirectly , through the induction of pro - inflammatory cytokine production by binding to pattern recognition receptors ( prrs ) on innate immune cells , or directly , by binding to pathogen - derived peptide specific receptors on t cells ( mills , 2011 ) . second , the progressive phase features the persistence of this pamps / mamps attack , stimulation by molecular - mimicking antigen , perhaps released as damps , exposure to or stimulation from commensal flora possessing the same antigenic epitope , and/or non - specific activation of innate immunity . these slowly progressive steps lead to a breakdown in immunological tolerance and the development of self antigen - specific t cell and antibody responses ( mills , 2011 ) and eventually establish the autoimmune diseases . throughout these processes , tlrs might participate as the key member of prrs involved in driving autoimmune inflammation ( mills , 2011 ; figure 1 ) . taking these considerations together , the pancreas is the field of the specific inflammation which would be triggered by the interaction with microorganisms , and a provider of the autoantigen to induce the production of autoantibodies . . however , additional factor(s ) such as genetic factors would be synchronized with the existence of microorganisms , and the progression switch to generate the status of aip would be turned on . during the initiation phase , pamps and/or non - pathogenic mamps trigger and second , the progressive phase features the persistence of this pamps / mamps attack or stimulation by a molecular - mimicking antigen , perhaps released as damps and/or exposure to or stimulation from commensal flora possessing the same antigenic epitope that the initial pathogen possessed , thereby upregulating the host immune response to the target antigen . these slowly progressive steps eventually lead to the development of autoimmune diseases . modified from previous reports ( haruta et al . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . aip , autoimmune pancreatitis ; anas , antinuclear antibodies ; ca - ii , carbonic anhydrase ii ; cps , capsular polysaccharide ; ctla-4 , cytotoxic t - lymphocyte antigen-4 ; damps , damage - associated molecular patterns ; fcrl3 , fc receptor - like gene 3 ; gbs , guillain barr syndrome ; gel , granulocyte - epithelial lesions ; hla , human leukocyte antigen ; hsp , heat shock protein ; idcp , idiopathic duct - centric pancreatitis ; lf , lactoferrin ; lps , lipopolysaccharide ; lpsp , lymphoplasmacytic sclerosing pancreatitis ; mamps , microorganism - associated molecular patterns ; pamp , pathogen - associated molecular patterns ; pbc , primary biliary cirrhosis ; pbp , plasminogen - binding protein ; poly i : c , polyinosinic : polycytidylic acid ; prr , pattern - recognition receptor ; treg , regulatory t cell ; tgf , transforming growth factor ; tlr , toll - like receptor ; ubr2 , ubiquitin - protein ligase e3 component n - recognin 2 .
little data exist on the management of intravenous drug use ( ivdu ) when treating severe infectious diseases ( i d ) , which necessitate parenteral antimicrobial therapy in rural communities . rural communities lack specialists in infectious diseases , addiction , or ethics to guide quality initiatives in managing infectious diseases in ivdu . standard guidelines offer appropriate antimicrobial therapy recommendations ; however , the means for optimization of treatment adherence and to prevent use of central access for intravenous ( iv ) drug injection is much less clear . quality initiatives to improve the transition of care of ivdus with infections into the community to receive parenteral therapy are likewise lacking . identifying patients with ivdu mertz et al . reported that appropriate antimicrobial therapy in hospitalized patients with ivdu is practical and successful though disease courses are complicated and readmissions are common . the absence of an established approach in managing such patients is daunting where the availability of rehabilitative and supportive resources in the community for addicts is scarce . the cost of not having a transitional mechanism in place has not been previously reported . we report a case of a patient who had 4 episodes of endocarditis within a 2-year time period . her nonadherence with ivdu treatment recommendations adversely affected her outcome from a drug addiction perspective and had a negative financial impact on the institution and providers . this case of serial episodes of endocarditis in one ivdu exemplifies the importance of an institution s need to design protocol driven , pre - emptive transitional plans addressing management of life - threatening infections in its community before the need for out - patient parenteral therapy ( opat ) arises in ivdus . the patient was a 45-year - old woman with a history of active oral and parenteral opioid drug use and chronic hepatitis c infection . she presented to our rural community hospital in southeast georgia 7 times over a 2-year time period with 4 different episodes of native valve endocarditis as defined by duke criteria , . in table 1 , the hospitalizations comparing i d diagnosis , psychiatric evaluations and adherence are summarized.table 1hospitalizations comparing i d diagnosis , psychiatric evaluations , and adherence.table 1hospitalizationid diagnosismicroorganismpsych evaluation / clearance for picc - opatid recommendationid compliancepsych compliancefirst hospitalizationmitral valve endocarditisgroup a streptococcusno / no42 days oral antibioticsnon / asecond hospitalizationmitral valve endocarditisgroup a streptococcusno / no42 days oral antibioticsnon / athird & fourth hospitalization(including transfer to tertiary care facility and return)native valve endocarditismrsayes / no56 days iv antibioticsyes ( in restricted setting)nofifth hospitalizationmssa bacteremiamssayes / no28 days iv antibioticsno , and left ama on day 17nosixth hospitalizationmssa endocarditismssayes / no42 days iv antibioticsno , and left ama on day 35noseventh hospitalizationstreptococcus viridans endocarditisstreptococcus viridansno / yes with contract42 days iv antibioticsyes ( in restricted setting)noabbreviations : ama : against medical advice ; compliance : adherence ; i d : infectious diseases ; iv : intravenous ; mrsa : methicillin - resistant staphylococcus aureus ; mssa : methicillin sensitive staphylococcus aureus ; n / a : not available ; opat : outpatient parenteral antibiotic therapy ; picc : peripherally inserted central catheter . hospitalizations comparing i d diagnosis , psychiatric evaluations , and adherence . abbreviations : ama : against medical advice ; compliance : adherence ; i d : infectious diseases ; iv : intravenous ; mrsa : methicillin - resistant staphylococcus aureus ; mssa : methicillin sensitive staphylococcus aureus ; n / a : not available ; opat : outpatient parenteral antibiotic therapy ; picc : peripherally inserted central catheter . in july 2014 , she presented with group a streptococcal endocarditis . she received parenteral ampicillin for 3 weeks and was discharged home on oral levofloxacin to complete a 42-day - course of treatment . she was readmitted with altered mental status 2 weeks later only to be discharged again on levofloxacin . the patient did not return for scheduled follow - ups . in december 2014 , she presented with methicillin - resistant staphylococcus aureus ( mrsa ) mitral valve endocarditis and had evidence of septic emboli and paraspinal / gluteal abscesses . she underwent incision , drainage , and washout of a pinned knee at a tertiary center . on her return to the rural hospital to complete the 42 days of parenteral vancomycin , psychiatry was consulted . she was diagnosed with a severe opioid use disorder according to diagnostic and statistical manual of mental disorders-5 and was considered to be high - risk for manipulation of a peripherally inserted central catheter ( picc ) line in the outpatient setting . post discharge she failed to enter addiction treatment . in june 2015 , she presented with methicillin sensitive staphylococcus aureus ( mssa ) septicemia . given her non - adherence with prior recommendations , antimicrobial management was again in the hospital . only 17 days of the recommended 28 day of oxacillin therapy was completed because she left against medical advice . commitment for addiction treatment was entertained by the psychiatrist ; however legal counsel of the hospital did not favor such an approach . she favored buprenorphine / suboxone agonist treatment , but such a provider ( within a 50 mile radius ) was not accepting new patients . in october 2015 , the patient was hospitalized with mssa endocarditis and treated for only 35 of the 42 recommended days of oxacillin therapy . the antibiogram of the mssa was similar to the strain isolated in june 2015 , suggesting a relapse of that infection . in april 2016 , she presented directly to a tertiary center and was diagnosed with a penicillin - nonsusceptible alpha hemolytic streptococcus endocarditis of the mitral valve , which was treated with parenteral gentamicin for 10 days and parenteral ceftriaxone for 42 days . a contract between the i d consultant and the patient was signed which delineated that she would be adherent with home care visitations , would not access the picc line for drug use , and would allow guardianship by her father . she arrived to the rural hospital overdosed on opioids requiring mechanical ventilator support and vasopressor support . she was transferred to third hospital for decompensated congestive heart failure from severe mitral regurgitation and underwent a tissue mitral valve replacement . the hospital expenses ( medicare part a and medicaid but not medicare part b component of charges ) were approaching $ 380,000 , including 20 encounters and 5 hospitalizations . providers fees and cardiothoracic surgery costs , which were performed in an outside facility , were not included in this sum of monies . little has been written on the opat options for patients who have active iv drug use disorders in rural communities where resources are especially limited . the infectious disease society of america ( idsa ) practice guideline for opat of endocarditis centers on the timing of complications such as embolization of the vegetation and the pathogens responsible for the infection , . advice is rendered that injection drug use or alcohol use problems should be specifically evaluated before opat therapy is initiated . indeed a safe and successful treatment of ivdu in a patient with a picc in an outpatient treatment center was reported by ho et al . . also , a retrospective observational study of patients with ivdu on opat demonstrated high cure rates ( 73.3% ) . i d providers are equipped to potentially handle these infections in restricted and outpatient settings . however , identifying and managing the addiction is the focal point of all medical care among ivdus . involvement of psychiatry early in the hospitalization and the creation of a transitional care plan are critical for successful medical care within the restraints of local resources and monies . our patient had more successful i d treatment courses while in a restricted setting where illicit drugs were not accessible , but it was costly to the institution at $ 380,000 excluding cardiac surgery and provider fees . as the opioid addiction epidemic has expanded to all communities , medical specialists need to lead their institutions to design locally a transitional care guideline to aid the management of ivdu patients with severe infections into the community . risk stratification in regards to the degree of addiction and probability of compliance should be addressed . if risk is deemed at an acceptable level to discharge the patient from the hospital , an oversight management team and plan should exist . unfortunately , most rural communities lack the specialist providers to initiate such a quality initiative which means practice guidelines need to be established which can be modified locally . that assessment would include:(1)the likelihood of outpatient compliance with abstinence and the treatment program designed by the psychiatrist and i d specialist.(2)risk assessment of line manipulation for drug use is indicated.(3)commitment for addiction treatment by the patient which may or may not include a contract.(4)define alternative routes of management options available in the community and institution in the event the patient is defined high - risk for outpatient care . a consensus in management by providers would facilitate the optimization of care in this challenging population and limit the cost to institutions . the likelihood of outpatient compliance with abstinence and the treatment program designed by the psychiatrist and i d specialist . commitment for addiction treatment by the patient which may or may not include a contract . define alternative routes of management options available in the community and institution in the event the patient is defined high - risk for outpatient care . a consensus in management by providers would facilitate the optimization of care in this challenging population and limit the cost to institutions . this case report illustrates the concept that much of adult infectious diseases are determined by the underlying medical conditions , complications of medical treatments or procedures . if a physician does not manage those underlying conditions and predisposing risks , management and prevention of the secondary infection are prone to fail . in this case the lack of a transitional care plan into the community to rehabilitative services rendering parenteral antimicrobials cost the institution $ 380,000 . all infections were cured but the primary disorder was not able to be managed in the community . practice guidelines and/or institutions need to address this gap in care of ivdus with life - threatening infections to reduce cost and address the primary disorder . this research did not receive any specific grant from funding agencies in the public , commercial , or not - for - profit sectors .
intracellular signalling cascades are the primary routes of communication between the plasma membrane and regulatory targets in various intracellular compartments . sequential activation of kinases is a widely conserved mechanism of signal transduction in many cellular processes . protein kinases are ubiquitous enzymes that modulate the activities of other proteins through the addition of phosphate groups to tyrosine , serine , or threonine amino acid residues , a process referred to as phosphorylation . over the last decade , several related intracellular signalling cascades , collectively known as mitogen - activated protein kinase ( mapk ) signalling cascades , have been characterized . mapks belong to a large group of serine / threonine protein kinases which have been shown to be conserved in organisms as diverse as yeast and humans [ 13 ] , and which can be activated by numerous extracellular stimuli [ 4 , 5 ] . in combination with several other signalling pathways , mapks can differentially alter the phosphorylation status of numerous proteins including transcription factors , cytoskeletal proteins , kinases , and other enzymes and can significantly influence gene expression , metabolism , cell division , cell morphology , and cell survival . epigenetic aberrations of these enzymes , or of the signalling cascades that regulate them , have been implicated in a variety of human diseases including cancer , inflammation , and cardiovascular disease . dysregulation of normal mapk signalling has also been implicated in both acute and chronic kidney disease . in this paper we focus on the role of mapks in kidney disease , and in particular , the role that mapk signalling plays in drug - induced kidney injury and disease . the transmission of extracellular signals to various intracellular targets is a multifaceted process which often involves the activity of one or more mapks . the process begins in response to external stimuli such as binding of a growth factor to its associated receptor on the cell surface . the resulting activation of the integral or associated protein tyrosine kinases contained within the intracellular domain of the receptor then initiates intracellular signalling events . activation of a mapk employs a core three - kinase cascade whereby a mapk kinase kinase ( map3k or mapkkk ) phosphorylates and activates a mapk kinase ( map2k or mkk ) which in turn phosphorylates one or more mapks [ 68 ] . once activated , mapks can phosphorylate several different intracellular targets including transcription factors , nuclear pore proteins , membrane transporters , cytoskeletal elements , and other protein kinases [ 810 ] . uniquely , mapks themselves can be activated by addition of phosphate groups to both their tyrosine and threonine amino acids ( dual phosphorylation ) after stimulation of a receptor by growth factors , mitogens , hormones , cytokines , or environmental stresses . these various members of the mapk family have been duplicated with slight variations , allowing cells to instigate multiple biological responses through a set of map kinase networks . in the early 1980s , the 42 kda extracellular signal - regulated protein kinases 1 and 2 ( erk1/2 ) were the first members of the mapk family to be identified and cloned from a vertebrate species [ 1216 ] . erk1/2 functions have been linked to the regulation of growth and differentiation [ 17 , 18 ] . erk1/2 activation is mediated by the specific protein kinases mapk / erk kinases ( mek ) 1/2 , which are members of the mapk supergene family mek2 . mek1 and mek2 are encoded by different genes , but are very similar in terms of sequence , substrate specificity , and regulation [ 19 , 20 ] . mek1/2 themselves are activated through phosphorylation by three distinct mapkk kinases ( map3k or mapkkk ) , raf , c - mos , and mek kinase ( mekk ) . the major pathway by which tyrosine kinase mediated signals are directly conveyed to the erk1/2 cascade is through ras - mediated recruitment of raf to the plasma membrane . ras activation is dependent on the tyrosine - kinase - mediated membrane translocation of the guanine nucleotide exchange factor sos by the grb2 adapter protein , which connects sos to the tyrosine - phosphorylated receptor . elk-1 and srf are target genes regulated by erk1/2 . erk signalling plays a major role in mediating renal cell responses to a diverse range of stimuli and has previously been shown to be involved in compensatory renal hypertrophy and pathological conditions such as glomerular and tubulointerstitial diseases . robust erk activation has been detected in the cyst epithelium in polycystic kidney disease ( pkd ) , while inhibition of the erk pathway led to inhibition of cyst - induced gain in kidney mass and improved renal function . in instances of drug - induced nephropathy such as kidney injury associated with cisplatin administration , stimulation of erk occurs via the egf - r / src cascade . conversely , inhibiting erk in vivo , in a rat model of progressive membranous nephropathy ( phn ) , was demonstrated to worsen dna damage observed in the podocytes and resulted in an upregulation of p21 , suggesting a protective role of erk in this model . this suggests a dual nature of this particular mapk : on the one hand mediating a kidney injury response while on the other hand playing a role in renal defence . the jnk cascade was first discovered through studies on the oncogenic cooperation between ras and the target jnk transcription factor c - jun , and on the activation of transcription factor ap-1 by uv irradiation . both studies indicated that phosphorylation of a component of ap-1 at ser63 and ser64 occurred in response to either ras activation or uv irradiation such phosphorylation , especially at ser73 , enhances the ability of c - jun to activate gene transcription . the protein kinases that bind to c - jun and phosphorylate it at ser63 and ser73 were subsequently identified as jnks . there are two jnk polypeptides , the products of two distinct genes , and they share 81% sequence identity . like all mapks , consistent with the different sequences surrounding their activating phosphorylation sites , the jnks are not phosphorylated by mek1/2 but by another mapkk , named sek 1 , mkk4 or jnkk1 . similar to other mapkks , jnkk1 is phosphorylated and activated by a map3k called mekk1 . very strong jnk activation is observed after irradiation of cells with either uv light or treatment with certain translational inhibitors such as anisomycin , exposure to il-1 , costimulatory activation of t - cells , ischemia reperfusion , and exposure to alkylating agents . jnk activation is also observed after treatment of certain cell types with growth factors such as egf and ngf . the majority of experimental analysis has focused on the mechanisms of jnk activation by growth factors and members of the src family of tyrosine kinases [ 28 , 30 ] . in the context of renal injury , jnk signalling is mediated by different insults including ischaemia / reperfusion ( i / r ) , ureteric ligation , immune - mediated injury , and hyperglycaemia [ 31 , 32 ] . jnk activation has been demonstrated in several glomerulonephritides , and jnk inhibition suppresses inflammation in rat antiglomerular basement membrane disease and also suppresses tubular apoptosis and interstitial fibrosis in unilateral ureteral obstruction ( uuo ) models . demonstrated that acute activation of jnk signalling occurs following i / r , with higher jnk activation detected in deceased donor compared to live donor allografts , thus suggesting that increased jnk reflects greater ischaemic damage . administration of a jnk inhibitor prior to i / r injury prevented tubular damage and renal dysfunction , suggesting involvement of jnk activation in both cellular rejection and acute tubular necrosis . p38 is another mapk protein , which is most similar to the yeast mapk hog-1 which is activated in response to osmotic shock . like hog-1 , p38 is also activated in response to osmotic shock , as well as by lps and il-1 . for the most part the similarity between the regulation of p38 and jnk is not surprising since jnkk1 ( sek1 ) is also a direct activator of p38 . downstream targets of p38 include the genes mapkap kinases 1 and 2 and atf-2 [ 38 , 39 ] . p38 mapk is an important regulator of senescence growth arrest due to its ability to activate both the p53 and prb / p16 growth arrest pathways . furthermore , p38 mapk activity is required for the senescence arrest caused by oncogenic ras , and constitutive p38 mapk activity can induce a growth arrest in normal human cells [ 41 , 42 ] . p38 mapk is known to upregulate specific cytokines such as il-6 , il-8 , and tnf in several biological contexts including kidney damage [ 39 , 43 , 44 ] . p38 mapk activity has been shown to be necessary and sufficient for the development of a senescence - associated secretory phenotype ( sasp ) . sasp is a persistent , nonacute inflammatory response in cells which have been induced to senescence by direct dna damage or oncogenic ras [ 4547 ] . it has been reported that inhibition of p38 mapk in autoimmune renal disease reduced the severity of the disease , resulting in a prolonged life span in mrl - fas mice . this protection against renal injury in vivo resulted from reduced infiltration of leukocytes , diminished expression of cytokines which are known to promote renal injury , and reduced production of ig , leading to the conclusion that activation of p38 mapk is required to promote cytokine / chemokine and ig production , which in turn result in lethal autoimmune renal injury in vivo . stambe et al . localized components of the p38 mapk pathway to podocyte - like cells , endothelial cells , and infiltrating neutrophils in a model of acute renal inflammation . it was shown that blockade of the p38 pathway significantly inhibited acute renal failure and proteinuria in rat anti - gbm glomerulonephritis via a neutrophil - platelet and p - selectin - dependent mechanism , thus suggesting that blockade of the p38 mapk pathway may be a novel therapeutic strategy for the treatment of acute renal inflammation . ras is a small gtp - binding protein with multiple effector molecules , each of which defines a pathway with specific functions . ras has been implicated , to different extents , as a mediator of erk , jnk , and p38 activities . p38 has been implicated in cell motility stimulated by pdgf , in a ras - dependent pathway . phosphatidylinositol 3-kinases ( pi3k ) have been suggested to be involved in cell motility through ras - mediated activation of rac . rac is a member of the ras superfamily of small gtp proteins which has a well - established role in cell migration and invasiveness [ 5153 ] . rac can be activated by ras directly via recruitment to the cell membrane or via ras activation of pi3kinase . in contrast to its functions in motility , rac has been shown unambiguously to strengthen cell - cell adhesion thereby preventing tumour cell invasiveness [ 54 , 55 ] . studies have shown these opposing effects of rac arise due to dependency on the cell substrate . on substrates permissive for locomotion , expression of active rac promotes motile behaviour , whereas , on substrates impeding cell motility , rac - dependent cell - cell adhesion is favoured . other members of the ras superfamily of small g proteins are rho and cdc42 . along with rac , rho and cdc42 control different aspects of the cytoskeleton and seem to act in a cascade in which cdc42 acts upstream of rac which in turn acts upstream of rho . rho displays complex functions in cell scattering and it is involved in the assembly of focal contacts and actin stress fibres in fibroblast cells [ 57 , 58 ] . rho plays a positive role in colony - stimulating factor-1 induced macrophage translocation and in the migration and metastatic properties of human hepatocellular carcinomas . rho carries out these actions by stimulating the phosphorylation of myosin light chain and adducin , an actin - binding protein . like rac , rho can also have antagonistic effects on epithelial cell scattering by reinforcing cell - cell adhesion sites . activated ras is sufficient for full erk activation but is only a weak jnk activator producing about one - third of the jnk activity observed after treatment with egf or expression of v - src . it is proposed that rac in turn can stimulate pak1 via cdc42 , which could induce phosphorylation of myosin light chain , thus linking this ras / rac - induced pathway to proteins directly affecting cell movement [ 37 , 62 ] . pak1 has been demonstrated to induce modest activation of jnk suggesting autocrine upregulation of jnk signalling . src tyrosine kinase is another positive regulator of growth - factor - induced cell scattering . these enzymes have a pivotal role in the regulation of a variety of biological functions which are associated with changes in morphology , including malignant transformation [ 64 , 65 ] , epithelial plasticity , and modulation of intercellular adhesion . in addition , the src family is required during mitogenesis induced by egf , pdgf , and colony - stimulating factor-1 [ 68 , 69 ] . there are nine members in the src family ; src , fyn , and yes are ubiquitously expressed while the other members have a more restricted pattern of expression . there is a possibility of redundancy of function amongst the src family members so the specificity of action of each individual member is not clear . this redundancy can arise by the phosphorylation of common substrates important for signalling , and it is probable that src and yes have redundant functions during cell scattering . firstly , src may phosphorylate specific substrates that are mainly cytoskeletal - based components , and molecules localised in cell - cell and cell - substrate adhesion sites , tyrosine phosphorylation of which could in turn alter cellular architecture [ 7275 ] . secondly , by inducing myc via a specific transduction pathway , src may also participate in entry into s - phase . finally , src could interact with other signalling pathways , as it has been shown that it is capable of binding shc , an early element of the ras cascade leading to the activation of this pathway [ 77 , 78 ] . a role for src signalling in the repair mechanisms of acute kidney injury has also been indicated . a recent study by takikita - suzuki et al . has provided evidence to suggest the importance of active src kinase in the early phase of pdgf - b - dependent nephrogenic repair after acute ischemia / reperfusion injury and has identified the distribution of active src kinase in the normal and reperfused kidney . the anatomical , biochemical , and physiological specialisations which permit the kidney to perform its vital roles in homeostasis may increase the risk of drug exposure to the components of the nephron , and its ancillary structures . l of plasma , producing 1.5 l of urine each day . as a result , high levels of drugs may be concentrated , leaving the epithelium of the nephron at a significantly greater risk of damage from pharmaceutical agents . in addition , the kidney is a metabolically active organ which contributes significantly to metabolism of xenobiotics . while renal metabolism usually contributes to the detoxification of xenobiotics , there are instances whereby substances undergo bioactivation to more toxic metabolites . in addition to glomerular filtration , removal of xenobiotics and waste products from the blood can occur via transcellular transport , which transports compounds directly from the blood into the lumen by organic ion transporters . this organic ion transport system may allow toxic compounds to accumulate in the cells of the nephron which may otherwise not gain access to the cytosol . antibiotics are a group of drugs used to treat various infections caused by bacteria and other microorganisms . the first antibiotic was discovered by sir alexander fleming in 1928 in a significant breakthrough for medical science . the development of antibiotics is probably the largest advance in medicine in the 20th century and has saved millions of lives worldwide from infections such as tb . gentamicin is an aminoglycoside broad spectrum gram - negative antibiotic which is routinely used in clinical practices for the treatment of gram - negative infections alone or in synergy with beta - lactam antibiotics in adults , children , and neonates . gentamicin use is however associated with significant nephrotoxicity it is estimated that roughly 30% of patients receiving gentamicin for 30 days show some signs of renal impairment [ 8284 ] . gentamicin - induced nephrotoxicity is characterized by morphological alterations including destruction of cell organelles and necrosis , lysosomal swelling and mitochondrial vacuolisation preceding functional alterations marked by proteinuria , increased levels of serum urea and creatinine , which lead to acute kidney injury ( aki ) . the site of gentamicin nephrotoxic action is the kidney cortex , especially the proximal tubules . animal models of aminoglycoside nephrotoxicity also present areas of interstitial fibrosis in the renal cortex and progressive tubular injury [ 85 , 86 ] . vancomycin is a cationic glycopeptide antibiotic used in the treatment of penicillin - resistant gram - positive infections , methicillin - resistant staphylococcus aureus infections , and clostridium difficile infections , which has seen a resurgence in use due to the emergence of -lactam - resistant gram - positive organism - associated infections . vancomycin is known to be both ototoxic and nephrotoxic , with vancomycin - induced nephrotoxicity reported to occur in up to 25% of patients [ 88 , 89 ] . studies carried out in animal models showed increased urinary excretion of proximal tubule cells and in the activity of malate dehydrogenase ( mdh ) following vancomycin administration [ 90 , 91 ] . furthermore , a recent study also suggested that oxidative stress might underlie the pathogenesis of vancomycin - induced nephrotoxicity . however , given the diversity of the mechanisms of action of the most commonly used antibiotic therapies , it is perhaps not immediately clear whether this injury occurs as a result of a defined process . there is evidence however to implicate involvement of the mapk signalling cascade in antibiotic - induced renal injury initiated by several distinct classes of antibiotics . a study carried out by volpini et al . in 2006 showed that mapks may be involved in the pathogenesis of acute renal failure following gentamicin treatment . since there is evidence that both the mapk and nf-b systems can be activated by oxidative stress in gentamicin - treated animals , the expression of p - p38 mapk and nf-b in the kidney during the evolution of tubulointerstitial nephritis and its relationship with histological features and renal function was investigated in gentamicin - treated rats in the presence or absence of an nf-b inhibitor . western blot analysis demonstrated the presence of the 43-kda phosphorylated p38 mapk and the 65-kda nf-b proteins in the renal cortex from all treatment groups compared to control . the gentamicin - treated animals showed a greater p38 expression than the control and nf-b inhibitor and gentamicin - treated animals . data obtained in this study showed that p38 mapk expression is increased during the development of gentamicin - induced interstitial nephritis and that such alteration is associated with enhancement of nf-b expression and the inflammatory process in the renal cortex , suggesting that the p38 mapk pathway may be involved in the renal lesions induced by gentamicin . other studies verify this involvement of mapk in antibiotic - associated nephrotoxicity ; p38-mapk has been found to be upregulated in rat kidneys following gentamicin treatment , and it has been shown that combination treatment with the lipid - lowering drug , atorvastatin , ameliorated gentamicin - induced nephrotoxicity , through inhibition of p38-mapk and nf-b expression . previous studies have also demonstrated that the proliferative response observed after sublethal toxicant - induced renal injury may be mediated by activation of the mapk signalling pathway , which is ultimately regulated by bioenergetic capacity [ 9598 ] . a study carried out investigating the effects of vancomycin exposure in renal llcpk1 cells on cell proliferation showed a dose- and time - dependent increase in cell number and total cellular protein . these effects were inhibited by pretreatment with a mapk inhibitor , pd098059 , preventing vancomycin - induced entry into the cell cycle , thus suggesting an association between the cell proliferative effects of vancomycin and the induction of mapk signalling cascades . in 1954 , the first successful transplantation of a human kidney was performed between identical twins ; the success of which was based on the lack of significant rejection between genetically identical twins , thus circumventing the requirement for immunosuppression . solid - organ transplantation was not truly implemented until the 1970s following significant technical and pharmacological advances , in particular , the discovery and development of the calcineurin inhibitors ( cnis ) . despite the advances over the past four decades , the majority of renal allografts fail after a period of progressive functional decline which is associated with glomerulosclerosis , tubular atrophy , interstitial fibrosis , and arteriosclerosis , in a process referred to as chronic allograft injury ( cai ) or chronic allograft nephropathy ( can ) . the deterioration of renal allograft function and structure associated with cai can occur due to immunological processes ( i.e. , chronic rejection ) and/or a range of simultaneous nonimmunological factors such as cni - induced nephrotoxicity , hypertension , and infection . the two most commonly employed immunosuppressants are the cnis cyclosporine a ( csa ) and tacrolimus ( fk506 ) . csa , also known as sandimmune , is a 1203 dalton , lipophilic , cyclic compound , derived from fungal origins which was discovered in 1976 . the first incidence of csa employed as an immunosuppressant was in 1978 , revolutionizing the field of organ transplantation . csa is primarily renowned as a powerful immunosuppressant for use in organ transplantation to prevent graft rejection in kidney , liver , heart , lung , and combined heart - lung transplants . tacrolimus , also known as fk506 or prograf , is a 822 da , 23-membered macrolide compound ( c44h69no12 ) that was isolated from a soil microorganism streptomyces tsukubaensis in japan in 1984 , [ 104 , 105 ] . fk506 is a potent immunosuppressive agent employed worldwide since the early 1990s that is effective in allograft prophylaxis after organ transplantation , for therapy of acute rejection and in treatment of different immune diseases [ 106 , 107 ] . in 2003 , fk506 was used as initial immunosuppression in 67% of kidney recipients and 89% of liver recipients ( unos united network for organ sharing 2004 ) . fk506 is up to 100 times more potent than csa , which has significantly reduced the incidence and severity of acute rejection rates in organ transplantation [ 109 , 110 ] . both cnis follow similar molecular pathways , with both fk506 and csa eventually inhibiting nfat - dependent production of il-2 and other cytokines and prevention of t - cell growth [ 111 , 112 ] . recently , alternative molecular pathways have been identified for csa , which has been found to inhibit the jnk and p38 signalling pathway activity triggered by antigen recognition in t cells ( figure 5 ) . csa and fk506 are widely used in transplant organ recipients , but in the kidney allograft , they may cause tubulointerstitial as well as mesangial fibrosis . the fibrogenic effect of cnis in the renal allograft is predominantly mediated by elevated intrarenal expression of tgf- [ 57 , 115 ] , and subsequent excessive extracellular matrix ( ecm ) generation [ 116 , 117 ] . using the rmc cell line , rat kidney mesangial cells , it has been shown that csa and fk-506 induce an extremely rapid and dose - dependent increase of y - box - binding protein-1 ( yb-1 ) content in a cell type - specific manner . the highly conserved yb-1 is a member of the family of cold - shock proteins with mitogenic properties which play a role in cellular stress responses and tumourigenesis and also controls tgf-1 translation in proximal tubular cells [ 118120 ] . given the fact that yb-1 is a downstream target of mapk erk1/2 [ 121 , 122 ] , the study continued to investigate the involvement of erk1/2 in csa - triggered cell activation . previous studies have documented that yb-1 undergoes phosphorylation at serine 102 ( ser ) by activated serine / threonine protein kinase akt / protein kinase b [ 114 , 123 ] . showed that inhibition of akt / erk signals upstream of yb-1 activation prevents its phosphorylation at ser and abolishes the csa - mediated yb-1 protein increase demonstrating that csa - induced yb-1 accumulation was dependent on mapk / erk and pi3k / akt signalling . also verified these results in vivo , treating mice either with a vehicle control or csa and analysing the renal yb-1 content . immunoblotting indicated elevated yb-1 protein content in the csa - treated mice , localised in the mesangial compartment . chemotherapy continues to play a crucial role in the management of cancer , with the basic aim to kill cancerous cells whilst causing minimal damage to the other healthy cells in the body . cancer chemotherapeutics are divided into different categories with several members in each category , including alkylating agents ( e.g. , cyclophosphamide ) ; antimetabolites ( e.g. , methotrexate ) ; plant alkaloids ( e.g. , etoposide ) ; anthracyclines(e.g . , doxirubicin ) ; antitumor antibiotics ( e.g. , mitomycin c ) ; platinum compounds ( e.g. , cisplatin ) ; taxanes ( e.g. , taxol ) . cancer chemotherapeutic agents can cause nephrotoxicity in various ways , with some drugs exerting immediate effects on renal function while others are known to have cumulative effects , causing renal injury after long periods of use . it is used to treat a wide variety of solid tumours , and successful cure rates for certain cancers such as testicular cancer are as high as 90% following cisplatin treatment . one of the major limiting factors in the use of cisplatin however is development of acute kidney injury , with clinically measureable nephrotoxicity usually detected 10 days after administration . it is estimated that 20% of patients receiving high doses of cisplatin develop renal dysfunction . the kidney is particularly susceptible to cisplatin - induced toxicity due to the high concentration of the organic cation transporter 2 ( oct2 ) , which is expressed mainly in the kidney and facilitates cellular entry of the cisplatin compound . the exact mechanisms governing cisplatin - induced nephrotoxicity are not completely understood ; however , it is believed that mapk plays a pivotal function . indeed several studies both in vitro and in vivo have shown that pharmacological inhibition of erk1/2 ameliorates cisplatin - induced nephrotoxicity . there is conflicting evidence about the role that both p38 and jnk / sapk play in cisplatin - induced kidney injury . . showed that erk , and not p38 or jnk / sapk inhibition , prevented cisplatin induced toxicity . however , other studies have shown that pharmacological inhibition of p38 both in vitro and in vivo prevented toxicity [ 129131 ] . the role that jnk / sapk plays in acute kidney injury following cisplatin exposure has been less well characterised ; however , it has been shown that jnk / sapk inhibition resulted in a significant reduction in cisplatin - induced nephrotoxicity in vivo . a study by pabla et al . identified pkc as a critical regulator of cisplatin nephrotoxicity , which can be effectively targeted for renoprotection during chemotherapy . the data showed that during cisplatin nephrotoxicity , src interacted with , phosphorylated , and activated pkc in mouse kidney lysates . after activation , pkc regulated mapks , but not p53 , to induce renal cell apoptosis . thus , inhibition of pkc , pharmacologically or genetically , attenuated kidney cell apoptosis and tissue damage , preserving renal function during cisplatin treatment . conversely , inhibition of pkc enhanced cisplatin - induced cell death in multiple cancer cell lines and , remarkably , enhanced the chemotherapeutic effects of cisplatin in several xenograft and syngeneic mouse tumour models while protecting kidneys from nephrotoxicity . together these results demonstrate a role of pkc in cisplatin nephrotoxicity and support targeting pkc as an effective strategy for renoprotection during cisplatin - based cancer therapy . the nsaid family includes several classes of drugs such as the carboxylic acids , for example , aspirin ; acetic acids , for example , diclofenac ; propionic acids , for example , ibuprofen and ketoprofen ; and cox-2 inhibitors , for example , celecoxib which are used worldwide as analgesics and antipyretics to combat pain , fever , and inflammation . they are especially effective for treating inflammatory diseases ( e.g. , arthritis ) through nonspecific inhibition of cyclooxygenase ( cox ) enzymes which limits production of prostaglandins . serious gastrointestinal side effects have been minimized with the advent of selective and specific cox-2 inhibitors and misoprostol . the spectrum of nephrotoxicity includes acute tubular necrosis , acute tubulointerstitial nephritis , glomerulonephritis , renal papillary necrosis , chronic renal failure , salt and water retention , hypertension , and hyperkalaemia [ 135 , 136 ] . hou et al . investigated the molecular basis of the renal injury by evaluating the expression of the stress marker , haeme oxygenase-1 ( ho-1 ) , in celecoxib - stimulated glomerular mesangial cells . conversely treatment with n - acetylcysteine , a free radical scavenger , strongly decreased ho-1 expression , suggesting the involvement of reactive oxygen species ( ros ) . following treatment with various mapk inhibitors , the study showed that only a specific jnk inhibitor attenuated celecoxib - induced ho-1 expression , and kinase assays demonstrated increased phosphorylation and activation of c - jnk following nsaid treatment . the presence of a free radical scavenger reduced the stimulatory effect of celecoxib on stress kinase activities , suggesting an involvement of jnk in ho-1 expression . treatment with a pi-3k specific inhibitor prevented the enhancement of ho-1 expression , which correlated with inhibition of the phosphorylation of the pdk-1 downstream substrate akt / protein kinase b ( pkb ) . the data presented in this study suggested that celecoxib - induced ho-1 expression in glomerular mesangial cells may be mediated by ros via the jnk - pi-3k cascade . the complex nature of critical illness often necessitates the use of multiple therapeutic agents , many of which may individually or in combination have the potential to cause kidney injury . drugs known to cause nephrotoxicity have been shown to exert their toxic effects through one or more common pathogenic mechanisms . it is important to appreciate that a single drug causing renal toxicity can involve multiple pathophysiological pathways and that predisposing factors are common to virtually all causative agents mediating kidney injury . various studies , both in vitro and in vivo , have shown that the administration of certain drugs acts as the stimulus to trigger various mapk cascades , thus mediating cellular responses to kidney injury . indeed , activation of these central pathways is evidenced in both acute and chronic kidney injury . data from renal biopsies in humans have shown upregulation of mapks in a variety of renal conditions , suggesting involvement in human renal disease , and may provide a new target for intervention . interventions aimed at providing renoprotection , such as ace - inhibition or statin therapy , can reduce the renal mapk expression , suggesting that increased renal mapk expression is involved in the pathophysiology of kidney damage . animal data presented in this paper suggests that mapk inhibition may be of use in acute inflammatory renal disorders , and in chronic conditions characterized by fibrosis . in order to explore the potential of mapks as a novel intervention strategy in kidney disease , it is important to establish the renal conditions that can specifically benefit from mapk inhibition , since studies have shown that not all conditions can be improved through inhibition of the mapk signalling cascade . however , since the mapk cascades have been implicated in numerous studies in the development of kidney damage and disease , continued research in this area will hopefully highlight novel therapies or mechanisms of prevention of kidney injury .
the cox maze procedure is the most effective procedure for eliminating atrial fibrillation ( af ) and restoring a normal sinus rhythm , and it has evolved from a cut - and - saw technique to ablation using alternative energy sources such as cryothermia and radiofrequency [ 1 - 3 ] . as the operative techniques continue to advance , a minimally invasive approach for surgical af ablation has also been developed . the wound from minimally invasive cardiac surgery ( mics ) is cosmetically superior , and earlier recovery is expected compared to a sternotomy approach . however , there are concerns about a minimally invasive approach to af ablation surgery in that the rhythm outcomes may be poorer than the sternotomy approach because the completeness of the transmural lesions may be disturbed by limited incisions . in patients with af associated with mitral valve ( mv ) disease , the port - access approach can establish a complete endocardial lesion set under cardiopulmonary bypass ( cpb ) support , which easily enables the combination of the maze procedure with mv surgery . the aim of this study is to evaluate the clinical and rhythm outcomes of the af ablation procedure thorough a port access mini - thoracotomy approach compared with conventional sternotomy in patients with af associated with mv diseases . between february 2006 and december 2009 , a total of 199 patients underwent mv surgery with biatrial af ablation in our institution . excluding 64 patients who underwent aortic valve replacement or coronary artery bypass grafting surgery resulted in a final total of 135 subject patients . among them , 78 underwent surgery through the port - access approach ( the mics group ) by an automated endoscope system using an optimal positioning aesop 3000 system ( computer motion inc . , santa barbara , ca , usa ) , whereas 57 patients underwent a median sternotomy ( the sternotomy group ) . the operative technique was chosen according to the surgeon 's preference . in the sternotomy group , conventional aortic and bicaval cannulation was used , and in the mics group , the right femoral artery , right femoral vein , and right internal jugular vein cannulation procedure was used . about a 4 to 6 cm main mini - thoracotomy incision with an intercostal muscle division was made over the 4th intercostal space and another three small port incisions were made for the insertion of a chitwood clamp , a thoracoscopy , and a vent sucker . the af ablation was performed using a flexible cryoablation system ( surgifrost ; medtronic , minneapolis , mn , usa ) . the right side ablation included cavo - tricuspid isthmus isolation and a line toward the superior vena cava . the left side ablation included a box lesion for isolation of the pulmonary veins , a line toward the left atrial appendage , and a line toward the mitral annulus , extending posteriorly ( fig . the cryoablation was conducted at -120 for 1 or 2 minutes . during the hospitalization , standard 12-channel surface electrocardiography ( ecg ) patients with postoperative af , atrial flutter , or atrial tachycardia were treated with amiodarone . the amiodarone therapy was initiated with 900 to 1,200 mg intravenous loading for 24 hours followed by 600 to 900 mg per day for 1 to 2 weeks as a maintenance therapy . the patients with rapid ventricular rhythm despite the amiodarone medication were treated with a beta - blocker , calcium - channel blocker , or digitalis . in the valve repair or bioprosthetic valve insertion patients , warfarin was administered for three to six months with a target international normalized ratio ( inr ) of 1.5 to 2.5 . late af events were defined as the episodes of af , atrial tachycardia , or atrial flutter after the initial 3 month period . the continuous variables are presented as meanstandard deviation or median and range and were compared using the student 's unpaired t - test or the mann - whitney u - test . the categorical variables were compared using the chi - square test or fisher 's exact test . kaplan - meier curves were generated to delineate freedom from af or freedom from major adverse events . for comparisons of the incidence of time - related events between the two groups , a log - rank test was performed . to reduce the effect of a treatment selection bias and potential confounding , we performed an adjustment for the differences in the baseline characteristics by use of propensity score analysis . the propensity scores were estimated without regard to outcome variables , with multiple logistic regression analysis . the prespecified covariates listed in table 1 were included for the calculation of the propensity scores . the discrimination and calibration abilities of the propensity score model were assessed by means of c statistics and the hosmer - lemeshow test . the model had a c statistic of 0.842 and a hosmer - lemeshow goodness - of - fit p - value of 0.55 , indicating the model was well calibrated with strong discrimination . there was limited overlap in the propensity scores between the two groups ; therefore , the propensity score was used as a covariate in statistical models . between february 2006 and december 2009 , a total of 199 patients underwent mv surgery with biatrial af ablation in our institution . excluding 64 patients who underwent aortic valve replacement or coronary artery bypass grafting surgery resulted in a final total of 135 subject patients . among them , 78 underwent surgery through the port - access approach ( the mics group ) by an automated endoscope system using an optimal positioning aesop 3000 system ( computer motion inc . , santa barbara , ca , usa ) , whereas 57 patients underwent a median sternotomy ( the sternotomy group ) . in the sternotomy group , conventional aortic and bicaval cannulation was used , and in the mics group , the right femoral artery , right femoral vein , and right internal jugular vein cannulation procedure was used . about a 4 to 6 cm main mini - thoracotomy incision with an intercostal muscle division was made over the 4th intercostal space and another three small port incisions were made for the insertion of a chitwood clamp , a thoracoscopy , and a vent sucker . the af ablation was performed using a flexible cryoablation system ( surgifrost ; medtronic , minneapolis , mn , usa ) . the right side ablation included cavo - tricuspid isthmus isolation and a line toward the superior vena cava . the left side ablation included a box lesion for isolation of the pulmonary veins , a line toward the left atrial appendage , and a line toward the mitral annulus , extending posteriorly ( fig . patients with postoperative af , atrial flutter , or atrial tachycardia were treated with amiodarone . the amiodarone therapy was initiated with 900 to 1,200 mg intravenous loading for 24 hours followed by 600 to 900 mg per day for 1 to 2 weeks as a maintenance therapy . the patients with rapid ventricular rhythm despite the amiodarone medication were treated with a beta - blocker , calcium - channel blocker , or digitalis . in the valve repair or bioprosthetic valve insertion patients , warfarin was administered for three to six months with a target international normalized ratio ( inr ) of 1.5 to 2.5 . late af events were defined as the episodes of af , atrial tachycardia , or atrial flutter after the initial 3 month period . the continuous variables are presented as meanstandard deviation or median and range and were compared using the student 's unpaired t - test or the mann - whitney u - test . the categorical variables were compared using the chi - square test or fisher 's exact test . kaplan - meier curves were generated to delineate freedom from af or freedom from major adverse events . for comparisons of the incidence of time - related events between the two groups , a log - rank test was performed . to reduce the effect of a treatment selection bias and potential confounding , we performed an adjustment for the differences in the baseline characteristics by use of propensity score analysis . the propensity scores were estimated without regard to outcome variables , with multiple logistic regression analysis . the prespecified covariates listed in table 1 were included for the calculation of the propensity scores . the discrimination and calibration abilities of the propensity score model were assessed by means of c statistics and the hosmer - lemeshow test . the model had a c statistic of 0.842 and a hosmer - lemeshow goodness - of - fit p - value of 0.55 , indicating the model was well calibrated with strong discrimination . there was limited overlap in the propensity scores between the two groups ; therefore , the propensity score was used as a covariate in statistical models . the patients in the mics group were younger than the patients in the sternotomy group . the af duration was longer and the fine af wave had more frequency in the sternotomy group . all these findings were suggestive of a higher risk of postoperative af recurrence in the sternotomy group than the mics group . the cpb time and aortic cross clamping ( acc ) time were approximately 20 to 30 minutes longer in the mics group ( cpb time : 176.049.5 minutes vs. 150.051.9 minutes , p=0.045 ; acc time : 115.028.5 minutes vs. 98.333.9 minutes , p=0.037 ) . a total of 4 patients were required to have a permanent pacemaker implanted due to sick sinus syndrome or a complete atrioventricular block . there was only one early mortality in the mics group ( n=1 , 1.3% ) ; the cause of death was rupture of the left ventricle following a tissue valve replacement for rheumatic mitral stenosis . there were no significant differences between the two groups in the rate of reoperation for bleeding ( 6 vs. 2 , p=0.47 ) , requirement for permanent pacing ( 1 vs. 3 , p=0.31 ) , stroke ( 0 vs. 1 , p=0.42 ) , or wound problems ( 1 vs. 0 , p=0.49 ) . a rhythm follow - up more than three months after surgery was possible in 131 patients ( 96.3% ) with a median follow - up period of 18.3 months ( range , 0.1 to 44.7 months ) , during which a total of 758 ecgs ( 5.7/patient ) and 120 holter monitoring data ( 0.9/patient ) were acquired for analyses . early af events occurred in 17 patients ( 5 [ 6.5% ] in the mics group and 9 [ 16.3% ] in the sternotomy group , p=0.40 ) . in addition , 31 patients experienced late af events during the follow - up period ( 13 [ 18.5% ] in the mics group and 18 [ 31.3% ] in the sternotomy group , p=0.64 ) . the rhythm outcomes at 3- , 6- , 12- , and 24-month points in time after operation are summarized in table 3 and are illustrated in fig . 2 . there were no significant differences in the rate of the normal sinus rhythm or af between the two groups over the course of the 2 years postsurgery . the two groups also had no significant differences in the freedom from af without anti - arrhythmic agents on crude and adjusted analyses for two years after surgery ( fig . when the freedom from recurrent af was estimated with the kaplan - meier method , the two groups showed similar rates of freedom from af in the simple comparison ( log - rank p=0.37 ) and in an adjusted analysis ( adjusted hazard ratio , 2.57 ; 95% confidence interval , 0.98 - 7.60 ; p=0.088 ) ( fig . a clinical follow - up was completed in 129 patients ( 95.6% ) with a median follow - up period of 27.1 months ( range , 0.1 to 58.1 months ) . there were 9 late deaths ( 4 [ 5.1% ] in the mics group and 5 [ 8.8% ] in the sternotomy group ) . in the mics group , there were unknown causes for the following conditions : death in 1 patient , aortic pseudoaneurysm rupture in 1 patient , metabolic encephalopathy in 1 patient , and sepsis in 1 patient . in the sternotomy group , there were unknown causes for the following conditions : death in 3 patients , sequelae after stroke in 1 patient , and intracranial hemorrhage in 1 patient . hemorrhagic events occurred in 7 patients ( 2 [ 2.6% ] in the mics group and 5 [ 8.8% ] in the sternotomy group ) . there was only one reoperation in the sternotomy group due to an infective endocarditis at 30 months after a mechanical valve replacement . there were no significant differences in the rate of death and major complications in the propensity score adjusted comparison between the two groups ( table 5 ) . the patients in the mics group were younger than the patients in the sternotomy group . the af duration was longer and the fine af wave had more frequency in the sternotomy group . all these findings were suggestive of a higher risk of postoperative af recurrence in the sternotomy group than the mics group . the cpb time and aortic cross clamping ( acc ) time were approximately 20 to 30 minutes longer in the mics group ( cpb time : 176.049.5 minutes vs. 150.051.9 minutes , p=0.045 ; acc time : 115.028.5 minutes vs. 98.333.9 minutes , p=0.037 ) . a total of 4 patients were required to have a permanent pacemaker implanted due to sick sinus syndrome or a complete atrioventricular block . there was only one early mortality in the mics group ( n=1 , 1.3% ) ; the cause of death was rupture of the left ventricle following a tissue valve replacement for rheumatic mitral stenosis . there were no significant differences between the two groups in the rate of reoperation for bleeding ( 6 vs. 2 , p=0.47 ) , requirement for permanent pacing ( 1 vs. 3 , p=0.31 ) , stroke ( 0 vs. 1 , p=0.42 ) , or wound problems ( 1 vs. 0 , p=0.49 ) . a rhythm follow - up more than three months after surgery was possible in 131 patients ( 96.3% ) with a median follow - up period of 18.3 months ( range , 0.1 to 44.7 months ) , during which a total of 758 ecgs ( 5.7/patient ) and 120 holter monitoring data ( 0.9/patient ) were acquired for analyses . early af events occurred in 17 patients ( 5 [ 6.5% ] in the mics group and 9 [ 16.3% ] in the sternotomy group , p=0.40 ) . in addition , 31 patients experienced late af events during the follow - up period ( 13 [ 18.5% ] in the mics group and 18 [ 31.3% ] in the sternotomy group , p=0.64 ) . the rhythm outcomes at 3- , 6- , 12- , and 24-month points in time after operation are summarized in table 3 and are illustrated in fig . there were no significant differences in the rate of the normal sinus rhythm or af between the two groups over the course of the 2 years postsurgery . the two groups also had no significant differences in the freedom from af without anti - arrhythmic agents on crude and adjusted analyses for two years after surgery ( fig . when the freedom from recurrent af was estimated with the kaplan - meier method , the two groups showed similar rates of freedom from af in the simple comparison ( log - rank p=0.37 ) and in an adjusted analysis ( adjusted hazard ratio , 2.57 ; 95% confidence interval , 0.98 - 7.60 ; p=0.088 ) ( fig . a clinical follow - up was completed in 129 patients ( 95.6% ) with a median follow - up period of 27.1 months ( range , 0.1 to 58.1 months ) . there were 9 late deaths ( 4 [ 5.1% ] in the mics group and 5 [ 8.8% ] in the sternotomy group ) . in the mics group , there were unknown causes for the following conditions : death in 1 patient , aortic pseudoaneurysm rupture in 1 patient , metabolic encephalopathy in 1 patient , and sepsis in 1 patient . in the sternotomy group , there were unknown causes for the following conditions : death in 3 patients , sequelae after stroke in 1 patient , and intracranial hemorrhage in 1 patient . hemorrhagic events occurred in 7 patients ( 2 [ 2.6% ] in the mics group and 5 [ 8.8% ] in the sternotomy group ) . there was only one reoperation in the sternotomy group due to an infective endocarditis at 30 months after a mechanical valve replacement . there were no significant differences in the rate of death and major complications in the propensity score adjusted comparison between the two groups ( table 5 ) . in this study , the clinical and rhythm outcomes of the mics and sternotomy groups were comparable . port - access mitral valve operations have been reported to provide acceptable results , showing excellent cosmetic outcomes , shorter hospitalization , and reduced surgical trauma . the benefits of this approach may be maximized in patients with poor pulmonary function because it is reported to better preserve pulmonary function compared to conventional sternotomy . in a study involving patients who underwent video - assisted pulmonary vein isolation using a bipolar radiofrequency ablation system , 72.5% of the patients were found to have recovered normal sinus rhythm postoperatively . however , in the cited study , only 40% of the patients with long - standing af had a normal sinus rhythm at discharge . this limited success rate may be attributable to the incomplete lesion sets and questionable transmurality of the ablation lesions . recent studies suggest that adding a maze operation to mv surgery does not increase operative mortality and morbidity . moreover , this procedure is reported to result in better rhythm outcomes and consequently reduce thromboembolic events in the long - term follow - up . in our study , the af recurrence rate of patients who underwent the port - access mini - thoracotomy approach was not higher than those in the sternotomy groups . these results may be attributable to the completeness of lesion sets ( biatrial full maze ) and transmurality of the lesions comparable to a standard sternotomy under the endocardial approach and full cpb support . although there were no significant differences in the patient profiles of two groups except age and af duration , these two variables affected the surgical outcomes . af tends to recur in patients who have factors such as an la size larger than 60 mm , patients older than 60 years , and fine af . a limitation of this study is that it is a retrospective work with a non - randomized design . the relatively small number of patients in this study is another limitation , but data from a larger population of patients with a longer follow - up duration might resolve this limitation . in conclusion , the port - access af ablation is an effective and safe approach compared with the sternotomy approach in patients undergoing biatrial af ablation combined with mv surgery . therefore , the port - access approach may offer an additional surgical option for patients , with the benefit of having superior cosmetic results or less surgical trauma for the treatment of af associated mv disease .
intraorally , the tongue is the most common location . here , a case of a schwannoma of the floor of the mouth in a 28-year - old male a 28-year - old male patient presented with a complaint of enlarging swelling of 1 month duration in the right anterior floor of the mouth which was otherwise asymtomatic . on intra - oral examination , a 4 3 cm swelling in the right anterior floor of the mouth , with smooth surface , well - defined border , covered with normal appearing mucosa , was noted . on palpation , it was firm and tender . computed tomography ( ct ) scans ( axial view ) showed a well - defined heterodense mass measuring 3.5 2.5 2.5 cm , with specks of calcification in the right sublingual region . there was a hypodense rim around the swelling causing smooth erosion of the adjacent mandible . furthermore , multiple enlarged , submental and bilateral submandibular lymph nodes were seen [ figure 1 ] . computed tomography scan in axial view showed a well - defined heterodense mass with specks of calcification in the right sublingual region based on the clinical findings , the ct scan features and anatomical location a provisional diagnosis of salivary gland tumor involving sublingual gland was given . the patient underwent surgical excision of the mass under general anesthesia ; the post - surgical course was uneventful . macroscopically , the resected mass was encapsulated , greyish - white in color , measuring 4 4 3 cm . microscopic examination revealed a well encapsulated tumor exhibiting areas of organized spindle - shaped cells in palisading arrangement around acellular , eosinophilic areas forming verocay bodies giving antoni type a pattern . b pattern exhibited less cellularity , less organized cells , which were plump , spindle - shaped and were generally seen adjacent to densely vascular areas [ figure 3 ] . immunohistochemical investigation of the tumor cells showed diffuse , strongly positive staining for s-100 protein [ figure 4 ] . were compatible with the diagnosis of schwannoma . gross appearance of the resected tumor mass depicts antoni type a tissue with spindle - shaped cells , palisading nuclei and verocay bodies , ( h and e , 10 ) immunohistochemical staining of the tumor cells showing diffuse , strong positivity for s-100 protein , ( 10 ) neurilemoma / schwannoma , is a benign tumor arising from and consisting solely of schwann cells . extracranially , about a quarter of all schwannomas occur in the head and neck region . schwannomas are usually solitary lesions ; however , some are seen as multiple lesions as part of neurofibromatosis type i. the solitary neurilemoma is a slow growing , encapsulated tumor that typically arises in association with a nerve trunk . usually the mass is asymptomatic , although tenderness or pain may occur in some instances . in the present case the schwannoma presented as an asymptomatic , enlarging , well circumscribed mass in the right anterior floor of the mouth in a 28-year - old male . schwannoma in the floor of the mouth or tongue has an intact overlying epithelium and , therefore , resembles any of the benign lesions known to occur in this region . the histological finding of the present case consists of a well - defined fibrous capsule having two patterns . a areas are composed of compact spindle - shaped cells with twisted nuclei , indistinct cytoplasmic borders and occasional clear intranuclear vacuoles arranged in bundles or inter lacing fascicles . in the antoni a areas there was nuclear palisading , whirling of cells and verocay bodies formed by two compact rows of well aligned nuclei separated by fibrillar cell processes . the spindle or oval cells were arranged haphazardly in the loose textured matrix , which was punctuated by microcystic change , inflammatory cells and delicate collagen fibres . these tumors may undergo degenerative changes in the form of cyst formation , hyalinization , calcification , hemorrhage , and nuclear atypism , but , are nonetheless benign . the observation of tumor acquiring such a large size within duration of 1 month is conflicting with routinely observed slow growing nature of schwannoma . however , its inconspicuous location in the floor of the mouth and asymptomatic behavior combined with well encapsulated nature and some areas of degenerative changes in the form of hemorrhage indicate that the tumor mass is of long standing nature . s-100 is strongly expressed by most cells in schwannoma in contrast to cells of neurofibromas , which variably expresses the antigen . although the expression of s-100 is diminished in antoni b areas , immunostaining for this protein is so consistent and of such intensity that it serves as an important diagnostic tool . in our patient , almost all tumor cells stained strongly for the s-100 protein , presenting as a proliferative lesion of schwann cells . s-100 staining and the characteristic hematoxylin and eosin staining pattern confirmed the diagnosis of schwannoma . the solitary schwannoma is treated by surgical excision ; the lesion normally will not recur . the extensive size of this lesion , occurrence in an uncommon location and in a short period of time led to a clinical diagnosis of a malignant lesion . a and b type compelled us to include this large - sized tumor under the benign category as one of the diagnosis .
esophageal ph monitoring was first used by tuttle and grossman in 1958 ( 1 ) . miller first reported in 1964 the use of an indwelling ph electrode positioned above the lower esophageal sphincter ( les ) to continuously monitor the intraesophageal ph ( 2 ) . this technique overcame many of the weak points of the other tests that had been used to detect gastroesophageal reflux . ambulatory 24-hr esophageal ph monitoring is increasing in popularity as the standard method for measuring esophageal exposure to gastric acid . it provides quantitative data on esophageal acid exposure as well as the temporal correlation between symptoms and actual acid reflux events . since acid reflux into the esophagus is a physiological event even in normal subjects , discrimination between physiological and pathological reflux is often difficult ( 3 ) . good temporal correlation between a distinct pathological event such as chest pain and a ph drop on the recording provides evidence of pathogenicity ( 4 ) . however , if any symptomatic correlation is not present , then only the quantifiable parameters can be used to determine the presence of any abnormality . as this technique has become widely available , questions have been raised regarding the best parameter to use to measure esophageal acid exposure , the optimal thresholds to differentiate normal from abnormal and the influence of gender and age on the measurement ( 5 - 8 ) . most studies concerned with the standard values of esophageal ph monitoring have been done in western countries ( 5 , 6 , 8 - 10 ) . because the genetic and environmental factors are different between western and eastern countries , there might also be a difference in the degree of gastroesophageal reflux between them . actually , the reported ph standards have shown a considerable variability between western countries and even among regions of the same country ( 6 , 7 , 10 , 11 ) . establishing the normal values of esophageal ph monitoring for eastern countries different in many aspects from western countries would be of great benefit for better understanding of gastroesophageal reflux disease ( gerd ) and its proper treatment . there have been a few previous reports about the normal reflux parameters in eastern countries such as china ( 12 , 13 ) . therefore , the aim of this study was to establish the normal values for gastroesophageal acid exposure in healthy koreans . this study was performed at 7 university hospitals in korea from may to october 2007 . each volunteer was carefully interviewed and when appropriate , he or she underwent a physical examination and laboratory studies to exclude systemic disorders that might alter esophageal motility or predispose to gerd . volunteers were excluded if there was any history of heartburn , regurgitation , chest pain , dysphagia for solids or liquids , odynophagia or use of antacids , promotility drugs , histamine-2 receptor blockers or proton pump inhibitors . additionally , none of the volunteers had a history of esophageal or gastric surgery , diabetes mellitus , alcoholism , neurological disorders or collagen vascular disorders . the medication histories were closely reviewed and none of the volunteers were taking any drugs that would influence acid secretion or esophageal function at the time of the study . upper endoscopy was performed on all the volunteers to exclude hiatal hernia , reflux esophagitis or other organic diseases such as peptic ulcer . informed written consent was obtained from each subject prior to the study and this study was approved by the institutional review board at each university hospital . ambulatory 24-hr esophageal ph monitoring was performed immediately after standard esophageal manometry with using a single - use monocrystalline antimony ph probe ( zinetics 24 , medtronic inc . , all the electrodes were initially calibrated in buffer solution of ph 7 and then in buffer solution of ph 1 . the ph catheter was introduced transnasally into the stomach and then it was withdrawn back into the esophagus until the electrode was 5 cm above the proximal margin of the les . the subjects were encouraged to eat regular meals with restrictions for the intake of drink or food with a ph below 4 . all the subjects recorded their meal times ( start and end ) , body position ( supine and upright ) and any symptoms in a diary . the data were collected using a portable data logger ( digitrapper mark iii , synetics medical co. , stockholm , sweden ) with a sampling rate of 4 sec , and the data was then transferred to a computer for analysis with using polygram for windows ( release 2.04 , synetics medical co. ) . a decrease in ph below 4 , which was not induced by eating or drinking , was considered the beginning of a reflux episode , and the following rise to ph above 4 was considered to be the end of such an episode . all the tracings were inspected by one of the authors to confirm the computerized calculations and to assure the quality of the recordings . the six parameters assessed for gastroesophageal reflux were the total percentage of time the ph was < 4 , the percentage of time the ph was < 4 in the supine and upright positions , the number of episodes the ph was < 4 , the number of episodes the ph was < 4 for 5 min , the duration of the longest episode the ph was < 4 and the composite score ( 14 ) . to obtain the composite score , a scoring system based on the standard deviation of the mean of each of the six components was used ( 5 , 10 ) . the simplified formula for scoring each of the six components is : component score = ( patient value - mean / standard deviation ) + 1 then , the composite score was obtained by adding the scores calculated for each of the six components ( 5 , 10 ) . the normal values for each parameter were assessed by calculating the 95th percentile for the subject group . the age of 40 was used to dichotomize the subject sample because visual inspection of the data suggested it would maximize the probability of finding an age effect on the ph variables . the mann - whitney test and the kruskal - wallis test were used to assess the effect of age and gender on each esophageal ph parameter . statistical calculations were performed using the spss version 10.0 for windows software ( spss inc . , chicago , il , u.s.a . ) . this study was performed at 7 university hospitals in korea from may to october 2007 . each volunteer was carefully interviewed and when appropriate , he or she underwent a physical examination and laboratory studies to exclude systemic disorders that might alter esophageal motility or predispose to gerd . volunteers were excluded if there was any history of heartburn , regurgitation , chest pain , dysphagia for solids or liquids , odynophagia or use of antacids , promotility drugs , histamine-2 receptor blockers or proton pump inhibitors . additionally , none of the volunteers had a history of esophageal or gastric surgery , diabetes mellitus , alcoholism , neurological disorders or collagen vascular disorders . the medication histories were closely reviewed and none of the volunteers were taking any drugs that would influence acid secretion or esophageal function at the time of the study . upper endoscopy was performed on all the volunteers to exclude hiatal hernia , reflux esophagitis or other organic diseases such as peptic ulcer . informed written consent was obtained from each subject prior to the study and this study was approved by the institutional review board at each university hospital . ambulatory 24-hr esophageal ph monitoring was performed immediately after standard esophageal manometry with using a single - use monocrystalline antimony ph probe ( zinetics 24 , medtronic inc . , minneapolis , mn , u.s.a . ) . a cutaneous reference electrode placed on the upper chest was also used . all the electrodes were initially calibrated in buffer solution of ph 7 and then in buffer solution of ph 1 . the ph catheter was introduced transnasally into the stomach and then it was withdrawn back into the esophagus until the electrode was 5 cm above the proximal margin of the les . the subjects were encouraged to eat regular meals with restrictions for the intake of drink or food with a ph below 4 . all the subjects recorded their meal times ( start and end ) , body position ( supine and upright ) and any symptoms in a diary . the data were collected using a portable data logger ( digitrapper mark iii , synetics medical co. , stockholm , sweden ) with a sampling rate of 4 sec , and the data was then transferred to a computer for analysis with using polygram for windows ( release 2.04 , synetics medical co. ) . a decrease in ph below 4 , which was not induced by eating or drinking , was considered the beginning of a reflux episode , and the following rise to ph above 4 was considered to be the end of such an episode . all the tracings were inspected by one of the authors to confirm the computerized calculations and to assure the quality of the recordings . the six parameters assessed for gastroesophageal reflux were the total percentage of time the ph was < 4 , the percentage of time the ph was < 4 in the supine and upright positions , the number of episodes the ph was < 4 , the number of episodes the ph was < 4 for 5 min , the duration of the longest episode the ph was < 4 and the composite score ( 14 ) . to obtain the composite score , a scoring system based on the standard deviation of the mean of each of the six components was used ( 5 , 10 ) . the simplified formula for scoring each of the six components is : component score = ( patient value - mean / standard deviation ) + 1 then , the composite score was obtained by adding the scores calculated for each of the six components ( 5 , 10 ) . the data were expressed as median values ( range ) unless otherwise noted . the normal values for each parameter were assessed by calculating the 95th percentile for the subject group . the age of 40 was used to dichotomize the subject sample because visual inspection of the data suggested it would maximize the probability of finding an age effect on the ph variables . the mann - whitney test and the kruskal - wallis test were used to assess the effect of age and gender on each esophageal ph parameter . statistical calculations were performed using the spss version 10.0 for windows software ( spss inc . , chicago , il , u.s.a . ) . fifty healthy volunteers ( 24 males and 26 females ; mean age , 45 yr ; range , 19 - 66 yr ) were recruited for this study . table 1 summarized the demographic data of the 50 volunteers who constituted the study population . the values for the composite score and the six parameters used to express the esophageal acid exposure in the 50 healthy volunteers are shown in table 2 . the median and 95th percentile values for the percent time ph < 4 for the total monitoring period and the composite score were 1.1 , 4.3 , 4.7 , and 14.2 , respectively . there was no difference in all the ph parameters between male volunteers and female volunteers ( table 3 ) . in addition , there was no difference in all the ph parameters between young volunteers and old volunteers ( table 4 ) . the interaction of gender and age did not show any significant difference for all the ph parameters ( table 5 ) . the results of our multicenter study for the amount of physiologic gastroesophageal acid reflux in 50 healthy koreans can be summarized as follows : 1 ) gender does not independently influence the ph parameters ; and 2 ) age also does not influence the ph parameters . men are generally known to secrete more gastric acid than women and this is explained in part by the larger body mass of men ( 15 , 16 ) . in a previous report , male volunteers showed higher median values than women volunteers for all the ph parameters except for the percentage time ph < 4 in the supine position ( 6 ) . in another report , there was no difference in esophageal acid exposure between males and females when the results of 24-hr ph monitoring were expressed as the composite score ( 5 ) . they suggested that using the composite score to determine when a patient had increased esophageal exposure to gastric juice could eliminate the necessity of having separate normal values for men and women . in present study , there was no difference in all the ph parameters between males and females . in one previous prospective study about acid output , gender had no significant effect on the basal output of acid and it had only borderline significant effects on the peak acid output ( 17 ) . this fact could explain our results , even though differences of genetic and environmental factors between koreans and western populations do exist . in the present study , there was no reliable association between the esophageal ph parameters and age . schlesinger et al . first raised the possibility that increasing age had an effect on the esophageal ph parameters ( 7 ) . it was also reported that older subjects showed significantly higher values for the total and upright percentage of time of ph < 4 as well as the total number of reflux episodes > 5 min ( 18 ) . but in that study , the sample size was relatively small and older controls were obtained from a hospitalized veteran population , and this veteran population had a high prevalence of hypertension and diabetes mellitus . on the other hand , the results of a large population study on 110 healthy subjects did not show an independent effect of age on the ph parameters ( 6 ) . thus , for clinical purposes , it seems that age generally does not have an important effect on the physiologic parameters of acid reflux . first , in our study , all the healthy volunteers underwent upper endoscopy to exclude asymptomatic esophagitis and hiatal hernia . in two large western studies concerned with the normal ambulatory esophageal ph values ( 5 , 6 ) therefore , they could not exclude the subjects with asymptomatic esophagitis or hiatal hernia . in the present study , we excluded these subjects by performing endoscopy . second , the distribution of gender and age of the healthy volunteers in our study was uniform ( data not shown ) . first , the number of subjects in this study was not large ; this could raise the possibility of a type-2 error . however , to be included in the study , the volunteers were required not to have any clinical evidence of gerd and they were also without endoscopic documentation of reflux esophagitis and hiatal hernia . these facts reduced the number of subjects for the study , so as to render our healthy volunteers as being appropriate . second , although our study population was carefully screened to be healthy and asymptomatic and we excluded the subjects who had reflux esophagitis or hiatal hernia , many of the subjects might have h. pylori infection with or without chronic atrophic gastritis . there is a higher prevalence of h. pylori infection in koreans than in western populations ( 19 ) . mild h. pylori - associated chronic active superficial gastritis had little effect on gastric acid output , whereas severe chronic active gastritis was associated with a lower gastric acid output ( 17 ) . this fact could also explain the lower values of the reflux parameters in our study as compared to the western studies . finally , because this study was performed at 7 medical centers , there would be bias of the reflux parameters among each center . to lessen the bias , we defined a standard method of ambulatory 24-hr esophageal ph monitoring and then we started this study . in addition , the coefficient of variation for the reflux parameters among each center was less than 40% , except the percentage of time the ph was < 4 in the supine positions . the normal values of the ph parameters , as 95th percentiles in 3 previous western studies ( 5 , 6 , 10 ) and in our study , are shown in table 6 . almost all the parameters except the number of reflux episodes were lower in our study than those in the western studies . many factors , including the differences in the age distribution of the control subjects , the real population differences for gastroesophageal reflux and the different incidence of h. pylori infection , could have contributed to these results . in conclusion , our multi - center data were based on 50 healthy volunteers , and these data provides the normal values for esophageal ph monitoring in koreans . however , no database is perfect because of the well - known variability of acid exposure from day to day and the inherent problems with the ph probe ( i.e. , it may get buried in the esophageal mucosa and so miss reflux episodes ) ( 8 , 20 ) . on the basis of our results , these normal esophageal ph values can provide reference data for future clinical and research studies in korea .
human herpes virus 6 ( hhv-6 ) and human herpes virus 7 ( hhv-7 ) during childhood are really widespread ( 1 , 2 ) , and the same as other herpesviruses , they are latent infections throughout life . salivary glands act as major sites harbouring persistent hhv-6 infection when hhv-6 is frequently isolated from the saliva of healthy individuals ( 3 , 4 , 5 ) . however , some studies represented that hhv-7 , instead of hhv-6 , is isolated from saliva frequently ( 6 , 7 , 8 , 9 ) , contradict previous reports . tumours of the salivary glands are an important area in the field of oral and maxillofacial pathology . the annual incidence of salivary gland tumours shows in isfahan , iran is far greater ( 1.13% ) than the world incidence that is about 1 to 6.5 cases per 100,000 people ( 10 ) . 20% of human malignancies are due to persistent viral infections , and tobacco is the second major risk factor for human carcinomas . herpes viruses have recognized to be the reason of several malignant and benign oral lesions ( 11 ) . in the present study , to declare the major sites of persistent infection with hhv-6 and hhv-7 , the existence of hhv-6 and hhv-7 genomes in formalin - fixed paraffin embedded tissue samples of salivary gland tumours were examined through highly sensitive real time pcr method . this analytical , descriptive study was performed in 60 formalin - fixed paraffin embedded tissue samples of salivary gland tumours , 23 benign salivary gland tumours including pleomorphic adenoma . monomorphic adenoma and 37 malignant salivary gland tumours including mucoepidermiod carcinoma and adenoid salivary gland specimens were obtained from surgery or autopsy from adult patients with oral cancer , sialoadenitis , pleomorphic adenoma of the salivary glands , and other diseases . first , 5 - 10 m tissue sections of formalin fixed , paraffin wax embedded tissue blocks ( depending on tissue size ) were transferred into 1.5 ml eppendorf tubes for dna isolation . to avoid cross - contamination , a new , sterile , and disposable microtome blade was used immediately before cutting each block for cleaning purposes . subsequently , dna extraction was performed by using the procedures according to the manufacturer s protocol described in the high pure nucleic acid extraction kit ( roche , germany ) . first , the paraffin wax was dissolved in 300 l of citrisoly ( xylene substitute ) and was washed with ethanol to remove the citrisoly . then cell lysis buffer ( 200 l ) and 20 l proteinase k solutions ( 20 mg / ml ) after the solution had cooled down to room temperature , 200 l binding buffer was added and the high pure filter tube was combined with collection tube . they were centrifuged and the flow - through was discarded , then 500 l inhibitor removal buffer was added followed by centrifuging . in the next step , the dna was washed with 500 l washing buffer and eluted with the elution buffer . the absorbance of a sample of dna solution was measured for concentration at 260 nm using an ultraviolet spectrometer . the absorbance ratio at 260 and 280 nm ( a260/280 ) was used to evaluate dna purity . the procedure was carried out with some minor modifications , for instance , longer incubation times ( overnight , approximately 16 hours ) and doubling proteinase k concentration ( 12 ) . the real - time pcr mixture contained 50 mm kcl , 10 mm tris - hcl ( ph=8 . 25 m mgcl2 as a 1x reaction buffer , 200 mm of each dntp , 10 m of each of primers and 1 u taq polymerase ( roche , germany ) . real time pcr was performed with hhv-7 and hhv-6 primers including forward and reverse primer with labels probe for hhv-7 and hhv-6 ( table1 ) . the pcr cycling temperatures for hhv-7 were 2 min of incubation at 50 c then followed by 2 min at 95 c ; the samples were subjected to 45 cycle s for 20 sec at 95 c followed by 1 min at 60 c ( 13 ) . primer and probe for pcr . * herpes virus 6- forward primer ( hhv-6-f ) , herpes virus 6- reverses primer ( hhv-6-r ) ; herpes virus 7- forward primer ( hhv-7-f ) , herpes virus 7- reverses primer ( hhv-7-r ) ; herpes virus 6-probe ( hhv-6-p ) , herpes virus7-probe ( hhv-7-p ) . amplification hhv-6 was carried out in a 25 l volume reaction mixture by use 1,100 nm each primer , and 200 nm probe . the reaction mixtures were incubated at 95 c for 10 min , followed by 45 cycles of 95 c for 15 sec and 60 c for 1 min ( 14 ) . the relationship of two groups was analysed through mc - nemar test by spss software ( version 11 , chicago , il , usa ) . the persistent infection with hhv-6 and hhv-7 , the existence of hhv-6 and hhv-7 genomes in formalin - fixed paraffin embedded tissue samples of salivary gland tumors is shown in tables 2 , 3 4 and 5 . of the 60 paraffin blocks of malignant and benign neoplasms of both major and minor salivary gland with equal chances of presence of hhv-7 and hhv-6 in the samples were compared . out of the 60 samples , 18 were positive for both hhv-7 and hhv-6 while 25 were only positive for hhv-7 , 10 samples were positive for hhv-6 but negative for hhv-7 , and 7 samples were reported negative for both hhv-6 and hhv-7 . it can , therefore , be suggested that a relationship is likely to exist between these two viruses in salivary glands neoplasms . the hhv-6 and the hhv-7 have been identified as one of the most important oncogenic viruses capable of producing a number of oncogenic factors such as bcl2 , bcl10 . a number of studies have suggested the presence of numerous oncogenic zones in the genome of this virus . the role of this virus as an etiological agent in cancer requires further research for validation . di - luca at 1995 ( 7 ) , reported the presence of hhv-6 genome in 63% of their healthy salivary glands and only in 3% of their saliva samples . levy at 1997 ( 16 ) , proposed hhv-6 as the agent for certain neoplasms such as lymphoma , leukemia , and cervical carcinoma . this is while hhv-6 has been recognized to have the capacity to activate other hhvs such as ebv and cmv as well as papilloma viruses . zhou et al 2007 ( 17 ) , reported a relationship between ebv and hhv-6 infections and the histological progress of angio - immunoblastic t - cell lymphoma . chen and hudnall ( 18 ) , in 2006 examined 8 autopsy samples from all body parts including 4 males and 4 females in the presence of 8 types of herpes virus ( ebv , cmv , vzv , hsv-2 , hsv-1 , hhv-6 , hhv-7 , and hhv-8 ) and only ebv , hhv-6 , and hhv-7 in all their samples were found . based on these findings , we decided to investigate , for the first time , the presence of hhv-7 and hhv-6 in salivary gland neoplasms in order to determine the relationships , if any , between these viruses and the related salivary gland tumours . the hypothesis of no correlation between hhv-7 and hhv-6 genomes in salivary gland neoplasms was refuted using the mcnemar s statistical test .
dieter gallwitz was the first to discover a yeast - encoded member of the rab family in the 1980s , when dna sequencing was just beginning to be widely adopted and required significant effort . it was related to ras but could not complement the loss of the yeast ras1 and ras2 genes and thus likely played a different role ( schmitt et al . , 1986 ; see also segev et al . , 1988 ) . for his phd thesis with randy schekman , peter novick had isolated and described the sec genes responsible for secretion in yeast ( novick et al . , 1980 ) . after a postdoctoral fellowship on the yeast cytoskeleton with david botstein at mit , novick returned to the secretory pathway for his own lab 's research and focused on the sec gene products that are required for the delivery of material from the yeast golgi to the cell surface . the original screen for yeast mutants uncovered 10 genes that , when mutated , accumulated post - golgi transport vesicles at a nonpermissive temperature ( novick et al . sequencing of the sec4 gene revealed that it encoded a ras - related protein ( salminen et al . , 1987 ) that was present on secretory vesicles ( goud et al . , 1988 ) and was later shown to recruit the so - called exocyst tether ( guo et al . , 1999 ) to help deliver vesicles to the bud tip for fusion ( walch - solimena et al . , 1997 ) . this was the first hint that membrane traffic might require a ras - like gtpase . these exciting findings in yeast led several labs to begin the search for mammalian homologues , and armand tavitian and marino zerial and their colleagues began cloning these relatives . tavitian named them ras - like proteins from rat brain ( rabs ; touchot et al . , 1987 ; chavrier et al . , 1990a ) , in line with the three - letter nomenclature of other ras - like protein families ; the rabs were numbered in the random order in which their sequences were obtained ; ypt1 was the homologue of rab1 . eventually , it would become clear that yeast encode 11 rabs and human cells encode at least 63 ( colicelli , 2004 ) . most rabs are stably , c - terminally prenylated on one or ( more commonly ) two cysteine residues that permit their tight membrane association . prenylome - wide analysis later revealed that a single cell might contain at least 42 different rabs ( nguyen et al . , 2009 ) ; other rabs are expressed in a more tissue - specific manner . ( 1988 ) showed that ypt1 was on the golgi , in contrast to sec4 , which novick had shown was present on secretory vesicles . soon thereafter , zerial and coworkers raised specific antibodies to their newly discovered gene products and found that these proteins each localized to different membrane compartments ( chavrier et al . , 1990b ) . even more striking was their subsequent discovery , using live - cell microscopy of cells expressing green fluorescent protein ( gfp) and gfp - variant- tagged proteins , that a single compartment might harbor multiple rabs , each occupying a distinct microdomain of that compartment ( snnichsen et al . , 2000 ; see also barbero et al . , 2002 ) . the importance of these findings can not be overstated : this represented the first molecular distinction between membrane - bound compartments within the endocytic pathway and provided a framework for all future molecular analyses of receptor endocytosis and recycling or degradation . indeed , zerial and colleagues showed that , like sec4 , rab5 played a key role in membrane traffic , and its function is rate limiting for endosome fusion and endocytosis ( gorvel et al . , 1991 ) . he and his colleagues eventually reconstituted the entire process of early endosome fusion using exclusively rab5-binding proteins , snare ( soluble n - ethylmaleimide sensitive factor attachment protein receptor ) proteins and their priming factors , liposomes , and a factor ( yip3/pra1 ) to deliver prenyl rab5 onto the liposomes ( see later discussion ; ohya et al . , 2009 ) . yoshimi takai was studying the biochemistry of a small gtpase , smg p25 , which was later identified as rab3a . he discovered a rat brain cytosol protein that inhibited the ability of smg p35/rab3a to release gdp , the rate - limiting step in the gtpase cycle ( sasaki et al . , 1990 ) . he named this protein gdp - dissociation inhibitor ( gdi ) and showed that it could extract rab3a from membranes in vitro but only in the presence of gdp and not gtp ( araki et al . , 1990 ) . we now know that there are two gdis in humans and one in yeast ; these proteins have the capacity to bind to all rab gtpases , with strong preference for their gdp - bound states . at steady state , rabs distribute themselves about half on membranes and half in cytosol ( although this varies considerably between rab proteins ) ; all cytosolic rabs exist in complex with gdi . the crystal structure of yeast gdi bound to ypt1 confirmed that gdi binds to the so - called rab switch regions that report on the status of the bound nucleotide ; the so - called c - terminal hypervariable domain of rab proteins is draped along the surface , and the prenyl groups bind tightly at the bottom of gdi ( pylypenko et al . , 2006 ) . my lab was the first to report that gdi binds rab9 gtpase with 20 nm affinity ( shapiro and pfeffer , 1995 ) ; goody and coworkers confirmed this tight interaction for other rab proteins ( wu et al . , 2007 ) . with this in mind , we and zerial studied the delivery of rab proteins onto membranes and showed that purified complexes of rab proteins bound to gdi contain all of the information needed for accurate membrane delivery ( soldati et al . , rabs were delivered onto membranes in their gdp - bound forms and converted into their gtp - bound forms after a lag of 5 min in vitro ( soldati et al . , 1994 ; ullrich et al . , 1994 ) . because this delivery required protease - sensitive components on the surface of membranes , we postulated that it was catalyzed by a so - called gdi - displacement factor ( gdf ) that dissociates rab gdi complexes . we went on to show that a protein called yip3/pra1 can dissociate endosomal rabs and deliver them to membranes ( sivars et al . , 2003 ) ; the zerial lab 's complete reconstitution of rab5-mediated endosome fusion using purified components absolutely requires the presence of yip3/pra1 in membranes to accomplish rab5 membrane association , despite the presence of a rab5 gef , rabex 5 , in their reactions ( ohya et al . , 2009 ) . itzen , goody , and coworkers found a legionella protein , drra , that can relocalize rab1 from the early secretory pathway to the inner surface of the plasma membrane ( schoebel et al . , 2009 ) . drra is a rab1 gef that binds rab1 extremely tightly and was shown to thus bypass any need for a distinct gdf activity . no endogenous rab gefs bind their rab substrates as tightly as drra , and thus gdfs may still facilitate some rab delivery events . several labs artificially localized gefs to unusual membranes such as mitochondria and succeeded in moving rabs to those locations ( gerondopoulos et al . , 2012 ; blmer et al . , 2013 ; cabrera and ungermann , 2013 ) . the most important conclusion from such experiments is that gdi is promiscuous in delivering rabs to membranes ; gdi is also likely to be able to correct mistakes it might make in delivery of rabs to membranes where the cognate gef is not present and the rab is , with gdp bound . most characterized rab gefs are not membrane anchored ; they are cytosolic proteins that can associate with other rab effectors to activate locally specific rab proteins . thus one needs rab domains to maintain rab domains , and , in cells , rabs usually show precise localizations ( yoshimura et al . , 2007 ) . these data suggest that rab proteins are delivered to membranes by gdi and , in some cases , might use the help of gdf factors . subsequent gef action is also clearly important to enable rabs to be stabilized at the site of their activation by loss of susceptibility to gdi extraction , together with active stabilization by subsequent , gtp - dependent effector binding . several studies showed that effector binding stabilizes rabs on membrane surfaces ( aivazian et al . , 2006 ) . the role of rab proteins is to recruit effectors such as motor proteins and tethering factors to facilitate downstream membrane traffic events . if you imagine that membrane flow should go from point a to point b in a pathway , you need a mechanism to template rabb after raba on different membrane compartments . zerial was the first to discover that effector proteins can simultaneously bind two adjacently acting rab gtpases , thereby linking their respective localizations and functions ( vitale et al . nature has taken this concept one step further , using an incredibly elegant system by which raba actually recruits to membrane surfaces the gef that will activate the next acting rab in the pathway ( figure 1a ) . thus , in yeast , ypt31 binds the sec 2 gef for the subsequent - acting sec4 rab protein . active rabs are then stabilized on membranes by binding to their cognate effector proteins . in this manner , a membrane patch containing one rab will template the establishment of a nearby domain containing the next rab . even better is the fact that the subsequent acting rabb also recruits a gap for raba to clear the membrane domain of the prior acting rab ( figure 1b ) . because rab gtpases each have distinct gefs and gaps , evolution has created a system by which the selectivity of each of these proteins coordinates to template an entire membrane - trafficking pathway . although several labs were obtaining clues to the existence of such interactions , it was peter novick who discovered these cascades and revealed their existence in living yeast cells ( ortiz et al . , 2002 ; rivera - molina and novick , 2009 ; see also , for example , nottingham et al . , 2011 ; pusapati et al . , 2012 ; suda et al . , 2013 ; rana et al . , this represented a spectacular discovery because it provided the first molecular clues about how cells create and maintain polarized secretory and endocytic pathways . rab cascade model for the establishment and maintenance of the polarity of the secretory and endocytic pathways . ( a ) in the first scenario , a rab gef specific for rab a generates active rab a. that rab recruits a second gef , which activates rab b. similarly , rab b recruits a gef to activate rab c. in this model , a membrane could have rabs a c intermingled or at least on a single compartment . ( b ) here gap proteins are included to remove a previous - acting rab from a specific membrane domain . data of rivera - molina and novick ( 2009 ) support the model in b. reprinted from nottingham and pfeffer ( 2009 ) . a special topics subgroup at the 2016 american society for cell biology meeting discussed a number of surprises that await further analysis . we still do not know the localizations or roles of many human rab proteins , nor do we know all of their cognate gef or gap identities or partner effector proteins . rabs need to be studied in their correct tissue or cell type and at endogenous expression levels . this represents many proteins yet to be discovered because a given rab may have 30 specific effector proteins ( christoforidis et al . , 1999 ) . in addition , gefs and gaps can be more promiscuous in vitro than in vivo , and so great care must be taken in all of these analyses . are cascades the answer ? we need to connect all of the rab gefs and gaps to determine the order of membrane - trafficking events , and we need to test the consequences of altering cascade specificity to verify the predictions of this satisfying model in the broadest physiological sense . finally , we need to reconstitute a rab cascade de novo to fully understand how cells establish and maintain secretory and endocytic pathways . recently rab proteins have been shown to be the main phosphorylation substrates for the lrrk2 kinase that is implicated in parkinson 's disease ( steger et al . , 2016 ) . what is the physiological consequence of phosphorylation , and how is this modification normally regulated ? transforming growth factor-activated kinase 1 ( tak1 ) also phosphorylates gdp - bearing rab1 and blocks gdi interaction but not gef interaction ( levin et al . , 2016 ) . this presumably stabilizes active rab1 on membranes in a totally unanticipated manner that activates membrane trafficking . pathogens modify rabs in many unexpected ways , including by ampylation and phosphocholination ( mller et al . , 2010 ; do these posttranslational modifications also occur in uninfected cells , and how ( and why ) are these events regulated ? finally , rab gtpases also interact with other small gtpases via shared effector proteins , and their joint coordination is only beginning to be explored . rab gtpases remain central regulators of membrane - trafficking pathways in healthy cells and disease . their continued study promises to offer many more important surprises for those who study them .
pediatric health care providers use weight to assess normal growth and development and guide nearly all therapeutic and medical interventions that their patients require . as such , significant attention has been paid to weight estimation in settings where the use of a scale is impractical or unavailable . in fact , nearly 2 dozen weight estimation strategies have been devised to assist with pediatric weight estimation . one of every 847 children in the united states is born with down syndrome , and virtually all require medical care throughout their lives . importantly , children with down syndrome demonstrate differences in height and weight for age when compared with neurotypical children . these anatomic differences should influence the accuracy of different weight estimation methods to varying degrees depending on the variables incorporated into those strategies . to date , not a single study has evaluated the performance of weight estimation methods in children with down syndrome . this study was designed to evaluate the predictive performance of 4 representative weight estimation strategies in this special population . all children presenting to the research center were enrolled unless : ( a ) there were limb deformities , ( b ) they were unable to be positioned for height / length measurements , or ( c ) the parents and/or children were unwilling to provide permission and assent for participation . children were enrolled with informed permission , and assent where appropriate ( ie , > 7 years of age ) , under a protocol that was reviewed and approved by the children s mercy hospital institutional review board . anthropometric measurements required for application of the selected weight estimation strategies were obtained on each child . these included height , weight , humeral length , and mid - upper arm circumference . children who were able to stand unassisted were positioned against the height rule of a portable stadiometer to obtain their height . humeral length was measured from the upper edge of the posterior border of the acromion process , down the posterior surface of the arm , to the tip of the olecranon process using a standard vinyl tape measure . mid - upper arm circumference ( muac ) was measured at the midpoint of the humerus with the arm hanging down at the child s side . length and weight measurements were recorded to the nearest millimeter and tenth of a kilogram , respectively , with the exception of infants where weight was recorded to the nearest gram . all raters obtaining measurements were required to undergo a quality control assessment prior to their involvement with the study with intrarater reliability required to be less than 5% for each anthropometric measure . the anthropometric data were applied to 4 representative weight estimation strategies : one based on age ( advanced pediatric life support [ apls ] ) , a second based on length ( broselow ) , a third based on habitus ( cattermole ) , and a fourth based on both length and habitus ( mercy ) . data on age were applied to the revised apls equations where weight was estimated in children 1 to 5 years of age according to [ 2 ( age in years + 4 ) ] and children 6 to 12 years of age according to [ ( 3 age in years ) + 7 ] . the broselow tape ( 2007 edition b ) was used to generate a weight estimate based on the child s length . data on muac were applied to the cattermole equation [ ( muac in cm 10 ) * 3 ] for the range of ages defined by the author ( ie , 6 - 11 years ) . finally , data on muac and humeral length were applied to the mercy method as previously published . given that age may be unavailable at the time of weight estimation ( eg , in a trauma setting ) , the methods were initially applied to those children who fell within the bounds of the method , as defined in the literature , and then separately to all children to explore the impact of extrapolation beyond the bounds of each method . the difference between predicted and actual weight was used to determine residual error ( re ) . percentage error ( pe ) was calculated by dividing the actual weight into the re and multiplying by 100 . root mean square error ( rmse ) was calculated by taking the square root of the average squared error . accuracy was assessed by evaluating the percentage of estimated weights that fell within 20% of actual weight . differences in re , pe , and percentage within 20% of actual between methods was determined by analysis of variance ( anova ) . altman plots using log - transformed data were constructed to evaluate agreement between the various weight estimation methods and the observed weight and the 95% limits of agreement calculated accordingly . agreement was also assessed by calculating the intraclass correlation coefficient ( icc ) using a 2-way random effects model and an absolute agreement definition . statistical analyses were performed for the methods as published ( ie , with only those children who satisfied the criteria for that method ) ; however , graphical presentations depict the method applied with and without restrictions . all children presenting to the research center were enrolled unless : ( a ) there were limb deformities , ( b ) they were unable to be positioned for height / length measurements , or ( c ) the parents and/or children were unwilling to provide permission and assent for participation . children were enrolled with informed permission , and assent where appropriate ( ie , > 7 years of age ) , under a protocol that was reviewed and approved by the children s mercy hospital institutional review board . anthropometric measurements required for application of the selected weight estimation strategies were obtained on each child . these included height , weight , humeral length , and mid - upper arm circumference . children who were able to stand unassisted were positioned against the height rule of a portable stadiometer to obtain their height . humeral length was measured from the upper edge of the posterior border of the acromion process , down the posterior surface of the arm , to the tip of the olecranon process using a standard vinyl tape measure . mid - upper arm circumference ( muac ) was measured at the midpoint of the humerus with the arm hanging down at the child s side . length and weight measurements were recorded to the nearest millimeter and tenth of a kilogram , respectively , with the exception of infants where weight was recorded to the nearest gram . all raters obtaining measurements were required to undergo a quality control assessment prior to their involvement with the study with intrarater reliability required to be less than 5% for each anthropometric measure . the anthropometric data were applied to 4 representative weight estimation strategies : one based on age ( advanced pediatric life support [ apls ] ) , a second based on length ( broselow ) , a third based on habitus ( cattermole ) , and a fourth based on both length and habitus ( mercy ) . data on age were applied to the revised apls equations where weight was estimated in children 1 to 5 years of age according to [ 2 ( age in years + 4 ) ] and children 6 to 12 years of age according to [ ( 3 age in years ) + 7 ] . the broselow tape ( 2007 edition b ) was used to generate a weight estimate based on the child s length . data on muac were applied to the cattermole equation [ ( muac in cm 10 ) * 3 ] for the range of ages defined by the author ( ie , 6 - 11 years ) . finally , data on muac and humeral length were applied to the mercy method as previously published . given that age may be unavailable at the time of weight estimation ( eg , in a trauma setting ) , the methods were initially applied to those children who fell within the bounds of the method , as defined in the literature , and then separately to all children to explore the impact of extrapolation beyond the bounds of each method . the difference between predicted and actual weight was used to determine residual error ( re ) . percentage error ( pe ) was calculated by dividing the actual weight into the re and multiplying by 100 . root mean square error ( rmse ) was calculated by taking the square root of the average squared error . accuracy was assessed by evaluating the percentage of estimated weights that fell within 20% of actual weight . differences in re , pe , and percentage within 20% of actual between methods was determined by analysis of variance ( anova ) . altman plots using log - transformed data were constructed to evaluate agreement between the various weight estimation methods and the observed weight and the 95% limits of agreement calculated accordingly . agreement was also assessed by calculating the intraclass correlation coefficient ( icc ) using a 2-way random effects model and an absolute agreement definition . statistical analyses were performed for the methods as published ( ie , with only those children who satisfied the criteria for that method ) ; however , graphical presentations depict the method applied with and without restrictions . participants were evenly divided between the genders ( 51% male ) although they were more heavily distributed throughout the younger age brackets . their corresponding age , height , and weight distributions are detailed in figure 1 . body mass index ( bmi ) percentiles , as classified by the centers for disease control , favored children who were normal ( 41% ) , followed by obese ( 21% ) , overweight ( 15% ) , and underweight ( 1% ) . the remaining children ( 22.6% ) fell into the infant category and ranged from < 3rd to > 97th percentile in weight - for - height . as published , the mercy method could be applied to 99% of the enrolled children followed by 94% for broselow , 78% for apls , and 31% for cattermole ( table 1 ) . these rates would be expected to drop markedly for apls , broselow , and cattermole if enrollment had been balanced to include older children . histograms depicting the distribution of age , weight , and height among the study participants . the age - based ( apls ) and length - based plus habitus - based ( mercy ) methods showed the smallest degree of bias in children with down syndrome as reflected by the average re and average pe ( table 1 and figure 2 ) . the length - based method ( broselow ) demonstrated a tendency to underestimate weight , whereas the habits - based method ( cattermole ) overestimated weight ( table 1 and figure 2 ) . broselow suffered the most extreme underestimation ( 63% ) , whereas apls suffered the greatest degree of overestimation ( 107% ; table 1 ) . only mercy reflected balance with respect to the degree of overestimation and underestimation ( table 1 ) . altman plots depicting the log - transformed difference between predicted weight and actual weight versus average log weight . the black symbols represent participants who fell within the published criteria for the method , and the grey symbols reflect values for participants who exceed the bounds of the published methods . there were also significant differences in accuracy between all methods ( p < .01 ) . mercy demonstrated the highest icc ( 0.987 vs 0.867 - 0.885 ) and predicted weight within 20% of actual in a greater proportion of children ( 88% vs 40% to 76% ) when compared with the other methods ( table 1 ) . performance of the cattermole , and to a lesser extent broselow , suffered to a greater extent when applied beyond the bounds defined in the literature ( figure 2 ) . performance statistics remained unchanged for apls and mercy when extended beyond their published criteria ; however , it is unclear whether this finding would persist for apls were older children adequately represented . the availability of weight estimation tools in settings where there is no opportunity to obtain a child s weight can be critical for the immediate medical management of children . accurate weight estimation tools are also valuable for routine care in populations where obtaining a scale - based weight can be challenging . this may include children affixed to medical equipment ( eg , ventilators ) , children immobilized in casts , postsurgical children who can not be easily moved , and children with intellectual or cognitive disabilities who may be excessively anxious or uncooperative with weight assessment using a standard scale . there are established differences in stature for children with down syndrome and a higher incidence of developmental disabilities that pose a challenge to weight assessment . yet ours is the first study to examine whether existing weight estimation methods are valid for use in this special population . we chose to evaluate 4 weight estimation strategies , each relying on a different anthropometric or demographic surrogate . those strategies that failed to account for body length ( eg , apls , cattermole ) fared worse than those that incorporated some measure of length ( eg , broselow , mercy ) . not surprisingly , the method that incorporated 2 variables ( eg , mercy ) displayed the best overall performance characteristics relative to methods that incorporated a single variable . irrespective of their basis , each weight estimation method demonstrated poorer performance characteristics in children with down syndrome than reported for unaffected children . this finding would suggest that the predictive performance of these methods can be optimized by incorporating data that account for the growth patterns unique to children with down syndrome . at present , the mercy tape appears to offer the best option for weight estimation in children with down syndrome . additional anthropometric data collection should allow investigators to refine existing weight estimation strategies for use in children with down syndrome .
during the shock state , understanding the mechanisms of acidosis , its hemodynamic and inflammatory consequences as well as the best therapeutic approach remain an ongoing debate . in the previous issue , schotola and colleagues provide , for the first time , new and en - lightening data on the consequence of acidosis in isolated human failing myocardium . over the past two decades , the mortality rate from a shock state has decreased ; for instance , in septic shock from 30% to around 20% . this improvement stems from many factors , including early management , more aggressive resuscitation with specific goals ( volemic expansion , targeted main arterial pressure , blood transfusion , and so forth ) and a better identification of prognostic factors such as acidosis . as a major metabolic consequence of shock states , acidosis is also known to be a high predictor of in hospital mortality . both a cause and a consequence , this acidosis impairs cardiac and vascular function through various cellular and molecular mechanisms . intracellular acidosis , which reduces myofilament sensitivity to ca , is thus one of the crucial factors involved in myocardial dysfunction . a low ph value also induces vessel hyporeactivity mediated by a number of factors such as nitric oxide , peroxynitrite , activation of katp channels and modification of catecholamine signaling . although it has long been known that cardiovascular function is impaired by acidosis , the bulk of the studies have been mostly experimental , and only a few human studies have been relevant on this subject [ 8 - 10 ] . in the present study by schotola and colleagues , these trabeculae were mounted in a chamber in which the development of isometric force was recorded and the tissues superfused with a hepes solution at ph 7.20 and 7.40 . the response to incremental doses of isoproterenol , a b mimetic agent , was also recorded . their major finding was a reduction in cardiac contractility at ph 7.20 and a rightward shift of the ph 7.20 curve compared with the 7.40 curve in response to increasing doses of isoproterenol . therefore , without providing actual new pathophysiological information regarding cardiac depression in acidotic patients , this study definitely confirms the deleterious effects of a moderate and common acidosis on the failing human heart . obviously , these experimental conditions ( that is , ex vivo fragments of myocardium , artificial organic acidosis ) are far from the clinical situation furthermore , given that the hemodynamic consequences of common mild acidosis on altered myocardium are important , physicians should be alarmed in the presence of severe acidosis . in shock patients , hemodynamic treatment in order to treat acidosis should be a primary goal for the physician . finally , it would be of interest , albeit ethically complex , to determine whether this mild acidosis has the same impact not only on preserved human myocardium but also on both preserved and altered arterial vessels . the main treatment must involve the correction of the underlying disease at the origin of acidosis . however , buffering severe acidosis by sodium bicarbonate is known to be associated with severe deleterious effects . bollaert and colleagues demonstrated that sodium bicarbonate infusion in acidotic rats decreases the intracellular ph . moreover , sodium bicarbonate infusion increases carbon dioxide , which is highly diffusible in cells ( hco3 + h<= > h2co3 and h2co3 < = > co2 + h2o ) . carbon dioxide increases dramatically and exacerbates cardiovascular dysfunction . another deleterious effect is that hypocalcemia is worsened by bicarbonate infusion , which probably also impairs cardiovascular function . a final example is hydric and sodium overload , which often occurs during sodium bicarbonate infusion . two small randomized , controlled clinical studies describe the inefficiency of this treatment in restoring the hemodynamic balance in critically ill patients . other treatments such as carbicarb , an equimolar solution of sodium bicarbonate and sodium carbonate , or tham ( trometamol ; tris - hydroxymethyl aminomethane ) , a biologically inert amino alcohol of low toxicity that buffers carbon dioxide and acids in vitro and in vivo , have also been studied for the treatment of metabolic or respiratory acidosis , although not in shock state patients . for all of these reasons , in fact , the surviving sepsis campaign guidelines only recommend against its use for ph 7.15 . currently , there are no relevant published studies regarding the effect of bicarbonate therapy for ph < 7.15 . nevertheless , while the underlying disease remains the main course of treatment , there is an urgent need to research a new symptomatic approach to acidosis management .
the identification and characterization of circulating tumor cells ( ctcs ) in the blood of cancer patients can help to diagnose cancer more accurately . epithelial cell adhesion molecule ( epcam)-based platforms are usually used to capture and identify ctcs [ 13 ] . ctcs can be successfully characterized by fluorescence in situ hybridization ( fish ) following enrichment using the epcam - positive selection platforms . however , as ctcs undergoing epithelial - mesenchymal transition frequently lose the expression of epcam , technologies targeting only epcam - positive cells may underestimate the actual number of ctcs . once ctcs are enumerated , they have to be properly characterized to ensure the sensitivity and reliability of the ctc enumeration platform used . we have previously developed a highly sensitive method for detecting ctcs that employs size - based filtration and epcam immunofluorescence staining . ctcs of epithelial origin were distinguished from blood cells as all 4,6-diamidino-2-phenylindole ( dapi)-positive , leukocyte common antigen ( cd45)-negative , and epcam - positive cells . to characterize ctcs independently of cell surface protein expression , we incorporated a chromosomal fish assay to detect abnormal copy numbers of chromosomes in cells collected from peripheral blood samples using the size - based filtration platform . abnormal copy numbers of chromosomes were detected not only in tumor cells , but also in certain benign cells , thus highlighting the limitations of the chromosomal fish assay for cancer diagnosis . five patients with non - small cell lung cancer and 2 patients with benign lung diseases were enrolled in this study , and no patient was excluded . a 10-ml blood draw was collected from the patients and processed on a size - based filtration platform . the collected cells were divided and loaded onto 2 microscopic slides . on 1 slide , half of the collected cells were immunofluorescently stained with anti - human cd45 monoclonal antibody ( mab ) , anti - human epcam mab , and dapi to check for epcam+/cd45/dapi+ cells . the remaining cells on the other slide were stained with 4-color fluorescent - conjugated oligo - fish probes specific to chromosomes 1 ( aqua ) , 7 ( green ) , 8 ( gold ) , and 20 ( red ) . the patient characteristics , epcam+/cd45/dapi+ cell counts , and aneuploid cell counts of the collected cells are summarized in table 1 . unexpectedly , aneuploid cells were also detected in the peripheral blood of patients with benign lung diseases , such as empyema necessitatis and non - tuberculous mycobacterial lung disease , which served as negative controls . for better identification of ctcs in patients with lung cancer , we performed both immunofluorescent staining of epcam+/cd45/dapi+ cells and chromosomal fish assays on the cells collected from the size - based filtration platform . unexpectedly , aneuploid cells were also detected in the peripheral blood of patients with benign lung diseases , such as empyema necessitatis and non - tuberculous mycobacterial lung disease . furthermore , the enumeration of aneuploid cells did not correlate with the immunofluorescence staining results . pantel et al . have shown that circulating epithelial cells in patients with benign colon diseases were detected as false positives using both cellsearch and epithelial immunospot assays . positive events were detected most frequently in patients with benign colon diseases , such as diverticulosis and crohn disease . it has high sensitivity ( > 90% ) and specificity ( 100% ) , and it also has a low false - negative rate ( < 10% ) and false - positive rate ( 0% ) . thus , fish assays pose little potential for false positives in patients with benign lung disease . the link between aneuploidy and inflammatory diseases has been highlighted by numerous studies , indicating that aneuploidy is not a unique feature of cancer . according to tsai et al . , the copy numbers of chromosomes in mouse embryo cells infected with mycoplasma were observed to change with each passage , indicating that mycoplasma infection can induce aneuploidy in infected cells . in addition , lay et al . have shown that a high number of nuclei were cells with aneuploidy induced by mycobacterium tuberculosis infection . furthermore , granulomas in inflamed tissues have been observed to form giant cells that block mitotic cell division that would inhibit the inflammatory response . we report , for the first time , evidence that aneuploid cells can also be detected in the blood samples of patients with benign disease . , comparisons should be performed on peripheral blood and tumor tissue samples from patients with lung cancer as well as patients with infectious diseases to determine the actual origin of the aneuploid cells . our platform can detect ctcs , but the collected cells have to be characterized using other techniques to increase the accuracy of cancer detection . have reported that fish is agood prognostic marker for detecting ctcs , comparable to epcam - positive selection platforms in urothelial cancer patients . counts of ctcs collected by either density gradient centrifugation or filtration from the blood of prostate , ovarian , and colorectal cancer patients are correlated with the aneuploid cell counts from fish in the same patients . in our study , positive chromosomal abnormalities were not detected in healthy donor samples ( fig . 1 ) , but they were detected in both patients with cancer and patients with benign infectious diseases . our report thus suggests the risk of false positives and raises concerns regarding the reliability of the fish assay for cancer diagnosis . in conclusion , we evaluated epcam+/cd45/dapi+ cells and aneuploid cells from the cells collected by a size - based filtration platform . aneuploid cells were detected in the peripheral blood of patients with lung cancer and , unexpectedly , in the peripheral blood samples of patients with benign lung diseases , such as empyema necessitatis and non - tuberculous mycobacterial lung disease , which served as the negative control group . we conclude that chromosomal abnormalities can also be detected in patients with benign infectious diseases of the lung , thus indicating that they are not a unique property of tumor cells .
the venous system contains 70% of the cerebral blood volume and is of importance for normal cerebral circulation . although cerebral venous thrombosis ( cvt ) only accounts for approximately 1% of all stroke cases in addition , cvt is usually noted in young individuals , including children . of all cvt cases , however , the amount of research on cvt has long been far less than that on cerebral artery thrombosis . recently , a statement by the american heart association ( aha ) and the american stroke association ( asa ) , regarding the diagnosis and management of cvt , emphasized for the first time that more attention needs to be paid to cvt . cerebral venous thrombosis may cause serious neurological syndromes such as intracranial hypertension , seizures , motor defects , sensory loss , and loss of consciousness . after a first cvt , there is an increased risk of further venous thromboembolic events ( vte ) , and 213% of all patients suffer cvt recurrence . however , very few studies have been done to assess cvt recurrence and determine predisposing risk factors for recurrence . although cvt can be caused by a hypercoagulable condition ( acquired or hereditary thrombophilias ) , an inflammatory state , or collagen vascular diseases , in 30% of cases no underlying etiology can be identified . identification of risk factors for recurrence after a first cvt would help to more accurately determine the best duration for anticoagulation treatment and for appropriate precautionary measures . in clinical practice , the authors have noticed that patients with cvt tended to have baseline high fibrinogen and/or low high - density lipoprotein cholesterol ( hdl - c ) levels . high fibrinogen or low hdl - c levels can increase arterial thrombosis and cardiovascular disease ; hyperfibrinogenemia and low hdl - c levels may correlate with thrombophilia , but it is still unclear whether these two factors can increase the likelihood of recurrent cvt . therefore , we hypothesized that there is some association between baseline elevated fibrinogen or lowered hdl - c levels , or both in combination , with the recurrence of cvt . in this study , the clinical profiles and laboratory testing parameters at baseline ( including fibrinogen and hdl - c ) of patients with a first cvt were analyzed to ascertain this correlation , in order to explore potential cvt risk factors . all patients with a first episode of cvt admitted to xuan wu hospital , capital medical university , a tertiary care academic medical center , from august 2005 to september 2009 , were included in this study . patients were identified through a computerized search of discharge diagnosis codes ( icd-10 : g08.x06 , g08.x09 , g08.x10 , 167.651 , 167.652 ) in the hospital discharge database . diagnosis had been confirmed by cerebral digital angiography , or magnetic resonance imaging ( mri ) plus magnetic resonance venography ( mrv ) in all patients . exclusion criteria included medication for lipid - lowering or contraception , pregnancy , postpartum state , dehydration , trauma , heart failure , hepatic and renal diseases , or malignancy . the data related to recurrence were obtained partly by reviewing clinical histories of patients who had revisited xuan wu hospital as outpatients or who were readmitted to the hospital as inpatients , and those of others from a telephone survey . the endpoints of the study were recurrent symptomatic cvt , death from cvt , or end of the study ( september 2010 ) . recurrent symptomatic cvt refers to the development of new neurological symptoms , with a new cerebral venous or sinus occlusion on repeated mri tests , combined with mrv or cerebral digital angiography . follow - up studies were conducted from discharge to september 2010 . if the patient had multiple recurrent cvt , demographic and clinical variables were collected in a standardized file comprising clinical characteristics ( age , gender , body weight , systolic blood pressure , diastolic blood pressure , history of smoking , hypertension , diabetes mellitus , cardiac disease , previous venous thrombosis , and the duration of anticoagulant therapy ) , and laboratory parameters ( white blood cell count , red blood cell count , blood platelet count , hemoglobin , plasma fibrinogen , fasting blood glucose , serum total cholesterol , low - density lipoprotein cholesterol [ ldl - c ] , hdl - c , triglycerides , apolipoprotein a1 , and apolipoprotein b ) . all of the above testing parameters were determined based on the results of blood samples taken on day 1 of hospitalization , and all analyses were performed in our hospital laboratory . plasma fibrinogen levels were measured using the sta automate ( de - sta - co , paris , france ) . serum total cholesterol , ldl - c , hdl - c , triglycerides , apolipoprotein a1 , and apolipoprotein b were analyzed with an automatic biochemical analyzer ( hitachi 7170 , tokyo , japan ) . warfarin was used to prevent cvt recurrence ( target international normalized ratio , 2.03.0 ) . basic demographic data and biochemical characteristics of patients were expressed as mean standard deviation . factors that predicted time to total recurrent events were identified by univariate cox regression analyses . the multivariate cox proportional hazards model was used to evaluate the independent contribution of the risks to cvt recurrence , and to adjust for potential confounders . the risk factors , including those variables that had a p < 0.05 upon univariate cox proportional hazard analysis , and some confounding factors , such as gender , venous thrombosis history , and duration of anticoagulation treatment , were introduced in a multivariate cox regression model . all statistical analysis was performed with the use of spss version 16.0 software ( spss , inc . all patients with a first episode of cvt admitted to xuan wu hospital , capital medical university , a tertiary care academic medical center , from august 2005 to september 2009 , were included in this study . patients were identified through a computerized search of discharge diagnosis codes ( icd-10 : g08.x06 , g08.x09 , g08.x10 , 167.651 , 167.652 ) in the hospital discharge database . diagnosis had been confirmed by cerebral digital angiography , or magnetic resonance imaging ( mri ) plus magnetic resonance venography ( mrv ) in all patients . exclusion criteria included medication for lipid - lowering or contraception , pregnancy , postpartum state , dehydration , trauma , heart failure , hepatic and renal diseases , or malignancy . the data related to recurrence were obtained partly by reviewing clinical histories of patients who had revisited xuan wu hospital as outpatients or who were readmitted to the hospital as inpatients , and those of others from a telephone survey . the endpoints of the study were recurrent symptomatic cvt , death from cvt , or end of the study ( september 2010 ) . recurrent symptomatic cvt refers to the development of new neurological symptoms , with a new cerebral venous or sinus occlusion on repeated mri tests , combined with mrv or cerebral digital angiography . follow - up studies were conducted from discharge to september 2010 . if the patient had multiple recurrent cvt , demographic and clinical variables were collected in a standardized file comprising clinical characteristics ( age , gender , body weight , systolic blood pressure , diastolic blood pressure , history of smoking , hypertension , diabetes mellitus , cardiac disease , previous venous thrombosis , and the duration of anticoagulant therapy ) , and laboratory parameters ( white blood cell count , red blood cell count , blood platelet count , hemoglobin , plasma fibrinogen , fasting blood glucose , serum total cholesterol , low - density lipoprotein cholesterol [ ldl - c ] , hdl - c , triglycerides , apolipoprotein a1 , and apolipoprotein b ) . all of the above testing parameters were determined based on the results of blood samples taken on day 1 of hospitalization , and all analyses were performed in our hospital laboratory . plasma fibrinogen levels were measured using the sta automate ( de - sta - co , paris , france ) . serum total cholesterol , ldl - c , hdl - c , triglycerides , apolipoprotein a1 , and apolipoprotein b were analyzed with an automatic biochemical analyzer ( hitachi 7170 , tokyo , japan ) . warfarin was used to prevent cvt recurrence ( target international normalized ratio , 2.03.0 ) . basic demographic data and biochemical characteristics of patients were expressed as mean standard deviation . factors that predicted time to total recurrent events were identified by univariate cox regression analyses . the multivariate cox proportional hazards model was used to evaluate the independent contribution of the risks to cvt recurrence , and to adjust for potential confounders . the risk factors , including those variables that had a p < 0.05 upon univariate cox proportional hazard analysis , and some confounding factors , such as gender , venous thrombosis history , and duration of anticoagulation treatment , were introduced in a multivariate cox regression model . all statistical analysis was performed with the use of spss version 16.0 software ( spss , inc . two patients died of other diseases ( trauma or cardiac disease ) during the follow - up period . an additional four patients were lost to follow - up , and two refused to participate in the study . the remaining 95 patients were eligible for the study , and their baseline demographic data and biochemical parameters are presented in table 1 . of these 95 patients , 47 were men and 48 were women , with a median age of 35.3 years ( range : 1569 years ) . the duration of disease was 2 days to 1.3 years , with 13.7% ( 13/95 ) within 1-week after onset and hospitalization . follow - up was conducted until september 30 , 2010 ( analysis cut - off date ) with a duration ranging between 1 and 61 months ( median of 27 months ) . baseline characteristics of patients with first cvt and univariate cox regression analysis of factors affecting recurrence * percentage calculated on the total number of patients ; p<0.05 indicates statistical significance . pvt : previous venous thrombosis ; ldl - c : low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . at the end of the follow - up period , of the recurrence rate of cvt was 2.76 per 100 patient - years ( 95% ci : 1.035.21 ) . elevated fibrinogen level was present in 41.1% of patients and lowered hdl - c level in 36.8% [ table 2 ] in the study . the risk factors affecting recurrence on univariate cox proportional hazards analysis are shown in table 2 . cvt patients with a smoking habit had a higher recurrence rate ( hazard ratio [ hr ] : 3.84 ; 95% ci : 1.2512.08 ; p < 0.05 ) . specifically , high fibrinogen or low hdl - c alone were not factors associated with recurrence ( p > 0.05 ) . interestingly , only when high fibrinogen and low hdl - c occurred together did patients have a higher recurrence risk of cvt ( hr : 4.93 ; 95% ci : 1.2719.07 ; p < 0.05 ) . univariate cox regression analysis of predictors for recurrence after first cvt according to the main clinical parameters * percentage calculated on the total number of patients ; p<0.05 . hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . multivariate cox regression analysis [ table 3 ] was used to search for possible independent predictive risk factors for the recurrence of cvt , including smoking and the coexistence of high fibrinogen and low hdl - c , and confounding factors such as gender , venous thrombosis history , and duration of anticoagulation . surprisingly , the result was that coexisting high fibrinogen and low hdl - c levels is the only independent predictive variable for recurrent cvt ( hr : 4.69 ; 95% ci : 1.1020.11 ; p < 0.05 ) . the cumulative recurrent risk affected by coexisting high - level fibrinogen and low - level hdl - c was calculated using kaplan patients with both high fibrinogen and low hdl - c levels had a cumulative risk of recurrent cvt , compared to that in patients with both normal fibrinogen and normal hdl - c , high fibrinogen alone , or low hdl - c alone ( = 6.61 , p < 0.01 ; = 3.86 , p < 0.01 ; = 5.61 , p < 0.01 , respectively ) . multivariate cox proportional hazards regression analysis for cvt recurrence * p<0.05 . hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . meier analysis for cumulative risk of recurrent cvt ( log - rank test ) : the cumulative risk of recurrent cvt in patients with both high fibrinogen and low hdl - c was significant difference from patients with both normal fibrinogen and normal hdl - c . hdl - c : high - density lipoprotein cholesterol , cvt : cerebral venous thrombosis . within the group of patients with recurrent cvt , all 7 took warfarin for a period from 3 to 12 months , and 6 patients ( 6/7 , 85.7% ) had recurrent cvt after discontinuation of anticoagulant treatment . two other patients with recurrent cvt who took warfarin for more than 3 months had high fibrinogen with normal hdl - c levels , and normal fibrinogen with normal hdl - c levels , respectively . the first patient had recurrent cvt during warfarin therapy , and the second had a cvt recurrence after stopping warfarin treatment . finally , two patients with normal fibrinogen and normal hdl - c levels experienced recurrence and had not taken any anticoagulant drugs . spearman 's correlation analysis was used to estimate the relationship between high fibrinogen and low hdl - c levels . the result showed that high fibrinogen levels were related to low hdl - c levels ( r = 0.205 , p < 0.05 ) . two patients died of other diseases ( trauma or cardiac disease ) during the follow - up period . an additional four patients were lost to follow - up , and two refused to participate in the study . the remaining 95 patients were eligible for the study , and their baseline demographic data and biochemical parameters are presented in table 1 . of these 95 patients , 47 were men and 48 were women , with a median age of 35.3 years ( range : 1569 years ) . the duration of disease was 2 days to 1.3 years , with 13.7% ( 13/95 ) within 1-week after onset and hospitalization . follow - up was conducted until september 30 , 2010 ( analysis cut - off date ) with a duration ranging between 1 and 61 months ( median of 27 months ) . baseline characteristics of patients with first cvt and univariate cox regression analysis of factors affecting recurrence * percentage calculated on the total number of patients ; p<0.05 indicates statistical significance . pvt : previous venous thrombosis ; ldl - c : low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . at the end of the follow - up period , of the 95 included patients , 12 ( 12.6% ) had recurrent cvt . the recurrence rate of cvt was 2.76 per 100 patient - years ( 95% ci : 1.035.21 ) . elevated fibrinogen level was present in 41.1% of patients and lowered hdl - c level in 36.8% [ table 2 ] in the study . the risk factors affecting recurrence on univariate cox proportional hazards analysis are shown in table 2 . cvt patients with a smoking habit had a higher recurrence rate ( hazard ratio [ hr ] : 3.84 ; 95% ci : 1.2512.08 ; p < 0.05 ) . specifically , high fibrinogen or low hdl - c alone were not factors associated with recurrence ( p > 0.05 ) . interestingly , only when high fibrinogen and low hdl - c occurred together did patients have a higher recurrence risk of cvt ( hr : 4.93 ; 95% ci : 1.2719.07 ; p < 0.05 ) . univariate cox regression analysis of predictors for recurrence after first cvt according to the main clinical parameters * percentage calculated on the total number of patients ; p<0.05 . hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . multivariate cox regression analysis [ table 3 ] was used to search for possible independent predictive risk factors for the recurrence of cvt , including smoking and the coexistence of high fibrinogen and low hdl - c , and confounding factors such as gender , venous thrombosis history , and duration of anticoagulation . surprisingly , the result was that coexisting high fibrinogen and low hdl - c levels is the only independent predictive variable for recurrent cvt ( hr : 4.69 ; 95% ci : 1.1020.11 ; p < 0.05 ) . the cumulative recurrent risk affected by coexisting high - level fibrinogen and low - level hdl - c was calculated using kaplan patients with both high fibrinogen and low hdl - c levels had a cumulative risk of recurrent cvt , compared to that in patients with both normal fibrinogen and normal hdl - c , high fibrinogen alone , or low hdl - c alone ( = 6.61 , p < 0.01 ; = 3.86 , p < 0.01 ; = 5.61 , p < 0.01 , respectively ) . hdl - c : high - density lipoprotein cholesterol ; cvt : cerebral venous thrombosis ; rr : relative risk ; ci : confidence interval . kaplan meier analysis for cumulative risk of recurrent cvt ( log - rank test ) : the cumulative risk of recurrent cvt in patients with both high fibrinogen and low hdl - c was significant difference from patients with both normal fibrinogen and normal hdl - c . hdl - c : high - density lipoprotein cholesterol , cvt : cerebral venous thrombosis . within the group of patients with recurrent cvt , 7 of 12 ( 58.3% ) had high fibrinogen and low hdl - c . all 7 took warfarin for a period from 3 to 12 months , and 6 patients ( 6/7 , 85.7% ) had recurrent cvt after discontinuation of anticoagulant treatment . two other patients with recurrent cvt who took warfarin for more than 3 months had high fibrinogen with normal hdl - c levels , and normal fibrinogen with normal hdl - c levels , respectively . the first patient had recurrent cvt during warfarin therapy , and the second had a cvt recurrence after stopping warfarin treatment . finally , two patients with normal fibrinogen and normal hdl - c levels experienced recurrence and had not taken any anticoagulant drugs . spearman 's correlation analysis was used to estimate the relationship between high fibrinogen and low hdl - c levels . the result showed that high fibrinogen levels were related to low hdl - c levels ( r = 0.205 , p < 0.05 ) . we found that the simultaneous presence of high fibrinogen and low hdl - c levels was a predictor of cvt recurrence compared to the presence of both normal fibrinogen and hdl - c levels , high fibrinogen level alone , or low hdl - c level alone . this study is the first to show an association between concurrent high fibrinogen and low hdl - c , and a risk for recurrent cvt . owing to the rarity of the disease , studies on cvt are mainly retrospective , with a small sample size and short follow - up . a larger retrospective cohort study from france included 77 cvt patients followed for 63 months . of these , 11.7% had recurrence of cvt , with 10.4% occurring during the first 12 months . this study is similar to our current study , in which we found a 12.6% rate of cvt recurrence over a median period of 27 months . the recurrent risk of cvt in our study was 2.76 per 100 patient - years . however , in the international study on cerebral vein and dural sinus thrombosis , only 2.2% of patients studied had an episode of recurrent cvt after a median follow - up of 13.9 months . of these cvt recurrences , 64.3% occurred within the 1 year . the lower recurrence rate of this study may be due to the shorter period of follow - up ( 13.9 months ) , as compared to 63 and 27 months in the studies mentioned above . the common risk factors for cvt in previous studies were prothrombotic conditions ; however , in the past , high fibrinogen or low hdl - c levels have not been described with regard to cvt . these two factors were also not included in the aha / asa statement about diagnosis and management of cvt . only a few studies have shown a correlation between high fibrinogen or low hdl - c with risk of vte or recurrent vte , but these findings were not consistent with other studies that suggested no correlation . until this study , there has been little attention paid to the prognostic importance of fibrinogen and hdl - c levels at baseline in cvt patients . however , there have been many reports about high fibrinogen and low hdl - c as prothrombotic factors promoting atherothrombotic diseases . fibrinogen is a circulating protein synthesized in the liver and is converted to fibrin by the action of thrombin . previous studies have demonstrated that fibrinogen was directly involved in a variety of mechanisms that mediate thrombotic processes . elevated fibrinogen levels can lead to increased intravascular fibrin deposition and thrombosis , and make clots more resistant to lysis . past studies have shown that hdl - c contributes an antithrombotic effect that may be a result of a reduction in viscosity , an inhibition of platelet aggregation , a decrease in inflammation , or the protection of endothelial cell function . low hdl - c level is associated with endothelial damage and the hypercoagulable state . in this study , 41.1% patients had elevated fibrinogen levels , and 36.8% had lowered hdl - c levels . these proportions are much higher than the 2% and 11.126.4% , respectively , in healthy subjects found in two previous studies . notably , although fibrinogen is an acute phase reactant , only 13.7% patients in our study were within 1-week after onset , while most other patients were in the subacute or chronic stage in the hospital ; therefore , we think that the effect of the acute phase response was not significant for the observations in the study . however , it was notable that elevated fibrinogen or lowered hdl - c alone had no significant correlation with the recurrence of cvt in this study . therefore , this implied that initial high fibrinogen or low hdl - c alone may not be independent risk factors for recurrent cvt . this is consistent with the viewpoint that thrombophilia alone is not sufficient to trigger recurrent cvt when it is not associated with other synergistic risk factors . to further explore a cooperative effect between fibrinogen and hdl - c , we examined cvt recurrence in patients with concurrent high fibrinogen and low hdl - c . surprisingly , we found that concurrent high fibrinogen and low hdl - c could increase the risk of recurrent cvt . we speculate that this is partially because of their similar or overlapping actions , such as their effect on blood viscosity , platelet aggregation , and inflammation , in promoting thrombosis . apart from that low hdl - c levels can lead to endothelial damage , which produces a more thrombogenic surface . elevated fibrinogen can then elicit augmented fibrin deposition at the vascular wall . therefore , elevated fibrinogen and low hdl - c could accelerate thrombosis , possibly by promoting and augmenting endothelial injury and fibrin deposition . this may help to explain the etiologic link between the additive effect of fibrinogen and hdl - c , which may predispose to recurrent cvt events . in addition , we found that high fibrinogen was also related to low hdl - c ( r = 0.205 , p < 0.05 ) . the levels of hdl - c dipped slightly when the concentration of fibrinogen rose above physiologic levels . it is likely that the impact of other parameters was more pronounced , and smoking was not an independent risk factor for cvt recurrence . although smoking is well - established as an important risk factor for atherosclerosis , a previous study found no association of smoking with vte . anticoagulant treatment is essential for the prevention of thrombosis extension and recurrence of the disease . the proper duration for administering oral anticoagulation after cvt has not been adequately studied , and the optimal time period of administration remains uncertain . guidelines have suggested a continuation of anticoagulation for a period between 3 and 12 months to prevent cvt recurrence . in our study , 75% ( 9/12 ) of recurrent patients were taking an anticoagulant ( warfarin ) for 312 months . however , we found no significant association between prolonging anticoagulant administration beyond 3 months and the recurrence of cvt . previous studies have also shown that the risk of venous thrombotic recurrence was not influenced by anticoagulant therapy or duration of anticoagulation . there have even been suggestions that routine use of long - term anticoagulant therapy or life - long secondary prevention is unnecessary for preventing venous thrombotic recurrence . however , this does not imply that prolonged anticoagulation is unnecessary for preventing recurrence of cvt in some patients with multiple risk factors . a total 58.3% ( 7/12 ) of recurrent patients had both high fibrinogen and low hdl - c in our study . prolonged administration of oral anticoagulation may be helpful for preventing recurrent cvt in these patients . besides , it is questionable whether prevention targeted at high fibrinogen and low hdl - c is as useful in reducing the recurrence of cvt as dietary modification , smoking cessation , weight loss , physical exercise , and even avoidance of potential air pollution exposure . recent epidemiological studies have demonstrated that air pollution is associated with elevated fibrinogen and low hdl - c . further research is required to explore the effect , pathogenesis , and preventive measures of the potential risk factors , including concomitant factors , related to atherothrombosis in cvt recurrence . there were some limitations in this study , including the small sample size , and data derived from only one center . in particular , inherited thrombophilia could not be completely assessed because of the retrospective nature of the study . in addition , because oral contraceptives , pregnancy , and the postpartum state may lead to a temporary increase in the risk for initial venous thrombosis or recurrence , we did not include such patients in our study . , we found that initial high fibrinogen level alone or low hdl - c level alone is not an independent risk factor for recurrent cvt ; however , concurrent high fibrinogen and low hdl - c levels at baseline may be associated with recurrence .
mesenchymal stem cells ( mscs ) are a subset of adult progenitor cells isolated from hematogenous bone marrow and tissues derived from mesoderm ( dominici et al . , 2006 ) . experimental studies provided evidence about the effect of bone marrow mesenchymal stem cells ( bmmscs ) on epidermal regeneration ( mcfarlin et al . , 2006 ; dash et al . , 2009 ; wu et al . , evidence from preclinical studies suggested the capacity of mscs to inhibit pathogenic immune responses and release neuroprotective and pro - oligodendrogenic molecules favouring tissue repair ( di nicola et al . small clinical studies showed safety and tolerability of mscs and stabilization or mild improvement in patients with multiple sclerosis ( karussis et al . , 2010 ; yamout et al . , 2010 ; bonab et al . , 2012 ; neuromyelitis optica ( nmo , devic 's syndrome ) is an inflammatory , demyelinating central nervous system disorder characterized by concurrence of optic neuritis and myelitis in a relapsing course . a humoral autoimmune mechanism is involved , targeting aquaporin 4 channels on astrocytes resulting in demyelination and axonal injury ( wingerchuk and weinshenker , 2008 ) . we report the suprising outcome of local application , on pressure ulcers , of autologous bmmscs in a case of severe nmo . in march 2003 , a 46-year - old male without previous medical and family history presented paraesthesia and pain in both legs followed by progressive paraparesis ( left leg 2/5 bmrc , right leg 3/5 bmrc ) . spinal cord magnetic resonance imaging ( mri ) revealed a lesion with edematous component ( t2 and flair hyperintensity and t1 hypointensity ) at t24 level , producing an increase in volume of the spinal cord and a reduction of the peri - spinal spaces , with central , ring - shape contrast enhancement corresponding to a demyelinating lesion . brain mri showed three small demyelinating lesions , with hyperintensity in t2 sequences , localized in the subcortical white matter of the left frontal lobe and right parietal lobe ( figure 1 ) . the patient tested negative for borrelia burgdorferi , human immunodeficiency virus , hepatitis b and c viruses . serum angiotensin converting enzyme , antinuclear antibodies , antineutrophil cytoplasmic antibodies , anti sm antibodies , and anti - double stranded dna antibodies were negative . until april 2008 the patient had three relapses : on september 2006 with paraplegia , on may 2007 with diminished visual acuity of the right eye , paraparesis ( 2/5 bmrc ) and in november 2007 with paraplegia . after recovery from the last relapse the patient 's expanded disability status scale ( edss ) score was 6.5 . the relapses have been treated with methylprednisolone 1 g intravenously once daily for 5 days and as chronic treatment : methylprednisolone oral , followed by pulse therapy with intravenous cyclophosphamide and then oral azathioprine associated to methylprednisolone . in april 2008 , the patient presented a new relapse with decreased visual acuity of the right eye and paraplegia . at the admission into the department of neurology of fundeni clinical institute , the neurologic examination showed in the right eye disc pallor and visual accuity ( corrected ) 20/40 and in the left eye visual accuity corrected 20/20 , paraplegia , bilateral pyramidal syndrome , superficial , vibratory and position sense hypoesthesia in right leg , anesthesia of left leg , urinary retention , with frequent urinary infections , sexual dysfunction , fatigue , anxiety , helpless bedridden patient , with no self - care ( edss 9 , hauser ambulation index ai 9 ) and three pus : two stage iv pus on the lateral left malleolus ( 8/7 cm ) and on lateral side of thigh ( 3 cm ) respectively and a stage iii pu on right calcaneum . brain and spinal cord mri showed hypotrophy of the spinal cord at t24 level , the three subcortical lesions were still present and no new demyelinating lesions appeared ( figure 2a c ) . veps showed a p100 latency of 115 ms and amplitude n7-p100 of 4 v in the right eye and a p100 latency of 112 ms and an amplitude n7-p100 of 12 v in the left eye . cerebrospinal fluid analysis showed 30 mg / dl protein , 52 mg / dl glucose , no oligoclonal bands and no cells . immunofluorescence on monkey tissue ( laboraf diagnostica e ricerca san raffaele milano , italy ) disclosed the presence of nmo igg anti - aquaporin-4 antibodies in serum sample . nmo was diagnosed based on clinical signs and course with relapses , the presence of a spinal cord mri lesion on three vertebral segments and nmo - igg seropositive status ( wingerchuk and weinshenker , 2008 ) . ( a d ) intramedullary t2 weighted hyperintense expansive lesion extended between t2 and t4 , with small central nodular enhancement ( arrows ) . magnetic resonance imaging ( mri ) evaluation in 2008 ( a c ) and 2009 ( d f ) . ( a c ) rare and small demyelinating subcortical bilateral frontal and right parietal lesions ( arrows ) . ( d f ) discrete progression of the demyelinating lesions in the frontal regions ( arrows ) . in order to treat the pus , the patient was referred to the department of general surgery and liver transplantation . systemic antibiotic therapy and local treatment with sorbalgon , atrauman ag , and tenderwet were conducted to clean the wounds . the patient was offered a plastic surgery consultation and the possibility to choose between skin graft and local application of autologous mscs for the treatment of pus . the approval by the ethics committee of fundeni clinical institute was obtained for application of biological dressing with autologous bmmscs . the patient signed the written informed consent for the treatment of pus with autologous bmmscs . the patient was investigated to exclude any ongoing malignant disease with the standard check - up procedure used for transplanted patients . the bone marrow aspirate was obtained from the right posterior superior iliac spina for bmmscs isolation . mononuclear cells were separated by centrifugation over a biocoll gradient ( biochrom , germany ) and suspended in alpha - mem containing 10% autologous serum and penicillin - streptomycin mixture ( biochrom , germany ) , followed by plating at an initial seeding density of 1 10 /cm . after 3 days , the nonadherent cells were removed and monolayers of adherent cells were cultured until confluence . cells were trypsinized with 0.25% trypsin and subcultured at densities of 5,0006,000 cells / cm . after the first passage , the culture of mscs became homogenous and demonstrated a high proliferation potential . the cell surface marker identification was performed by epics xl facs analyzer ( beckman coulter ) using fluorescein isothiocyanate ( fitc ) or phycoerythin ( pe)-labeled monoclonal antibodies . phenotypic characterization confirmed mscs : cd105 , cd90 , cd34 , cd45 , cd3 , cd14 ( figure 3 ) . the biological dressing was made of 8 10 bm - mscs seeded into a 30 cm collagen - agarose sponge placed on a decellularized human arterial fragment using a previous protocol ( sirbu - boeti et al . , 2009 ) . ( a ) culture of bone marrow mesenchymal stem cells ( bmmscs ) , phase contrast 100 ; ( b ) collagen scaffold ( green autofluorescence ) embedded with bmmscs stained with propidium iodide ( orange ) , fluorescence microscopy 400 ; ( c ) mscs enriched collagen - agarose sponge laid on aortic wall fragment , hematoxylin - eosin 40 ; ( d ) phenotype of cultured undifferentiated bmmscs ; cd3 cd14 cd34 cd45 cd90 cd105 . the evolution of the wounds was followed daily for the first 2 weeks and then every 2 days for other 2 weeks . the healing of the left malleolar pu was completed 5 months later with normal function of ankle joint . macroscopic aspect of pressure ulcers ( pus ) of the patient . left lateral malleolar pu at the admission in the department of neurolgy before ( a , b ) and after the application of the biological dressing embedded with autologous bone marrow mesenchymal stem cells ( bmmscs ) ( c h ) . left antero - lateral thigh pu at the admission in the department of neurology , before ( i , j ) and after the application of the biological dressing embedded with autologous bmmscs ( k p ) . concomitent with pressure ulcers healing the authors noticed a progressive improvement in the neurological status of the patient . since this change was not anticipated before the procedure , topical application of mscs being made with the intention to treat the pressure ulcers , the authors had not planned to assess the immunological status of the patient before and after the procedure . however the neurostatus was daily recorded by the neurologist and noticed in a dairy by the patient 's spouse and later by the patient himself . one month later the patient recovered his sensitivity for both legs , was able to move in bed by himself and to sit , the paraparesis was 3/5 bmrc for the left and right legs , but spasticity of both legs occurred . patient 's examination revealed : in the right eye disc pallor and visual acuity 20/40 , in the left leg bmrc 3 for hip flexors , knee flexors and extensors , plantar flexion and extension , in the right leg bmrc 3 for hip flexors , bmrc 2 for knee flexors and extensors , bmrc 2 for plantar flexion and extension , spasticity of both legs , moderate limb ataxia of both legs , moderate decrease of superficial and mild decrease of vibration sense of left leg , urinary retention , fatigue and the patient was able to walk 5 meters with constant bilateral assistance ( edss 7.0 , ai 7 ) . at four months neurologic examination showed : in the right eye disc pallor and visual acuity 20/40 , superficial hypoesthesia on left leg , paraparesis ( left leg bmrc 3/5 , right leg bmrc 4/5 ) , spasticity on flexors of the left inferior limb , no ataxia , 40 meters ambulation with constant bilateral assistance ( edss 6.5 , ai 6 ) . eight months later the patient was ambulatory with unilateral assistance ( cane ) for 150 m without rest ( edss 6.0 , ai 4 ) . in may 2009 , at 11 months , brain and spinal cord mri showed some new t2 lesions in the frontal regions and unchanged aspect at thoracic spinal cord ( figure 2d f ) . the patient was ambulatory without aid or rest for more than 300 m , edss 4.5 , ai 3 . at 32 months the patient was able to ride bicycle and to shovel snow ( edss 3.5 , ai 2 ) . at 76 months after mscs therapy the patient was free of relapse , with edss 3.5 , ai 2 , needs only 4 mg methylprednisolone every other day and had negative malignancy screening . neurological evolution of the patient after local biological dressing with mesenchymal stem cells ( mscs ) culture . ( a ) and ( b ) neurostatus after 2 months : edss = 7 , ai = 7 , ( c ) neurostatus after 5 months : edss = 6.0 , ai = 4 , ( d ) neurostatus after 12 months : edss = 4.5 , ai = 3 . ai : hauser ambulation index ; edss : expanded disability status scale . the successful healing of both pus was expected . other studies reported rapid wound healing with reduced scarring after administration of mesenchymal stem cells . they showed that mscs stimulate angiogenesis and induce fibroblast migration due to a paracrine effect ( rodriguez - menocal et al . , 2015 ) . the innovation consisted in the application of bm - mscs embedded in a collagen - agarose sponge that has the advantage of alleviating the cell damage which is associated with injection barotrauma and avoiding the side effects secondary to other administration routes ( e.g. , intravenous , intra - arterial ) . however the spectacular and long - term improvement of disability can not be explained only by healing of pus since the patient was paraplegic long before the development of pus . several studies showed that mesenchymal stem cells modulate the innate and adaptive immunity mostly through the secretion of paracrine factors ( bystander effect ) ( krampera et al . , 2011 ; keating , 2012 ) . in our case , a possible explanation of the improvement of disability secondary to the local application of bmmscs on pus might be that local inflammation facilitated the migration of bmmscs into the peripheral lymph nodes producing a peripheral immunomodulation since the recovery lasted . . however the interpretation of the outcome must be made with caution since , appart from the neurological evolution of the patient , we do not have other arguments to sustain that the long term remission was a consequence of the local application of mesenchymal stem cells . this is the first report of topical application of a biological dressing embedded with autologous in vitro expanded bmmscs in an nmo patient with stage iv pus that accelerated the healing of pus and was associated with a spectacular and long term improvement of patient 's disability ( from edss 9 to edss 3.5 ) . this curious association could be explained by a promotion / optimization of recovery mechanisms in the central nervous system even if alternative hypothesis should be considered . further studies are needed in order to assess the safety and efficacy of mesenchymal stem cells in nmo treatment .

they are most numerous in the distal appendages where they play a role in heat exchange . the majority of glomus tumors ( gts ) , which are mesenchymal in origin , are therefore found in the hands and feet . the incidence of gastric gts is 100 lower than gastrointestinal stromal tumors ( gists ) . in the largest study of gastrointestinal gts ( 32 cases ) , miettinen et al . found a female preponderance of 72% , and median age of 55 . of the 31 patients with gastric gts , 11 presented with upper gi bleeding , 9 with dyspepsia and 1 with perforation . the only non - gastric gt was in the cecum , and presented as appendicitis . an 88-year - old female presented with 5 days of weakness and dyspnea , severe anemia ( hemoglobin of 46 physical examination was remarkable for a blood pressure of 83/48 , and black stool on rectal examination . ct demonstrated a 2.5 1.9 cm well - circumscribed , partially exophytic , arterially enhancing lesion . pathology found a lobulated tumor composed of glomus cells in the submucosa and muscularis propria . a 70year - old male presented with light headedness , shortness of breath and 2 days of bright red blood per rectum progressing to melena . upper endoscopy demonstrated a 3-mm polyp in the duodenal cap ( biopsy would be reported as a completely excised carcinoid tumor ) . ct demonstrated a 2.3 1.6 cm ascending colon lesion with hyperenhancement in the arterial phase and persistent enhancement in the portal venous and delayed phases . on octreotide scan final pathology demonstrated a gt with negative margins , 26 negative lymph nodes , 1 mitosis per 50 hpf , no vascular invasion and absence of necrosis . follow - up for the colonic gt will include a colonoscopy and abdominal ct 1 year following surgery , as well as chromogranin a level every 6 months . , he will be followed with esophagogastroduodenoscopy + / endoscopic ultrasound ( eus ) every 36 months initially . endoscopic findings of gastric gts are that of a submucosal mass , typically in the antrum or distal body , with either normal mucosa or ulceration [ 3 , 4 ] ( fig . 1 ) . ct shows a well - defined submucosal tumor with a clear margin , strong enhancement in the arterial phase , and prolonged enhancement in the delayed phase [ 4 , 5 ] ( fig . 2 ) . often confused with gist , the distinguishing features are that the density of gists is lower than that of gts , and gists do not exhibit prolonged enhancement in the delayed phase . on eus , the gt is found in the fourth endosonographic layer , with a heterogeneous appearance . endoscopic biopsies of submucosal lesions are typically superficial and non - diagnostic . however , having a preoperative diagnosis could lead to a change in surgical planning , given the benign nature of gts . eus - guided biopsy is now a well - established modality for sampling submucosal lesions . there are case reports in the literature of eus - guided biopsy being diagnostic for gts . note the well - circumscribed submucosal lesion surrounding small vessels ( h&e stain , 20 ) . note the well - circumscribed submucosal lesion surrounding small vessels ( h&e stain , 20 ) . the cellular nodules are separated by streaks of gastric smooth muscle , which also surrounds the tumor . histologic features are central round to oval nuclei with inconspicuous nucleoli and clear to eosinophilic cytoplasm with distinct cell borders ( figs . 3 and 4 ) . they are most often negative for cd117 , cd34 , chromogranin and synaptophysin [ 3 , 6 ] . note central round or oval nuclei with clear , eosinophilic cytoplasm ( h&e stain , 200 ) . note central round or oval nuclei with clear , eosinophilic cytoplasm ( h&e stain , 200 ) . gts can present with chronic bleeding , often occult . while life - threatening hemorrhage is rare , there is minimal literature about colonic gts ; however , one patient in the miettinen study presented as appendicitis secondary to a cecal gt . the disease most often follows a benign course ; however , there are very rare case reports of metastases . one 65-year - old female was found to have a gastric gt with synchronous metastases of the kidney , brain , lung and humeral head . another describes a 58-year - old male who presented with cutaneous metastases to the scalp 6 years after gastrectomy for a gt . given their benign course and small size , gastric gts are amenable to wedge resection . there are limited reports of endoscopic submucosal dissection ( esd ) being used in gastric gts . zhou et al . described 26 gastric submucosal tumors treated with esd , 3 of which were gts . no residual tumor or recurrence was found after a mean follow - up of 8 months . for symptomatic it seems reasonable to suggest regular follow - up with investigations based on symptomatology . in cases with high mitotic activity
spondylolisthesis is an unstable lumbar disease demonstrated in approximately 6% of the general population and occurs most often at l4-l5 and l5-s1 levels10,13,14 ) . anatomy research indicates that isthmic spondylolisthesis usually occurs within the scope of 2 to 9 mm beneath the pedicles , resulting in the defects in bony contacts in the pars interarticularis to form pseudarthrosis , the gap of which is filled with fibrous connective tissue and chondroid tissue , which we called pars interarticularis scar3 ) . symptomatic patients with isthmic spondylolisthesis usually require surgical intervention , the goals of which include the stabilization of the motion segment , the decompression of neural elements , the reconstitution of disc space height , and the restoration of sagittal plane translational and rotational alignment6 ) . minimally invasive transforaminal lumbar interbody fusion ( tlif ) is a relatively novel spinal fusion technique which was developed in recent years on the basis of conventional tlif3 ) . but , little is known about the slip reduction according to the unilateral tlif methods . this study was to compare the slip reduction rate and clinical outcome between unilateral conventional tlif ( conventional tlif ) with pedicle screw fixation and unilateral minimal invasive tlif ( minimal tlif ) with pedicle screw fixation for treatment of one level low - grade symptomatic isthmic spondylolisthesis . transforaminal lumbar interbody fusion is a relatively new technique of lumbar arthrodesis via posterior transforaminal approach to the disc , indicated mainly in cases of degenerative disc disease , low grade spondylolisthesis and reoperation for disc herniation , especially when there is indication for interbody fusion and posterior decompression . the main advantage of tlif is that it allows the complete removal of the intervertebral disc through the vertebral foramen , decompression of the spinal canal and vertebral foramen with minimum risk of neural lesion , due to the access being lateral to the nerve roots . between february 2008 and april 2012 , 25 patients with low - grade isthmic spondylolisthesis underwent conventional tlif ( 12 patients ) and minimal tlif ( 13 patients ) in single university hospital by a single surgeon were included in the study - including 6 males and 19 females , whose average age was 51.96 years ( 21 - 72 years ) . all of them had preoperative radiographs , computed tomography scans , as well as magnetic resonance imaging , and all surgical procedures were performed by a single surgeon ( shy ) . lateral radiographs of lumbar spine were taken 12 months after surgery to analyze the degree of reduction . all measurements were performed by a single observer and were expressed as meansstandard deviations . to control for small variations , slip percentage was measured as a percentage of distance from the posterior border of the caudal to the posterior border of the rostral vertebra , normalized to the superior endplate diameter of the former as it presented in fig . clinical outcome was measured by visual analog scale ( vas ; score range : 0 to 10 , with 0 reflecting no pain ) . the minimal tlif procedure was performed on the side of radicular symptoms . if both the legs were symptomatic , the approach was from the side of more severe pathology and contralateral lamina and foramina decompressed by a unilateral exposure . sequential soft tissue dilators were inserted through the incision down to the facet complex until the desired working diameter was achieved . a facetectomy was then performed using a high - speed drill from lateral to medial side to expose the posterolateral aspect of the disc . an interbody graft was then placed in a direction anterior and contralateral to the annulotomy within the interbody space . if needed , the tubular retractor was angled contralaterally so that a more extensive boney decompression could be done . the tubular retractor was then removed and percutaneous pedical screws placed immediately above and below the interbody segment to be fused . under fluoroscopic guidance , a jamshidi needle was inserted into the pedicles . a k - wire was then passed through the jamshidi trocar into the pedicles . using cannulated instruments , a bone tap followed by cannulated screw compression was applied to the construct before final tightening , providing compression of the bone graft and maximizing lordosis . all wounds were copiously irrigated and the wounds were closed in layers . a midline skin incision was used in conventional tlif . bilateral pedicle screw rod constructs were inserted and laminectomy and unilateral facetectomy was then performed at that level . this was followed by unilateral anulotomy , discectomy , and placement of the interbody graft . similar to the minimal tlif approach , cartilaginous material was removed from the endplates using the endplate scraper . interbody graft was then placed anteriorly and contralateral to the annulotomy within the interbody space . for posteriorlateral arthodesis , local autogenous bone with or without bone extenders was used for bone grafting . chicago , il , usa ) was used throughout , and statistical significance was accepted for p values of < 0.050 . data were analyzed using the student , student paired t - test , and chi - square test . transforaminal lumbar interbody fusion is a relatively new technique of lumbar arthrodesis via posterior transforaminal approach to the disc , indicated mainly in cases of degenerative disc disease , low grade spondylolisthesis and reoperation for disc herniation , especially when there is indication for interbody fusion and posterior decompression . the main advantage of tlif is that it allows the complete removal of the intervertebral disc through the vertebral foramen , decompression of the spinal canal and vertebral foramen with minimum risk of neural lesion , due to the access being lateral to the nerve roots . between february 2008 and april 2012 , 25 patients with low - grade isthmic spondylolisthesis underwent conventional tlif ( 12 patients ) and minimal tlif ( 13 patients ) in single university hospital by a single surgeon were included in the study - including 6 males and 19 females , whose average age was 51.96 years ( 21 - 72 years ) . all of them had preoperative radiographs , computed tomography scans , as well as magnetic resonance imaging , and all surgical procedures were performed by a single surgeon ( shy ) . lateral radiographs of lumbar spine were taken 12 months after surgery to analyze the degree of reduction . all measurements were performed by a single observer and were expressed as meansstandard deviations . to control for small variations , slip percentage was measured as a percentage of distance from the posterior border of the caudal to the posterior border of the rostral vertebra , normalized to the superior endplate diameter of the former as it presented in fig . clinical outcome was measured by visual analog scale ( vas ; score range : 0 to 10 , with 0 reflecting no pain ) . the minimal tlif procedure was performed on the side of radicular symptoms . if both the legs were symptomatic , the approach was from the side of more severe pathology and contralateral lamina and foramina decompressed by a unilateral exposure . sequential soft tissue dilators were inserted through the incision down to the facet complex until the desired working diameter was achieved . a facetectomy was then performed using a high - speed drill from lateral to medial side to expose the posterolateral aspect of the disc . an interbody graft was then placed in a direction anterior and contralateral to the annulotomy within the interbody space . when necessary , the contralateral ligamentumflavum was resected to expose the contralateral exiting and traversing nerve roots . if needed , the tubular retractor was angled contralaterally so that a more extensive boney decompression could be done . the tubular retractor was then removed and percutaneous pedical screws placed immediately above and below the interbody segment to be fused . under fluoroscopic guidance , a jamshidi needle was inserted into the pedicles . a k - wire was then passed through the jamshidi trocar into the pedicles . using cannulated instruments , a bone tap followed by cannulated screw compression was applied to the construct before final tightening , providing compression of the bone graft and maximizing lordosis . bilateral pedicle screw rod constructs were inserted and laminectomy and unilateral facetectomy was then performed at that level . this was followed by unilateral anulotomy , discectomy , and placement of the interbody graft . similar to the minimal tlif approach , cartilaginous material was removed from the endplates using the endplate scraper . interbody graft was then placed anteriorly and contralateral to the annulotomy within the interbody space . for posteriorlateral arthodesis , local autogenous bone with or without bone extenders chicago , il , usa ) was used throughout , and statistical significance was accepted for p values of < 0.050 . data were analyzed using the student , student paired t - test , and chi - square test . the demographic data between conventional and minimal tlif were not different ( table 1 ) . slip percentage was reduced from 15.006.66% to 8.335.31% in conventional tlif , and from 14.153.76% to 9.625.42% in minimal tlif ( table 2 ) . in both groups , slip percentage was significantly improved postoperatively ( p=0.002 ) , but no significant intergroup differences of slip percentage in preoperative and postoperative were found ( table 3 ) . the reduction rate also not different between conventional ( 45.4128.80% ) and minimal tlif ( 32.9132.12% , p=0.318 ) . there were no signs of instrumentation failure and no visible subsidence of the cages until 12 months follow - up periods . all patients had their symptoms significantly improved , and the vas decreased from 7.002.69 before operation to 3.111.86 at 12 months after operation in conventional tlif , and from 7.143.15 before operation to 3.522.24 at 12 months after operation in minimal tlif ( table 3 ) . the advent of minimally invasive surgery had provided surgeons new techniques for treating clinical disease7 ) . minimally invasive spine surgery aims to reduce approach related morbidity , while producing clinical outcomes comparable to its open predecessors7 ) . one important example of this is the development of minimally invasive techniques for lumbar inter - body fusion , including tlif2,7 ) . the minimal invasive tlif technique , has displayed comparable outcomes to conventional tlif , while adding the benefits of less approach related morbidity , decreased intraoperative blood loss , and shorter hospital stays9,11 ) . however , critics of the technique have noted that the minimal tlif has longer operative times and exposes patients to increased fluoroscopic radiation . over the past decade minimal tlif has been shown to have a number of benefits , especially with regard to perioperative outcomes . ultimately , comparing the known literature of a conventional tlif approach to published reports on minimal tlif will identify the unique risks and benefits associated with each . generally , clinical result between conventional and minimal tlif were reported the similar or superior result in minimal invasive technique4,5,17 ) . all preoperative parameters except the operation time were superior in minimal tlif than the conventional tlif4,5,17,18,20 ) . but , all authors reported that radiation hazard is less effective in minimal tlif , and concluded the main disadvantage of minimal tlif4,5,17 ) . in radiological study , although many reported using the different criteria about the bone fusion , but all reported studies concluded the fusion rate between two tlif technique is similar17,18 ) . but , the comparisonal data slip reduction in patients with low - grade spondylolisthesis is lack of data , yet . in this study , the authors compared the slip reduction rate between two different unilateral tlif with pedicle screw fixation for treatment of one level low - grade symptomatic isthmic spondylolisthesis . to best knowledge to authors , this comparison is not published in literature yet , although the size of data was not sufficient to show the conclusive statistical evidence . in this small case series about 25 patients with low - grade isthmic spondylolisthesis underwent conventional tlif or minimal tlif showed , the slip percentage was significantly improved postoperatively in both tlif techniques ( p=0.002 ) . but , the slip percentage in pre / postoperative periods and reduction rate after operation also not different between conventional and minimal tlif . the similar results were also observed in the literatures , but most of them are focused only conventional or minimal tlif . recniket al.8 ) reported that significantly decreased translational slip from 20.7% to 14.9% after conventional tlif in 32 patients with single - level isthmic spondylolisthesis ( p<0.001 ) . yan et al.16 ) also reported the slip was reduced from 31.48.3 to 8.22.6% , and the average of reduction rate was 75.46.2 in conventional tlif . in minimal invasive techniques , pan et al.3 ) reported decreased translational slip from 24.2% to 10.5% after operation , and tsahtsarlis et al.19 ) also reported the anatomical reduction of the spondylolisthesis was complete in 16 patients ( 69.6% ) and incomplete in seven ( 30.3% ) . this result was similar to this data , but direct comparisonal result is observed in only this presented study . some authors reported surgical technique to increase the reduction rate among spondylolisthesis , the key technique is circumferential disc or ligament reconstruction which prevent the reduction1 ) . a bilateral traversing and exiting nerve roots decompression is a safer option prior to performing the deformity reduction and fixation , and the proposed minimally invasive technique may help in reducing surgical morbidity and improving postoperative recovery . the conventional and minimal tlif showed excellent slip reduction in patients with one level low - grade symptomatic isthmic spondylolisthesis and slip percentage and reduction rate were similar in the conventional and minimal tlif as we presented . but , as we commented , this study is small case series about 25 patients , so the statistical analysis had an opportunity into the trap of the type i error . indeed , we could not access the other meaningful clinical outcomes such as oswestrydisability index ( odi ) , neurogenic symptom score ( nss ) andshort form 36 ( sf-36 ) because of using retrograde data review . in the statistical analysis with tripling of this data showed statistical difference of reduction rate ( p=0.018 ) , so well - designed longitudinal prospective study with a larger number of isthmic spondylolisthesis could presented the different reductional result between conventional and minimal tlif . the conventional and minimal tlif with pedicle screw fixation showed excellent slip reduction in patients with one level low - grade symptomatic isthmic spondylolisthesis .
patients may file a claim if their medical treatment results in an injury or an unexpected side effect . in particular , this kind of risk for medical professional liability claims is a daily consideration for anesthesia providers . the collection and analysis of data relating to anesthesia - related injuries are thus important in decreasing clinicians ' fear of litigation , improving patient care , and decreasing medical costs . however , the rarity of severe anesthesia - related injuries renders retrospective or prospective clinical trials unwieldy and difficult or practically impossible to perform . thus , analysis of closed claims or expert consultation referrals on anesthesia - related issues is the typical method for studying the risk profile of severe and/or rare injuries ( 1 ) . the american society of anesthesiologists ( asa ) was the first professional organization to initiate a closed claims project ( asa - ccp ) , which has been ongoing since 1985 ( 1 , 2 ) . in korea , similar project was initially set up by the korean society of anesthesiologists ( ksa ) in 2009 ( 3 ) . the ksa database was constructed from expert consultation referrals on anesthesia - related issues , which were usually requested by police departments or courts . here , using the ksa database covering case files from july 2009 to june 2014 , the ksa legislation committee analyzed all surgical anesthesia cases , with the aim of identifying specific patterns of injury . we also explored the role of established practical guidelines in the prevention of anesthesia - related injuries . in july 2009 , the ksa legislation committee database was constructed to evaluate adverse anesthetic outcomes obtained from the case files of expert consultation referrals to the ksa . a detailed description of the data collection process has been reported previously ( 3 , 4 ) . briefly , each member of the ksa legislation committee reviewed a referred case file ( typically , medical records , testimony records of involved personnel , and/or autopsy reports , if performed ) . then , the reviewer completed a standardized data collection form plus a narrative summary for each case in which the sequence of events and nature of injury could be determined , together with an expert consultation service . since the first entry of a case file into the database in july 2009 , 182 cases had been collected as of june 2014 . of them , simple academic consultation cases with inadequate detail and non - anesthetic cases , including those arising in pain clinics , were excluded . although 128 cases were eligible for analysis , 23 cases were then excluded because of repeated consultation requests in the same case . finally , in total , 105 cases were included in the analysis . in each case , the standardized data set included information about patient characteristics , types of surgical procedure and hospital , anesthesia characteristics ( type of anesthesia , anesthesia provider , drugs used , intraoperative monitoring , and intra - procedural oxygen supply ) , timing and sequence of damaging events , complications , and the presence of relevant medical records ( pre - anesthetic evaluation record and anesthesia record ) . a ' damaging event ' refers to the primary mechanism causing the injury and a ' complication ' refers to the ultimate injury itself ( 2 ) . damaging events were grouped into broad categories based on the physiological system or anesthesia technique implicated in the injury ( 3 , 4 ) : respiratory events , cardiovascular events , nervous events , allergic or adverse drug reactions , equipment problems , hepatic events , renal events , endocrine events , thermal events , infectious events , or others . for further analyses , these 11 categories were subcategorized into more specific causative mechanisms , most of which are self - explanatory . complications were grouped into four categories ; temporary , permanent / minor , permanent / major , and death . while severe brain damage , quadriplegia , or paraplegia with lifelong care or fatal prognosis were considered to be ' permanent / major ' injuries , remaining permanent injuries were considered to be ' permanent / minor ' finally , each reviewer made a judgment as to the standard of care and preventability of adverse outcomes , based on reasonable and prudent practice at the time of the event . the appropriateness of anesthesia care was graded on a 9-point numerical rating scale ( nrs ) with 1 - 3 for ' avoidable ' , 4 - 6 for ' possibly avoidable ' , and 7 - 9 for ' probably unavoidable ' . this scale was made by the mix - up of 9-point nrs and 3-class coding system of preventability , which is known to have an acceptable inter - rater reliability ( 5 , 6 ) . this study was exempted from institutional review board approval because no human subject was studied and no patient health information was reviewed directly . this study was exempted from institutional review board approval because no human subject was studied and no patient health information was reviewed directly . of 105 cases included in the final analysis , 69 ( 65.7% ) were referred from police departments , and 34 ( 32.4% ) from civil or criminal courts . table 1 presents an overview of all cases that have been referred during the 5-yr study period . most patients were under the age of 60 yr ( 87/105 , 82.9% ) and were classified as asa physical status i or ii ( 95/105 , 90.5% ) . although cases related to general anesthesia were the most common , sedation cases were similarly prevalent , accounting for 37.1% of all cases . in 82 cases ( 78.1% of all cases ) , of 16 cases with permanent / major injuries , 13 resulted in severe hypoxic brain damage , two became paraplegic after spinal or epidural anesthesia , and one developed hemiplegia after general anesthesia due to a cerebral hemorrhage ( table 1 , 2 ) . of 4 cases with permanent / minor injuries , one resulted in urinary dysfunction after epidural anesthesia , another disclosed femoral nerve injury after general anesthesia , and two others disclosed incomplete brachial plexus injury after axillary block . temporary injuries ( n=3 ) consisted of a sexual harassment during propofol plus ketamine sedation , a recovered case of disseminated intravascular coagulation after liposuction under propofol sedation , and a minor traffic accident after propofol sedation due to hypoglycemia . the appropriateness of anesthesia care , graded using a 9-point nrs , was not high in a considerable number of cases ( table 1 ) . indeed , in 45 cases ( 42.9% ) , the resulting injuries were determined to be ' avoidable ' if an appropriate standard of care had been used ( table 2 ) . in - depth analysis found that the proportion of ' avoidable ' injuries was much higher in patient with asa physical status i or ii than in those with asa physical status iii or vi ( 45.3% [ 43/95 ] vs. 20.0% [ 2/10 ] ) . analysis of cases according to anesthetic technique revealed that , with the exception of 4 cases , both general anesthesia and sedation cases resulted in grave complications ( i.e. , permanent / major injuries or death ) . however , in contrast to the general anesthesia cases , most sedation cases ( 27/39 , 69.2% ) showed deviations from the appropriate standard of care ( i.e. , were determined to be ' avoidable ' ) . in sedation cases , no pre - procedural testing was performed at all in 32 of 39 patients ( 82.1% ) . pre - anesthetic evaluation records and anesthesia records were absent in 92.3% and 89.7% of the sedation cases , respectively . a significant lack of vigilance during procedures was found in the sedation cases ; six patients had virtually no monitoring , and 24 patients did not receive supplemental oxygen ( table 2 ) . most sedation ( 36/39 , 92.3% ) was provided simultaneously by the non - anesthesiologist(s ) who performed the surgical / diagnostic procedure(s ) . propofol - based regimens were used in the vast majority of sedation cases ( 35/39 , 89.7% ) ; propofol was used alone ( n=20 ) or in combination with midazolam , ketamine , or remifentanil ( n=15 ; fig . there were two cases relating to nurse anesthesia ( table 2 ) . specifically , one patient who underwent epidural anesthesia disclosed cauda equina syndrome and had a persistent voiding difficulty despite surgical intervention . the other patient became paraplegic after cesarean section under general anesthesia , in which the causative mechanism was uncertain ( injury due to incorrect patient position vs. surgical injury ) . table 3 presents the damaging events in cases with permanent ( minor or major ) injuries and death . of these , the three most common forms of respiratory events were hypoxia secondary to airway obstruction or respiratory depression ( n=31 ) , difficult intubation ( n=8 ) , and aspiration ( n=5 ) . the sec common damaging event was a ' cardiovascular events ' , which accounted for 29.3% of all deaths . in the subgroup ' cardiovascular damaging events ' , almost half the cases ( 12/26 , 46.2% ) were due to acute myocardial infarction , all of which resulted in death . analysis of all cases according to physician specialty revealed that orthopedics ( 24 cases , 22.9% ) was the most common department , and plastic surgery ( 18 cases , 17.1% ) and general surgery ( 18 cases , 17.1% ) were the next two ( fig . expert consultation , as analyzed in this study , is quite different from medical expert witnesses or testimony in that the former should not be coerced into participation of conclusions . consultation only involves answering questions raised by referrals through a review of the information pertaining to the medical dispute . on receiving each expert consultation referral , the ksa legislation committee has clarified that the consultants can not become involved in the legal interpretation of medical malpractice , and the case file will be used for academic purposes after protecting patient confidentiality . the magnitude of injuries in our database appeared to be considerably high than those of the asa - ccp database ; 90.5% of the cases were for injuries resulting in death ( n=82 ) or severe brain damage ( n=13 ) in the ksa database vs. 39.0% in the asa - ccp database ( 2 ) . it is evident that the medico - legal burden of procedures in medicine has been increasing steadily in korea ( 7 ) . worse outcomes did not correlate with preoperative physical status , patient age , or the invasiveness of the procedures . in the present study , the majority of cases involved patients under the age of 60 yr ( 82.9% ) , those with asa physical status i or ii ( 90.5% ) , and minor surgeries or diagnostic procedures such as gastroscopy ( 75 cases , 71.4% ) . because the inherent risk of such cases is essentially low , these findings primarily reflect increased dissatisfaction of patients or their relatives about adverse outcomes ( occurrence of adverse outcomes despite a previously healthy condition or the ' simple ' surgical procedure(s ) performed ) . additionally , it may be , in part , attributable to our findings that adherence to the standard of care is deliberately ignored in ' simple ' surgical procedures in apparently healthy patients . hypoxia secondary to airway obstruction or respiratory depression was a leading causative mechanism . in common with our first report ( 3 ) , the present analysis identified ' respiratory events ' as the leading damaging event ( 53.3% of all cases ) , all of which resulted in permanent / major injuries or death . specifically , the largest subclass of respiratory events was hypoxia secondary to airway obstruction ( ' ca n't breathe ' situation ) or respiratory depression ( ' wo n't breathe ' situation ) . of these cases , propofol was used alone or in combination with other sedatives , except in three cases . such a prevalence of non - anesthesiologist - administered sedation cases may lead to the high proportion of plastic surgical cases in our case files , which is obviously different from the previous report of kwon ( 8) . in that study , plastic surgical cases accounted for 3.6% of all surgical anesthesia dispute cases , although the evaluation period ( november 1994-october 2002 ) was different from this study . such a difference may be primarily due to a combination of an increase in the number of the denominator ( overall plastic surgical cases ) and the climate of an undervalued risk of sedation in local clinics or hospitals . although target levels of sedation have been defined , the actual level of sedation in individual patients may easily fluctuate . in particular , at similar depths of sedation , propofol is more likely than midazolam to cause airway obstruction or respiration depression ( 9 ) . in this regard , the ksa , similar to asa ( 10 ) and the european society of anesthesiologists ( 9 ) , has supported the food and drug administration regulation that propofol is restricted solely to personnel trained in general anesthesia . however , other societies of endoscopists ( 11 ) , plastic surgeons ( 12 ) , and emergency medicine doctors ( 13 ) have proposed that non - anesthesiologists are capable of administering propofol after the completion of specialized training . although the use of anesthesia personnel to deliver sedation for average - risk patients remains controversial , all published practice guidelines , regardless of clinical specialty , commonly recommend that sedation must be performed by an independent trained medical person not involved in the procedure ( 9 , 10 , 11 , 12 , 13 ) . however , our results showed that in 92.3% of sedation cases ( 36/39 ) , sedation was provided simultaneously by non - anesthesiologist(s ) who performed the surgical / diagnostic procedure(s ) . thus , from a medico - legal perspective , adherence to this single item of the guidelines alone ( i.e. , sedation by an independent trained medical person including a qualified nurse ) can help clinicians avoid many medico - legal pitfalls . the sec largest subclass of respiratory events was difficult tracheal intubation ( 14.3% of all respiratory events ) . of these , there were two cases of an anticipated difficult airway ( cervical fracture and ankylosing spondylitis with morbid obesity ) , in which the first strategy was persistent attempts at laryngoscopic intubation without any preparation for a difficult airway . none of the difficult intubation cases reflected the use of a supraglottic airway such as a laryngeal mask airway for providing rescue ventilation in difficult airway management ( 14 ) . in five cases , an emergency tracheostomy was finally attempted , but was unsuccessful , thereby resulting in death . in cases of difficult airway , repeated attempts at laryngoscopic intubation can lead to a ' can not intubate and can not ventilate ' situation , as well as airway and hemodynamic complications ( 15 ) . thus , familiarity with difficult airway practice guideline ( 14 ) and the skills required to anticipate and manage a difficult airway are essential for every clinician who performs anesthesia or sedation . one closed claims study ( 16 ) found that after publication of the guidelines , difficult airway claims associated with death or brain damage during induction decreased from 62% to 35% . aspiration was considered the primary damaging event in five cases , of which three cases did not follow the standard preoperative fasting time . during the last decade , policy and practice regarding ' nothing by mouth ' ( npo ) status before elective surgery have been relaxed , especially with regard to clear fluids . however , it is notable that the latest asa guidelines ( 17 ) maintain a npo time of more than 6 - 8 hr for solids before elective surgery , and this has been accepted rigidly in our courts . during the perioperative period of major non - cardiac surgery , the incidence of myocardial infarction has been reported to be 1%-3% ( 18 ) . in the present analysis , acute myocardial infarction was the most common cardiovascular event ( 12 cases , 44.4% of cardiovascular events ) , all of which resulted in death . of them , nine patients showed a new onset of acute myocardial infarction during or after the surgical procedures . the symptoms of myocardial ischemia are rare or often silent , making the detection of inducible myocardial ischemia , very difficult . however , in - depth analysis of our cases revealed disproportionate gender ( male dominance : 8 men , 4 women ) and age ( median : 50.0 yr ; interquartile range : 39.0 - 58.0 yr ) distributions . thus , in male patients aged 40 yr , more attention may be necessary to identify atherosclerotic risk factors in preoperative history taking and the physical examination , as recommended in the latest american college of cardiology / american heart association guidelines for perioperative cardiovascular evaluations ( 19 ) . korean national health insurance claim data also indicated that males and patients aged 45 yr represented 69.9% and 93.0% of newly hospitalized patients with acute myocardial infarction in 2010 , respectively ( 20 ) . because myocardial infarction can be brought on by ischemic imbalance other than coronary artery disease alone , the risk for myocardial infarction can be further reduced by avoiding myocardial supply / demand mismatch conditions ( e.g. , inadequate pain control , hypoxemia , anemia , or severe hemodynamic alterations ) . our analysis identified two cases of nurse anesthesia . unlike the certified registered nurse anesthetists ( crnas ) system in the united states ( 21 ) , nurse anesthesia is legally prohibited in korea . of course , the nurses can assist clinicians ' anesthetic practice in the limited area , which is legally deemed the nursing practice . although the korean government has still maintained nurse - anesthetists since 1973 , recent precedent of the supreme court clearly stated that even nurse - anesthetist can not perform anesthetic practice licensed to the doctors , and it is also a violation of medical law , even performed in the direction of the supervised doctor . in contrast to the cases related to non - anesthesiologists , most cases involved with anesthesiologists ( 49/61 , 80.3% ) was associated with general anesthesia . in 27 cases , adverse events occurred in the postanesthesia care unit or ward . legally , the responsibility for anesthesia providers holds until the regaining of consciousness in normal patients , not simply the restoration of normal respiration . especially , it is notable that in 3 cases , adverse respiratory events developed after anesthesiologists had left the hospital in office - based anesthesia setting . though the cases involved with anesthesiologists had a similar incidence of death to those related to non - anesthesiologists ( 78.7% vs. 81.0% ) , both groups showed a different distribution of damaging events : increased prevalence of cardiovascular events in the cases involved with anesthesiologists ( 20/61 [ 32.8% ] vs. 6/42 [ 14.3% ] ) . this result may be mainly attributed the findings that anesthesiologists were involved in higher asa patients undergoing more complex surgical procedures . however , this result highlights the importance of surveillance for cardiovascular events , especially in the anesthesia community . compared with the closed - claims analysis of the asa - ccp , the ksa legislation committee database enables us to identify recent changes in anesthetic injury trends . closed claims analyses are limited by a long time lag ( approximately 3 - 6 yr in korea ) between the adverse event and the closure of the claim , when it becomes available for further study . nonetheless , this study has some limitations in interpretation . first , only the injury cases that were referred to the ksa can be investigated ; the actual numbers and nature of all other injuries remain unknown . in the united kingdom , only 1.5%-3% of patients who experience possibly negligent care actually file a malpractice claim ( 22 ) . this may introduce a bias towards the inclusion of cases involving more severe injuries in our database . sec , clear cause and effect conclusions can not be drawn from our analysis due to inherent defects such as the retrospective nature , absence of denominator data , and no use of a standardized protocol for diagnosis or management . in particular , as mentioned in a previous report ( 3 ) , some data for several cases seemed doubtful in terms of authenticity . regarding the narrative statements from the healthcare personnel involved , they tended to lack impartiality . lastly , some criticisms may be raised about our evaluation scale for the appropriateness of anesthesia care ( i.e. , three nominal classes with an individual 3-point nrs score ) in that it was independently rated by different reviewers . however , the consistency with which appropriateness of care can be judged by different reviewers has been demonstrated previously ( 5 , 6 ) . actually , there are not enough authoritative guidelines currently in existence to cover the range of issues involved in medical malpractice cases . previous study ( 23 ) suggested that there were only enough guidelines to provide useful guidance in about 20% of malpractice cases . in conclusion , the present analysis using the ksa database of anesthesia - related medical disputes demonstrated several ' typical ' injury profiles : an apparent lack of vigilance in less invasive procedures in apparently healthy patients , hypoxia secondary to airway obstruction or respiratory depression in a poorly controlled sedation environment , a ' can not intubate and can not ventilate ' situation as a result of non - compliance with the guidelines for difficult airway management , and high proportion and mortality of perioperative acute myocardial infarction . in almost half of the cases , the resulting injuries were determined to be ' avoidable ' if an appropriate standard of care had been followed . therefore , patient safety and obviation of malpractice litigation can be increased by a clear understanding of these typical injury profiles and strict adherence to established practical guidelines ; careful pre - procedural evaluation , adequate level of intra - procedural monitoring , and proactive responses to adverse occurrences .
the raw reads and gene expression data have been deposited in the national center for biotechnology information ( accession no . the transcription factor slzfp2 negatively regulates aba synthesis during tomato fruit development and ripening , and further protein - dna binding assays identified the ( a / t)(g / c)tt motif as its core dna binding sequences . in the wild type , aba production is drastically reduced after fruit set , usually completed around 2 dpa , in tomato . when suppressing slzfp2 expression in tomato by rna interference ( rnai ) , the young developing fruits accumulated significantly higher aba content . to better understand the impact on global gene expression by down - regulation of slzfp2 expression and also to identify its direct targets during early fruit development , we conducted a high - throughput rna - seq analysis on 2 dpa fruits from the slzfp2 rnai line and its nontransgenic sibling ( as wild type control ) . the whole fruits collected from hand - pollinated inflorescences of 35 plants were used for rna - seq analysis and the experiment was conducted in three biological replicates . the representative rnai line ( 207 ) showed significant reduction of slzfp2 expression in the fruits as analyzed by quantitative rt - pcr . the rnai line 207 and its nontransgenic sibling ( 207n ) were grown in the phytotrons at 20 c to 25 c under a humidity of 70% to 80% , with daily illumination for 16 h by 150 me m s light from metal halide and high - pressure sodium lamps . the whole fruits from hand - pollinated flowers were collected and immediately frozen in liquid nitrogen and stored at 80 c . total rna was extracted whole fruits with trizol reagent ( invitrogen ) based on the method previously described . the integrity of isolated total rna was checked by gel electrophoresis in rna mops ( 3-n - morpholino - propanesulfonic acid ) buffer , then quantified using nanodrop 2000c ( thermo fisher scientific inc . , barcoded paired - end sequencing libraries were constructed for the six samples using the truseq stranded mrna kit ( rs-122 - 2101 , illumina ) based on the manufactory 's instructions . we sequenced the six libraries in one run on an illumina 's miseq system using the 500-cycles miseq reagent kit ( ms-102 - 2003 ) . after in - house quality control and removal of the index sequences , the 250-bp paired - end reads were mapped to the tomato reference genome ( itag2.5 ) using the tophat program version 2.0.12 . the mapping was guided by current genome annotation and following parameters were used : read - mismatches 5 read - gap - length 3 read - edit - dist 5 library - type = fr - firststrand splice - mismatches 0 and default settings for other parameters . in total , the numbers of read pairs mapped for each replicate were as follows : 1,497,635 ( 207n , replicate 1 ; 80.7% mapped ) , 1,191,334 ( 207n , replicate 2 ; 80.6% ) , 1,279,658 ( 207n , replicate 3 ; 83.2% ) , 573,982 ( 207 , replicate 1 ; 80.2% ) , 749,210 ( 207 , replicate 2 ; 80.2% ) , and 1,158,542 ( 207 , replicate 3 ; 80.5% ) . uniquely mapped reads were then assembled by cufflinks program 2.2.1 using following parameters : gtf - guide frag - bias - correct min - frags - per - transfrag 10 . then , differentially expressed genes ( adjusted p value of 0.05 or less ) were selected by comparison between the rnai line and its nontransgenic sibling ( wild type ) using cuffdiff command in the cufflinks program with following parameters used : frag - bias - correct multi - read - correct min - alignment - count 10 fdr 0.05 . the analysis identified a total of 2722 differentially expressed genes in the 2 dpa fruits between the slzfp2 rnai line 207 and the wild type . after further comparison with the gene list of putative slzfp2 targets based on bacterial one hybrid screening , 193 genes were identified as direct targets of slzfp2 in early fruit development . the transcription factor slzfp2 negatively regulates aba synthesis during tomato fruit development and ripening , and further protein - dna binding assays identified the ( a / t)(g / c)tt motif as its core dna binding sequences . in the wild type , aba production is drastically reduced after fruit set , usually completed around 2 dpa , in tomato . when suppressing slzfp2 expression in tomato by rna interference ( rnai ) , the young developing fruits accumulated significantly higher aba content . to better understand the impact on global gene expression by down - regulation of slzfp2 expression and also to identify its direct targets during early fruit development , we conducted a high - throughput rna - seq analysis on 2 dpa fruits from the slzfp2 rnai line and its nontransgenic sibling ( as wild type control ) . the whole fruits collected from hand - pollinated inflorescences of 35 plants were used for rna - seq analysis and the experiment was conducted in three biological replicates . the representative rnai line ( 207 ) showed significant reduction of slzfp2 expression in the fruits as analyzed by quantitative rt - pcr . the rnai line 207 and its nontransgenic sibling ( 207n ) were grown in the phytotrons at 20 c to 25 c under a humidity of 70% to 80% , with daily illumination for 16 h by 150 me m s light from metal halide and high - pressure sodium lamps . the whole fruits from hand - pollinated flowers were collected and immediately frozen in liquid nitrogen and stored at 80 c . total rna was extracted whole fruits with trizol reagent ( invitrogen ) based on the method previously described . the integrity of isolated total rna was checked by gel electrophoresis in rna mops ( 3-n - morpholino - propanesulfonic acid ) buffer , then quantified using nanodrop 2000c ( thermo fisher scientific inc . , barcoded paired - end sequencing libraries were constructed for the six samples using the truseq stranded mrna kit ( rs-122 - 2101 , illumina ) based on the manufactory 's instructions . we sequenced the six libraries in one run on an illumina 's miseq system using the 500-cycles miseq reagent kit ( ms-102 - 2003 ) . after in - house quality control and removal of the index sequences , the 250-bp paired - end reads were mapped to the tomato reference genome ( itag2.5 ) using the tophat program version 2.0.12 . the mapping was guided by current genome annotation and following parameters were used : read - mismatches 5 read - gap - length 3 read - edit - dist 5 library - type = fr - firststrand splice - mismatches 0 and default settings for other parameters . in total , the numbers of read pairs mapped for each replicate were as follows : 1,497,635 ( 207n , replicate 1 ; 80.7% mapped ) , 1,191,334 ( 207n , replicate 2 ; 80.6% ) , 1,279,658 ( 207n , replicate 3 ; 83.2% ) , 573,982 ( 207 , replicate 1 ; 80.2% ) , 749,210 ( 207 , replicate 2 ; 80.2% ) , and 1,158,542 ( 207 , replicate 3 ; 80.5% ) . uniquely mapped reads were then assembled by cufflinks program 2.2.1 using following parameters : gtf - guide frag - bias - correct min - frags - per - transfrag 10 . then , differentially expressed genes ( adjusted p value of 0.05 or less ) were selected by comparison between the rnai line and its nontransgenic sibling ( wild type ) using cuffdiff command in the cufflinks program with following parameters used : frag - bias - correct multi - read - correct min - alignment - count 10 fdr 0.05 . the analysis identified a total of 2722 differentially expressed genes in the 2 dpa fruits between the slzfp2 rnai line 207 and the wild type . after further comparison with the gene list of putative slzfp2 targets based on bacterial one hybrid screening , 193 genes were identified as direct targets of slzfp2 in early fruit development .
it is regarded as an unusual healing pattern of appendicitis in contrast to the conventional pattern . this type of tissue reaction can involve any organ but the most common sites are kidney and gallbladder . most of the patients present with a picture of acute or subacute abdominal pain and occasionally with a mass lesion in the right iliac fossa . mass lesions in the right iliac fossa can mimic locally advanced cancer but they have a benign course and can be cured by surgical resection . to avoid extensive resection , intraoperatively , imprint cytology can be used to decipher the cause of the mass . due to the rarity of xa itself and the use of imprint cytology for intraoperative diagnosis the case is being presented . a 47-year - old female was brought to the surgical emergency room with the complaints of severe pain abdomen , vomiting , and fever . on clinical examination , there was tenderness on the mcburney 's point , rebound tenderness , and a palpable lump . routine blood tests revealed a total leucocyte count ( tlc ) of 14.0 10/l and the erythrocyte sedimentation rate ( esr ) was 90 mm ist hour . ultrasonography remained inconclusive and computed tomography ( ct ) scan of the abdomen showed a large soft - tissue mass in the right lateral pelvis with adherent loops of intestine . the walls of intestine were thickened along with three enlarged lymph nodes in the vicinity . radiologically differential diagnosis of inflammatory versus neoplastic mass was given and the patient was taken up for exploratory laparotomy . on exploration , a jumbled up mass in the right iliac fossa with adherent and thickened large intestine and lymph nodes was identified . intraoperative imprint cytology was performed following which a limited right colon resection and lymph node removal were done . smears revealed benign glandular epithelial cell groups and sheets of xanthoma cells along with multinucleate histiocytic giant cells in the background of neutrophils and mononuclear inflammatory cells [ figure 1 ] . imprint cytology showing foamy histiocytes , lymphocytes , and histiocytic giant cells ( giemsa , 40 ) a jumbled up mass , comprising 2-cm part of small intestine , 5.5-cm long swollen appendix , adherent omentum , and 3-cm long large intestine , was received along with six lymph nodes . cut surface of the appendix showed markedly thickened wall with obliterated lumen showing gray white and yellow foci . microscopic examination revealed appendicular lining with focal ulceration and dense inflammatory cell infiltrate in the wall comprised of lymphocytes , plasma cells , histiocytes , eosinophils , foci of sheets of foamy macrophages , pools of extravasated mucin , marked foreign body giant cell reaction , and fibroblastic reaction against it [ figure 2 ] . microscopic view showing ulceration of lining epithelium with underlying inflammatory infiltrate ( h&e , 40 ) ( inset shows a high power view of foamy histiocytes and two giant cells ) smears revealed benign glandular epithelial cell groups and sheets of xanthoma cells along with multinucleate histiocytic giant cells in the background of neutrophils and mononuclear inflammatory cells [ figure 1 ] . imprint cytology showing foamy histiocytes , lymphocytes , and histiocytic giant cells ( giemsa , 40 ) a jumbled up mass , comprising 2-cm part of small intestine , 5.5-cm long swollen appendix , adherent omentum , and 3-cm long large intestine , was received along with six lymph nodes . cut surface of the appendix showed markedly thickened wall with obliterated lumen showing gray white and yellow foci . microscopic examination revealed appendicular lining with focal ulceration and dense inflammatory cell infiltrate in the wall comprised of lymphocytes , plasma cells , histiocytes , eosinophils , foci of sheets of foamy macrophages , pools of extravasated mucin , marked foreign body giant cell reaction , and fibroblastic reaction against it [ figure 2 ] . microscopic view showing ulceration of lining epithelium with underlying inflammatory infiltrate ( h&e , 40 ) ( inset shows a high power view of foamy histiocytes and two giant cells ) xanthogranulomatous inflammation is a well - documented but uncommon entity that occurs at many sites in the body . though initially described in kidney in 1944 , it occurs in other organs including fallopian tubes , endometrium , testicle , female and male genital tract , gallbladder , pituitary , colon , retroperitoneum , adrenal , and appendix . xanthogranulomatous appendicitis ( xa ) is a rare entity with only 11 cases reported in literature . of these three cases presented as mass lesion , chuang et al . in 2005 reported a case of a 39-year - old man who was admitted with fever , lower abdominal pain , and a mass in the right iliac fossa . with a suspicion of cancer , hemicolectomy was performed but on histopathological examination it turned out to be xa . omar et al . , in 2011 reported a rare case of xa and cecal angiolipoma in a patient who was admitted with acute appendicitis and an appendicular mass . in 2013 , mado et al . reported a case of a 78-year - old patient with abdominal pain , and the ct scan showed a 4-cm irregular mass near the caecum . the patient was operated with a clinical diagnosis of appendiceal mucocele , but the histopathological findings were consistent with xa . our case also presented with a mass lesion in the right iliac fossa and was explored with the possibility of carcinoma colon . the proposed mechanisms for the occurrence of xanthogranulomatous inflammation are defective lipid transport ; immunological disorders altering the chemotaxis of leucocytes and macrophages ; reaction to a specific infectious agent , such as proteus and escherichia species ; infection by low virulence organisms ; and lymphatic obstruction . the particular mechanism eliciting xa are organ obstruction , suppurative infection , hemorrhage , defective lipid transport , and local hypoxia . our case was remarkable for the finding of xa in a 47-year - old female who presented with acute abdomen and a mass lesion in the right iliac fossa that is suggestive of a cecal growth . this case is reported because of its rare presentation and the possibility that xa should be considered in the differential diagnosis of carcinoma colon . this case also shows the usefulness of on - site touch cytological evaluation for quick diagnosis .
the benefits of exercise in the prevention of chronic diseases including overweight and obesity are well documented . regular physical activity reduces blood pressure , creates a more favorable lipid profile , and reduces risk for stroke , coronary heart disease , hypertension , and colon cancer [ 1 , 2 ] . regular exercise also helps maintain healthy body weight and may aid in weight loss and weight loss maintenance . to help prevent weight gain ( or obesity ) , the 2008 physical activity guidelines for americans , sponsored by the centers for disease control and prevention and healthy people 2020 , suggests incorporating a minimum weekly total of two and a half hours of moderate - to - vigorous intensity physical activity , spread over most days of the week . working up to five or more hours per week ( ~60 min / day ) although the aforementioned recommendations , if followed , are likely to have a major impact on health , intervention studies find that exercise without intentional food restriction and/or behavior modification does not effectively promote weight loss , [ 5 , 6 ] , particularly in women [ 7 , 8 ] . this may be because exercise stimulates a compensatory ( relative to the energy expenditure of the activity ) or noncompensatory drive to eat that is either biologically(i.e . these studies , however , are not consistent with short - term experimental studies conducted mostly in men which have found reductions in appetite and relative food intake following moderately intense - to - vigorous exercise . this may be because the exercise - induced effect is influenced by factors including the intensity and mode of the exercise [ 911 ] , the sex , and body composition of the exerciser [ 9 , 10 ] . several previous studies found that hunger and/or food intake are suppressed following 3090 min of intense- but not necessarily light - to - moderate intensity exercise [ 1218 ] including cycling , running , and brisk walking . less is known concerning individual differences ; however , one study found suppressed hunger and food intake in lean but not overweight women following bicycle exercise . the recent discovery of several gut peptides involved in appetite regulation and energy homeostasis provides an attractive mechanism to explain how exercise reduces hunger / appetite in some conditions and increases it in others . alterations in circulating ghrelin , the only known orexigenic gut peptide , along with the anorexigenic gut peptides peptide yy ( pyy ) and glucagon - like peptide-1 ( glp-1 ) may work in concert to influence exercise - associated alterations in hunger and food intake . alterations in circulating gut peptides appear to regulate food intake for as long as 24 h and are not specifically controlled by body fat stores . a number of previous studies have found that these peptides are altered by an acute bout of exercise [ 16 , 17 , 2228 ] ; however , the majority of studies evaluated only a single mode of exercise compared to rest . in addition , only a few of these studies simultaneously evaluated both the orexogenic and anorexogenic gut peptides [ 17 , 27 , 28 ] , and few included women [ 23 , 27 , 28 ] . the purpose of this study was to assess the effect of a 60-minute bout of exercise on circulating concentrations of gut peptides ghrelin , pyy , and glp-1 ; appetite and ad libitum food intake among women . an additional purpose was to assess whether alterations in these gut peptides were associated with alterations in appetite following exercise . exercise was performed at a moderately hard intensity in two different modes : running and walking . we hypothesized that circulating ghrelin would be suppressed ; pyy and glp-1 concentrations elevated following both modes of exercise compared to rest . furthermore , we hypothesized that ghrelin concentration would be directly correlated with ratings of hunger and desire to eat and pyy and glp-1 concentrations would be indirectly correlated . nine endurance - trained female runners and ten habitual walkers between the ages of 1840 were recruited for the study . to qualify , participants had to be in good general health , have normal hemoglobin ( between 14.018.0 mg / dl ) and thyroid status ( thyroid stimulating hormone between 0.404.50 mlu / l ) , have regularly occurring menstrual cycles , and be of low exercise risk as per the american college of sports medicine ( acsm ) . the runners had to be currently running at least 32 km / wk , be performing runs of at least 60 min in duration as part of their training regiment , and have maximal aerobic capacity ( vo2max ) of at least 45 ml / kg / min . the walkers had to be performing walks of at least 60 min in duration three or more days / wk and have a vo2max of less than 40 ml / kg / min . participants were excluded if they smoked , were anemic , hyper - or hypo - thyroid , pregnant or postmenopausal , had renal , hepatic , endocrine , gastrointestinal , pulmonary , cardiac , or hematological diseases including high blood pressure ( > 120/80 mm / hg at rest ) , prediabetes / diabetes , demonstrated signs of significant depression , anxiety , other psychological problems , alcoholism or other substance abuse , used prescription or over the counter medications ( other than contraceptives ) , or herbal preparations that can influence metabolism , had food allergies , or were unwilling to consume all foods / beverages provided in the run - in diet . volunteers were fully informed of possible risks of all procedures before providing written informed consent . approximately two weeks before initiation of the experimental protocol , vo2max was determined on a motor - driven treadmill ( trackmaster tmx22 , newton , ks , usa ) in accordance with acsm recommendations . for most runners , testing was initiated at 6 mph ( 0% grade ) with the grade increasing by 1% every min until exhaustion . for the walkers , the test was initiated at 3.5 mph ( 2% grade ) with grade increasing by 1% every min until exhaustion . oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) were measured continuously using a metabolic cart ( parvomedics tureone 2400 , sandy , ut , usa ) , and heart rate ( hr ) was monitored by an electrocardiography machine ( quinton q-5000 , bothell , wa , usa ) . rating of perceived exertion ( rpe ) was assessed during the last 10 seconds of each stage using the modified borg scale . the highest 20-second vo2 and respiratory exchange ratio ( rer ) achieved in the final two min of exercise were recorded as the maximum values . to qualify as an acceptable maximum test , participants had to meet two of the four following criteria : ( 1 ) a leveling or plateau of vo2 ( defined as an increase of < 2 mlkgmin with increased workload ) ; ( 2 ) rer 1.10 ; ( 3 ) maximum heart rate within 10 beats of age predicted maximum [ 208 ( 0.7 age ) ] ; ( 4 ) rating of perceived exertion ( rpe ) 17 . after a 30 min recovery period , participants underwent a titration run / walk to determine the speed and grade required to elicit an oxygen uptake of 70% vo2 max . for descriptive purposes , body composition was measured using dual - energy x - ray absorptiometry ( dexa , ge lunar prodigy 8743 , waukesha , wi , usa ) . the study was a counterbalanced , cross - over study where participants completed an exercise and control ( rest ) test day . the two test trials were scheduled in the follicular phase of the participants ' menstrual cycle ( between days 1 and 11 ) and spaced either 2 to 10 days or 1 menstrual cycle ( 3 to 5 wks ) apart . the exercise test day consisted of a 60 min run / walk at 70% vo2 max followed by 2 h of rest , whereas the control day consisted of 3 h of rest . food intake was controlled for 24 h prior to each test day by providing participants with a controlled diet . the diet provided 2000 kcal ( 64% carbohydrate , 14% protein , and 22% fat ) from commercially available foods and beverages plus an optional additional 200 kcal provided as two 100 kcal snack bars ( 28.4 g , ~100 kcals ; clif bar and company , berkeley , ca , usa ) . volunteers were asked to consume the foods provided ( and nothing in addition other than water ) and to return empty wrappers and any food and beverages that could not be consumed . on both test days , participants consumed a standard breakfast ( boost smoothie , clif builder bar and 2 cups of water ; ~380 kcal ; 65% carbohydrate , 20% protein , 15% fat ) at 0630 prior to arriving in the laboratory . at 0730 , on the control visit , an intravenous indwelling ( iv ) catheter was inserted into an arm or hand vein and connected to a normal saline solution ( 0.9% saline solution ) that was slowly infused to keep the catheter patent . blood was drawn immediately before ( baseline , preexercise ) and immediately after one h of rest ( t = 0 min ) and every 30 min thereafter for 2 h ( t = 30 , 60 , 90 , 120 min ) . the first 3 cc at each time point draw was presumed to be diluted with saline and was discarded . resting energy expenditure ( ree ) and respiratory quotient ( rq ) were measured using a metabolic cart ( 160 lpm pneumatach , parvomedics , trueone 2400 , sandy , ut , usa ) while the subject lay motionless in the supine position , as previously described . the last 20 min of data was used to calculate ree . on the exercise day , baseline blood was obtained by venipuncture . an iv catheter was inserted immediately following the 60 min run / walk , and blood was drawn on the same schedule as the rest day . for 10 min at the beginning ( between 515 min after the start ) and end ( last 5 min ) of the run / walk session , vo2 , vco2 , rer and rpe , were measured using a metabolic cart ( 800 lpm pneumatach , parvomedics , trueone 2400 , sandy , ut , usa ) . exercise pace was adjusted , if necessary , during the first 10 min to achieve an oxygen cost as close to 70% vo2max as possible but was not further adjusted . hr was monitored continuously using a portable heart rate monitor ( polar s60i , polar , port washington , ny , usa ) . on both test days , hunger and satiety were assessed using 100 mm visual analogue scales ( vass ) , anchored at each end with a word describing the extremes of the appetite being measured . ( 2 ) how satisfied do you feel ? ; ( 3 ) how full do you feel ? hunger and satiety ratings were obtained 4 to 5 min before each blood draw ( t = 0 , 30 , 60 , 90 , and 120 ) and at 20 min after initiation of the ad libitum meal ( see below ) . at completion of the exercise and rest sessions ( 120 min ) , participants were offered an ad libitum , free - choice meal . the free - choice meal consisted of the following ( in weighed portions ) attractively and consistently arranged on the dining table : rigatoni pasta ( 140 g dry , cooked ) , marinara sauce ( 140 g ) , alfredo sauce ( 140 g ) , whole - wheat bread ( 2 slices ) , white bread ( 2 slices ) , hard boiled eggs ( 2 ) , apples ( 2 ) , oranges ( 2 ) , clif snack bar ( 2 ) , clif builder bar ( 2 ) , nonfat yogurt ( 1 , 6 oz ) , regular yogurt ( 1 , 6 oz ) , individual portions of margarine ( 4 ) , honey ( 4 ) , peanut butter ( 4 ) , assorted jellies ( 4 ) , lemon - lime gatorade ( 2 , 20 oz ) , 2% milk ( 2 , 8 oz ) , and water ( ~1500 g ) . participants were given 20 min to eat the meal and were instructed to eat until satiety . participants were not allowed to read or study during the meal or carry backpacks , purses , or coats into the room . they were discretely monitored by the same investigator who worked quietly on a computer in the back of the room ( with their back turned toward the participant ) . food and water consumption were determined by weighing remaining food ( to the nearest 0.1 g ) at cessation of eating . by difference , food / beverages consumed were analyzed for total energy , protein , fat , carbohydrate , simple sugars , and fiber using nutritionist pro ( axxya systems , stafford , tx , usa , 77477 ) . ad libitum water intake and intake of energy and macronutrients as solids and liquids were also assessed . relative food intake was calculated by subtracting estimated energy expenditure during the exercise ( 60 min exercise , 120 min rest ) or rest ( 180 min rest ) sessions from the respective free - choice energy intake . blood samples taken both before exercise and rest ( baseline ) were analyzed for serum concentrations of progesterone concentration using a human solid phase ria kit ( siemens diagnostics , los angeles , ca , usa ) . plasma samples were analyzed for total ghrelin , acylated ghrelin ( ghrelinacyl ) , pyy3 - 36 , glp-1 , glucose , lactate and hematocrit at pre - exercise / rest and for 120 min following exercise and rest were analyzed for total ghrelin , acylated ghrelin ( ghrelinacyl ) , pyy3 - 36 , glp-1 , glucose , lactate , and hematocrit . blood samples for total ghrelin , ghrelinacyl , pyy3 - 36 , and glp-1 were collected into edta - treated prechilled tubes . blood collected for analysis of plasma ghrelinacyl was collected into a chilled tube containing 100 l of 200 mm aebsf with 200 l of 1 n hcl added per ml of plasma following centrifugation . samples collected for analysis of pyy were treated with 150 l of aprotinin and 40 l dpp - iv . all plasma samples were cold - centrifuged ( 28c ) for 10 min at 3500 rpm . aliquots of supernatant were stored in cryovials at 80c and batch - analyzed in duplicate at study completion by radioimmunoassay using commercially available kits specific for humans ( millipore , st . blood samples for analysis of glucose and lactate were collected into 4 ml purple top vacutainers , centrifuged as above , and the plasma was stored at 80c until analysis . glucose and lactate were analyzed using a microstat multiassay analyzer ( analox instruments , lunenburg , ma , usa ) . hematocrit was analyzed as a marker of hemoconcentration and hemodilution using an autocrit ultra 3 ( clay adams , sparks , mi , usa ) at each blood draw . a sample size analysis conducted with mean and standard deviation estimates based on preliminary exercise - associated data from russel et al . in women ( n = 10 ) and martins ( n = 6 men , 6 women ) for the pre - to post - exercise change in pyy3 - 36 ( 15 to 25% increase with exercise ; sds proportional to means ; ratio of sd to mean = 0.26 ) and an alpha = 0.05 determined that a sample size of n = 8 was sufficient to detect , with 80% power , a minimal postexercise increase of ~20% ( http://www.statsalive.com/ ) . given this calculation , an n = 9 for each exercise group ( i.e. , one additional subject per group ) was selected . the sample size calculation was not performed using ghrelin or ghrelinacyl due to inconsistent results for ghrelin and lack of published results for ghrelinacyl . our first aim was to assess the effect of exercise on circulating concentrations of gut peptides , appetite , and ad libitum food intake among runners and among walkers . concentrations of the gut peptides ( total ghrelin , ghrelinacyl ; pyy3 - 36 and glp-1 ) , and the primary measure of appetite , hunger ratings were measured at baseline and five time points ( t = 0 , 30 , 60 , 90 , and 120 minutes ) following exercise and rest . as secondary outcomes relating to appetite , we also considered ratings of satiety , fullness , and desire to eat . for each subject , we summarized the repeated measures by calculating the slope or the rate of change in outcome per 30-minute interval , following exercise and rest . using the derived slopes as the outcome , linear regression models were fit to evaluate differences between exercise and rest responses over the entire period of 120 minutes following exercise or rest . we adjusted for baseline levels and included an interaction between exercise / rest and runner / walker group . we calculated robust standard errors that accounted for correlation between exercise and rest measures from the same subject . as a way of capturing total response over all time points , the area under the curve ( auc ) was also calculated for the 120 minutes following exercise and rest for the gut peptide concentrations and hunger ratings using the trapezoid method ( graphpad prism version 5.02 for windows , graphpad software , san diego , ca , usa , http://www.graphpad.com/ ) . for ghrelin , ghrelinacyl , and glp-1 which were observed to dip below baseline in the later postexercise period , negative auc was also calculated as the area above the curve and below baseline . paired t - tests were used to evaluate differences between exercise and rest responses over the entire period of 120 minutes after exercise or rest , within runners and within walkers . ad libitum food intake was measured at a single time point following the ad libitum meal , as absolute energy intake and relative energy intake . again , paired t - tests were used to compare exercise versus rest within runners and within walkers . as secondary outcomes , we also considered more specific components of energy intake , using three macronutrients : protein , carbohydrate , and fat . as an exploratory analysis relating to this aim , we also assessed the immediate effects of exercise versus rest on gut peptide concentrations and appetite . that is , rather than considering the trajectory of each outcome across all time points from t = 0 to t = 120 , we considered only the difference between the measurements at t = 0 and at baseline for exercise versus rest . with these differences as the outcomes , we fit linear regression models , including an interaction between exercise / rest and runner / walker group . again , we calculated robust standard errors that accounted for correlation between exercise and rest measures from the same subject . our second aim was to investigate whether changes in ghrelin , ghrelinacyl , pyy , and glp-1 were associated with changes in hunger following exercise . for each gut peptide , we fit a linear regression model with the peptide concentration as the predictor of interest and hunger as the outcome . additionally , we included an interaction between hormone level and runner / walker group and adjusted for baseline hunger rating and exercise / rest period . again , we calculated robust standard errors to account for multiple measures from the same subject . statistical analyses were performed using stata ( version 10 ) and r statistical software ( version 2.10.1 ) . all reported p values were two - sided , with statistical significance taken to be p value < 0.05 . the majority of the runners regularly competed in local road races and two were ncaa division i collegiate runners . the walkers walked regularly either for fitness and weight control , or as cross - training for other activities . data from blood samples are missing for several participants at one or more time points following the exercise or rest periods due to complications from obtaining blood via the indwelling catheter ( i.e. , occasional clotting inadequate venous return ) . data for ghrelinacyl are missing for several time points due to undetectable readings by the ria . table 1(a ) shows that the exercise groups were fairly comparable with respect to age and height . not surprisingly , the runners were leaner than the walkers and had a higher vo2max than the walkers . comparing exercise versus rest , table 1(b ) and figure 3 show unexpected differences at baseline ( preexercise or rest ) in mean ghrelinacyl only among runners , whereas mean ghrelin , pyy , and glp-1 were similar in both groups before exercise and rest periods . differences in mean appetite ratings were also observed prior to exercise versus rest periods among both runners and walkers . the energy and macronutrient intakes of the controlled diet were similar ( p > 0.05 ) before exercise and rest in both the runner and walker groups . runners averaged 1868 380 kcal ( 14.1 1.7% protein , 64.8 3.2% carbohydrate , and 21.4 2.3% fat ) before exercise and 2035 239 kcal ( 14 0.6% protein , 63.6 1.3% carbohydrate , and 22.4 1.1% fat ) before rest . walkers averaged 1770 400 kcal ( 14.7 1.5% protein , 63.8 2.7% carbohydrate , and 21.5 2.6% fat ) before exercise and 1811 357 kcal ( 14.9 1.8% protein , 62.3 1.8% carbohydrate , and 22.7 1.5% fat ) before rest . in the runners , the exercise test day fell on menstrual cycle day 5.9 3.1 , whereas the rest day fell on day 5.2 2.9 . in the walkers , exercise and rest days fell on 5.4 serum progesterone concentrations were less than 2 ng / ml for all participants during both exercise and rest and averaged 0.66 0.2 and 0.64 0.19 for runners and 0.63 0.33 and 0.46 0.3 for walkers during exercise and rest , respectively , confirming follicular status . absolute vo2 , hr , rer , rq , rpe , and energy expenditure ( ee ) during the 60 min bout of running or walking and rest along with concentrations of lactate and glucose are shown in table 2 . runners ran at an average pace of 2.9 0.18 m / s ( 6.5 mph ) , and walkers walked at an average of 1.69 0.1 m / s ( 3.77 mph ) . absolute vo2 was higher during exercise in runners versus walkers , but relative vo2 was similar ( p = 0.624 ) and averaged 70.4 4.1% and 68.6 6.4% vo2max during the run and walk , respectively . hr was also ~10 bpm higher during exercise in the runners compared to the walkers ( 172 bpm versus 162 bpm ) but was similar between the rest trials . energy expenditure was approximately 180 kcals higher during exercise in runners compared to walkers ( 483.1 kcal / h versus 305.1 kcal / h ) but was similar at rest . blood concentrationas shown in figure 2 , total hematocrit was fairly constant over time following both exercise and rest in the runners and walkers . however , because hemoconcentration was observed following exercise in quite a few of the walkers and an occasional dilute samples from saline infusion was observed in both groups , blood data were adjusted according to the methods of dill and costill and used in all statistical analyses . as a sensitivity analysis , we repeated all analyses using the unadjusted data and obtained similar results ( data not shown ) . as shown in figure 2 , total hematocrit was fairly constant over time following both exercise and rest in the runners and walkers . however , because hemoconcentration was observed following exercise in quite a few of the walkers and an occasional dilute samples from saline infusion was observed in both groups , blood data were adjusted according to the methods of dill and costill and used in all statistical analyses . as a sensitivity analysis , we repeated all analyses using the unadjusted data and obtained similar results ( data not shown ) . ghrelin figure 3 ( upper left panel ) illustrates the average trajectory over all time points ( one time point before exercise or rest and five time point after exercise or rest ) . average total ghrelin concentration drifted upward immediately after exercise in the runners and the walkers , and then leveled off in the runners , while demonstrating large variability in the walkers . table 3(a ) reflects these results , indicating that exercise may increase the overall rate of change of ghrelin concentration in runners . while the rate of change after exercise stay close to zero ( 2.1 pmol / l per minute ) , a positive average postexercise slope ( 10.0 pmol / l per 30 minutes ) and an overall positive difference between exercise and rest ( 12.9 pmol / l per minute , 95% ci [ 3.9 , 29.7 ] , p value = 0.12 ) are estimated . meanwhile , for walkers , the larger variability at later time points may obscure the true effect of exercise . figure 3 ( upper right panel ) shows patterns for average ghrelinacyl concentration that are somewhat similar to those for total ghrelin . table 3(a ) shows little evidence of an exercise effect on the rate of change of ghrelinacyl concentration over time . there is some indication , however , of a larger immediate increase in ghrelinacyl concentration after exercise versus after rest among runners ( 7.9 pmol / l , 95% ci [ 0.9 , 16.7 ] , p value = 0.075).the positive auc ( area above baseline ) for both total ghrelin and ghrelinacyl was found to be higher following exercise in the runners but not the walkers . together , these results indicated that total response as measured by total ghrelin and ghrelinacyl tended to be higher after exercise than after rest among runners ( table 4 ) . figure 3 ( upper left panel ) illustrates the average trajectory over all time points ( one time point before exercise or rest and five time point after exercise or rest ) . average total ghrelin concentration drifted upward immediately after exercise in the runners and the walkers , and then leveled off in the runners , while demonstrating large variability in the walkers . table 3(a ) reflects these results , indicating that exercise may increase the overall rate of change of ghrelin concentration in runners . while the rate of change after exercise stay close to zero ( 2.1 pmol / l per minute ) , a positive average postexercise slope ( 10.0 pmol / l per 30 minutes ) and an overall positive difference between exercise and rest ( 12.9 pmol / l per minute , 95% ci [ 3.9 , 29.7 ] , p value = 0.12 ) are estimated . meanwhile , for walkers , the larger variability at later time points may obscure the true effect of exercise . figure 3 ( upper right panel ) shows patterns for average ghrelinacyl concentration that are somewhat similar to those for total ghrelin . table 3(a ) shows little evidence of an exercise effect on the rate of change of ghrelinacyl concentration over time . there is some indication , however , of a larger immediate increase in ghrelinacyl concentration after exercise versus after rest among runners ( 7.9 pmol / l , 95% ci [ 0.9 , 16.7 ] , p value = 0.075 ) . the positive auc ( area above baseline ) for both total ghrelin and ghrelinacyl was found to be higher following exercise in the runners but not the walkers . together , these results indicated that total response as measured by total ghrelin and ghrelinacyl tended to be higher after exercise than after rest among runners ( table 4 ) . pyy and glp-1 in runners , pyy concentration peaked immediately after exercise ( figure 3 ) then gradually returned to baseline over the 120 min after exercise ; whereas in walkers , pyy concentration peaked at 30 min after exercise before returning to baseline 90 min after exercise . table 3(a ) shows evidence of the effect of exercise on the rate of change of pyy , with exercise causing a faster decline in pyy concentration over time among runners ( 2.0 pmol / l per 30 minutes , 95% ci [ 4.0 , 0.095 ] , p value = 0.041 ) and among walkers ( 6.7 pmol / l per 30 minutes , 95% ci [ 13.2 , 0.14 ] , p value = 0.43 ) , although the effect was not statistically significant in the walkers potentially due to high variability . immediate effects were also evident , but positive ( table 3(b ) ) , thus reflecting the observed pattern of an immediate rise in concentration followed by a decline among both runners and walkers . the positive auc for pyy tended to be higher after exercise versus rest in the runners . negative auc was also found to be higher after exercise versus rest in walkers ( table 4).similar to pyy , glp-1 concentration peaked immediately after exercise in both runners and walkers returning to preexercise concentrations at approximately 30 min after exercise . unlike pyy , glp-1 dipped visibly below baseline after 60 min after exercise in both groups ( figure 3 ) . table 3(a ) shows fairly large effects of exercise on the rate of change of glp-1 . again , exercise caused a faster decline in glp-1 concentration among both runners ( 10.7 pmol / l per 30 minutes , 95% ci [ 17.0 , 4.4 ] , p value = 0.002 ) and walkers ( 16.5 pmol / l per 30 minutes , 95% ci [ 28.0 , 5.0 ] , p value = 0.008 ) . there was evidence of an immediate effect of exercise among runners , but not in walkers . the positive auc for glp-1 was not different following exercise compared to rest in either the runners or the walkers , however , the area below baseline was greater in the walkers ( 1957 2058 and 1447 3459 , p = 0.05 ) but not the runners after exercise versus rest ( table 4 ) . in runners , pyy concentration peaked immediately after exercise ( figure 3 ) then gradually returned to baseline over the 120 min after exercise ; whereas in walkers , pyy concentration peaked at 30 min after exercise before returning to baseline 90 min after exercise . table 3(a ) shows evidence of the effect of exercise on the rate of change of pyy , with exercise causing a faster decline in pyy concentration over time among runners ( 2.0 pmol / l per 30 minutes , 95% ci [ 4.0 , 0.095 ] , p value = 0.041 ) and among walkers ( 6.7 pmol / l per 30 minutes , 95% ci [ 13.2 , 0.14 ] , p value = 0.43 ) , although the effect was not statistically significant in the walkers potentially due to high variability . immediate effects were also evident , but positive ( table 3(b ) ) , thus reflecting the observed pattern of an immediate rise in concentration followed by a decline among both runners and walkers . the positive auc for pyy tended to be higher after exercise versus rest in the runners . negative auc was also found to be higher after exercise versus rest in walkers ( table 4 ) . similar to pyy , glp-1 concentration peaked immediately after exercise in both runners and walkers returning to preexercise concentrations at approximately 30 min after exercise . unlike pyy , glp-1 dipped visibly below baseline after 60 min after exercise in both groups ( figure 3 ) . table 3(a ) shows fairly large effects of exercise on the rate of change of glp-1 . again , exercise caused a faster decline in glp-1 concentration among both runners ( 10.7 pmol / l per 30 minutes , 95% ci [ 17.0 , 4.4 ] , p value = 0.002 ) and walkers ( 16.5 pmol / l per 30 minutes , 95% ci [ 28.0 , 5.0 ] , p value = 0.008 ) . there was evidence of an immediate effect of exercise among runners , but not in walkers . the positive auc for glp-1 was not different following exercise compared to rest in either the runners or the walkers , however , the area below baseline was greater in the walkers ( 1957 2058 and 1447 3459 , p = 0.05 ) but not the runners after exercise versus rest ( table 4 ) . as shown in figure 4 ( top panel ) , ratings of hunger increased from baseline across the 120 min after exercise or after rest period in both the runners and walkers during the exercise and rest trials . similar results were observed for desire to eat , that is , how much you think you can eat ( data not shown ) . ratings of satiety ( figure 4 , bottom panel ) and fullness ( data not shown ) tended to decrease from baseline across the 120 min after exercise or after rest period in both groups during both trials . we see from table 5 that there was not enough evidence to show an effect of exercise on the rate of change over time for any of the four subjective appetite ratings in either group . the aucs for the four appetite ratings were also not found to be significantly different after exercise versus after rest for either group . as shown in table 5 , there was no evidence of a difference between absolute energy intake and macronutrient intake at the free - choice meal following running versus rest . however , absolute intake tended to be higher following walking compared to rest ( 73.2 kcals , 95% ci [ 11.0 , 157.5 ] , p value = 0.080 ) . interestingly , walkers ( but not runners ) tended to consume more protein ( in walkers : 3.4 g , 95% ci [ 0.043 , 6.8 ] , p value = 0.048 ) and fat ( in walkers : 4.1 g , 95% ci [ 2.2 , 6.0 ] , p value = 0.001 ) following exercise compared to rest ( figure 5 ) . after adjusting for the cost of exercise or rest , relative energy intake was lower following exercise compared to rest in both groups ( p 0.001 ) . in runners , relative energy intake was 477.7 kcals lower ( 95% ci [ 610.1 , 345.3 ] ) after exercise compared to after rest . the difference was smaller in walkers , with after exercise being 274.6 kcals lower ( 95% ci [ 385.0 , 164.3 ] ) than after rest . in the runners , the change in concentrations of pyy and glp-1 was predictive of the change in hunger ( table 6 ) . analogous results were obtained for satiety , fullness , and desire to eat ( data not shown ) . increases in pyy and glp-1 were positively associated with satiety and fullness and negatively associated with hunger and desire to eat . in the walkers , there may be some indication of an association between hunger ratings and ghrelinacyl ( 0.63 units , 95% ci [ 0.19 , 1.46 ] , p value = 0.12 ) and glp-1 ( 0.095 units , 95% ci [ 0.32 , 0.028 ] , p value = 0.095 ) concentrations . the purpose of the current study was to evaluate the effect of 60 min of moderately hard running and walking at the same relative intensity ( i.e. , 70% of maximal oxygen uptake ) on gut peptide concentrations , appetite , and food intake at a single ad libitum meal offered 2 h after exercise in habitually active women . short - lived increases in circulating concentrations of the anorexogenic peptides and a trend for an increase in ghrelinacyl following exercise were apparent in the runners but not the walkers . these alterations in circulating gut peptides were associated with lower relative energy intake after exercise compared to rest which created a negative energy deficit in the runners but not walkers . interestingly , the average rate of change in the anorexogenic peptides pyy and glp-1 but not the orexogenic peptide ghrelin over time was found to predict hunger in runners but not walkers . ghrelin is secreted by specialized cells in the stomach and is currently the only known orexogenic peptide [ 35 , 36 ] . circulating concentrations of ghrelin peak during fasting , drop after a meal and are thought to be involved in hunger and meal initiation . peripheral infusion of ghrelin increases food intake in animals and humans through interaction with neuropeptide y ( npy ) and agouti - related protein ( agrp)-expressing neurons of the hypothalamic arcuate nucleus ( arc ) and/or inhibition of vagal - afferent nerves . although ghrelin is present in circulation in acylated ( ghrelinacyl ) and desacyl forms , only ghrelinacyl is thought to cross the blood - brain barrier and exert orexigenic effects [ 35 , 40 ] . thus , measurement of ghrelinacyl , which accounts for ~10% of circulating concentration , is important particularly because ghrelinacyl responds more rapidly to glucose infusion and exercise . our finding that the total response ( i.e. , auc ) of both total ghrelin and ghrelinacyl was elevated above rest following running but not walking at the same relative intensity is intriguing . previous studies have observed decreases [ 17 , 22 , 26 , 43 , 44 ] , increases [ 24 , 45 ] or no alteration [ 18 , 23 , 27 , 46 ] in both total ghrelin and ghrelinacyl concentrations following exercise , but these inconsistent findings may be due to the intensity ( or energy cost ) of the exercise employed and/or the sex of the exerciser . for example , total ghrelin was not altered by 60 min of submaximal cycling but was increased following 3 h of prolonged cycling and ~2 to 2.5 h of intense running . ghrelinacyl was also found to increase at a meal following treadmill walking in overweight women but not men but only when the energy lost through exercise was not replaced . overall , these results suggest that the energy cost of the exercise ( which was ~38% higher during running versus walking ) may promote increased ghrelin secretion , perhaps more so in women . coupled with our finding that neither total ghrelin or ghrelinacyl correlated with hunger , the results also suggests that ghrelin is not a large contributor to postexercise food intake perhaps because the signal is dampened by increases in the anorexogenic peptides over the same time point . in contrast to ghrelin , peptide yy and glp-1 are satiety peptides which are secreted from the endocrine l cells of the distal gastrointestinal tract in response to a mixed meal . circulating concentrations of both pyy and glp-1 are low in fasting and increase following meal ingestion . peripheral infusion of both peptides at physiological concentrations markedly decrease food intake in humans [ 49 , 50 ] which appears to be additive when infused simultaneously . the action of pyy is thought to be via inhibition of npy / agrp neurons and/or stimulation of vagal - afferent nerves , whereas the action of glp-1 is thought to be via vagal mediation . both forms of pyy ( pyy3 - 36 and pyy1 - 36 ) and glp-1 are thought to serve as satiety signals , regulating the termination of individual meals . consistent with our findings , previous studies have found elevations in both pyy [ 25 , 27 , 52 ] and glp-1 [ 27 , 52 ] following different modes and intensities of exercise . a study by ueda and colleagues found that postexercise elevation of pyy but not glp-1 was dependent on exercise intensity and was elevated to a greater extent following 30 min of cycling at 75% compared to 50% vo2max . in another study , broom and colleagues found elevated pyy and suppressed hunger in the 2 h after a 60 min bout of running at 69% vo2max compared to both rest and a 90 min bout of resistance exercise . it is important to note , however , that the energy cost was 50% higher in the high- compared to the moderate intense cycling and ~260% greater with the running compared to the resistant training in the afore - mentioned studies . thus , our finding that both pyy and glp-1 were elevated immediately after running , and that only pyy was elevated after walking may also be explained by the greater energy cost of the run , which was ~37% greater than the walk . interestingly , the average rate of change in pyy and glp-1 after the run , and the rate of change in glp-1 after the walk was significantly greater relative to rest , indicating an average downward trend following exercise , particularly for glp-1 which dipped below baseline in the later postexercise period . while it is possible this dip in glp-1 , which had a more negative auc in walkers compared to runners , at least partially accounted for the higher ( less negative ) relative intake in the walkers compared to the runners , future research is needed to affirm that such a role is causal . our results concerning ad libitum food intake following exercise in women are in agreement with previous studies in both sexes which found either no difference or slightly higher absolute food intake after a bout of exercise compared to a noexercise control , but significantly lower relative energy intake when accounting for the energy cost of exercise [ 12 , 13 , 5355 ] . interestingly , in these studies , relative energy intake was lowest ( i.e. , creating a more negative balance ) when exercise intensity was high , and when foods offered in the subsequent ad libitum meal were low in fat [ 13 , 53 , 54 ] . imbeault and colleagues , for example , found lower relative energy intake after 34 min of running at 75% vo2max than after 72 min of walking at 35% vo2max , which elicited the same energy cost ( ~485 kcal ) . king and colleagues , who were first to introduce the concept of relative energy intake , have argued the greater relevance of relative rather than absolute energy intake because higher energy intake would be an expected compensatory mechanism of increased energy expenditure through increased physical activity . thus , if energy intake remains the same following exercise , as in the current study , it can be considered equivalent to a suppression of appetite relative to the intake expected to compensate for the exercise . unfortunately , the majority of studies , including the current study , have not measured energy intake for a long enough period after exercise to evaluate how compensation for negative energy balance occurs following different modes of exercise like running but not necessarily walking . total or partial compensation through altered energy intake and reduced energy expenditure are possible and likely , otherwise exercise would result in drastic reductions in body mass / body adiposity . although we did not find significant differences in perceived hunger at any point following running or walking compared to rest , small changes in hunger due to exercise rather than time ( observed in the nonexercise control condition ) may be difficult to detect using available methodology . indeed , only about half of the studies in men using designs similar to ours have observed differences in hunger using vas [ 12 , 14 , 16 , 17 , 22 , 23 , 27 , 53 , 56 ] , whereas very few studies in women have detected exercise - associated differences [ 55 , 57 ] . the lack of a strong exercise influence on appetite in all studies may be because vas are not sensitive enough to detect small changes following exercise using sample sizes typically employed for exercise studies . it also may be that only a small subset of subjects is in tune with biological hunger cues and respond instead to other signals including time of day or time past since the last meal . mattes , for example , observed that food intake often occurred when hunger was low or had not changed acutely . in our studies we did find , however , that vas track well with changes in both pyy and glp-1 in runners and tended to track with glp-1 in walkers which suggests a relation between appetite ratings and satiety peptides even if exercise - induced alterations in appetite were not observed . the current study used a unique complex modeling approach to evaluate whether changes in the gut peptides tracked with or predict changes in hunger and/or ad libitum food intake . collectively , our findings suggest that changes in pyy and glp-1 over time tracked indirectly with changes in hunger and desire to eat , and directly with changes in satiety . interestingly , the change in either total ghrelin or ghrelinacyl did not track with subjective ratings of hunger . this provides additional support for the hypothesis that signals from elevated concentrations of circulating ghrelin may be muted by elevated concentrations of satiety peptides . given that few [ 16 , 22 , 25 ] previous studies have found clear associations between gut peptide concentrations and appetite following exercise , it is probable that exercise - induced alterations in appetite are driven by complex changes in appetite - regulating hormones rather than a single gut peptide in isolation . a previous study by martins and colleagues , for example , observed an inverse temporal pattern between hunger and both pyy and glp-1 concentrations during 1 h of exercise but did not describe such a relation following exercise . ueda et al . observed direct associations between the auc for plasma ghrelinacyl and hunger , and indirect association between the auc for glp and postexercise energy intake . the discrepancy between the findings and published studies may be explained by the different exercise - induced patterns of gut peptide release . in the current study , we elected to evaluate the effect of walking and running on appetite and gut hormone responses because both weight - bearing activities are recommended for weight loss and weight loss maintenance . walking , however , is the most common exercise recommended and , unlike running , can be undertaken by the majority of the population because it does not require the fitness base or produce the biomechanical stress of running . our overall observation that walking did not elicit the same negative energy balance or increase in the satiety hormones as did running , yet promoted a slightly higher postexercise fat and protein intake , suggests that walking may create some challenges for long - term weight loss unless dietary restriction is employed . while our results appear to contradict those of king and colleagues who observed significantly lower relative energy intakes in men after a 60-min brisk walk at a self - selected pace ( ranging from 33.8 to 55.5% vo2max ) , the apparently discrepant results may help explain why exercise is less effective in promoting weight loss in women compared to men [ 7 , 8 ] . the mechanism , however , may not be easily identified because ghrelinacyl was not altered by walking in either study , and king and colleagues unfortunately did not simultaneously measure pyy , glp or other satiety peptides . in our study , we also observed a curious tendency for ghrelinacyl , total ghrelin and subjective hunger to be lower when subjects knew that they were going to exercise , which may have interfered with our ability to detect true changes with exercise compared to rest . the increased consumption of fat may be important given that a reversal of the energy deficit induced by previous exercise is noted when high - fat rather than low - fat foods offered after exercise [ 13 , 53 , 54 ] . finally , from our study design , it is impossible to determine whether our observed differences between running and walking are due to exercise mode or the physiological characteristics of the walkers who were on average fatter and had a lower vo2max ( i.e. , were less fit ) then the runners . although the current study did not measure any long - acting adiposity hormone such as leptin or insulin , it is possible that these hormones were higher in the walkers . emerging evidence suggests that long- and short - acting signals interact to alter hypothalamic sensitivity to satiation signals which could ultimately influence eating behavior following exercise . future studies should consider different modes of exercise along with sex and adiposity differences of the exerciser and measurement of short- and long - acting satiety signals .
it is a spontaneous rupture of the esophagus caused by impaired coordination of the act of vomiting and attempts to stop it . the classic symptoms of bs , described in 1952 and known as mackler 's triad , are observed in less than half of the affected patients ; they include : severe and profuse vomiting , acute sharp pain behind the sternum and/or in the epigastrium , and the appearance of subcutaneous emphysema on the chest wall , neck , and face [ 29 ] . a combination of these symptoms may appear with diverse intensity ( one of them may be dominant , e.g. strong substernal pain ) , which may consequently lead to diagnostic errors that often end in tragedy for the patient . this results primarily from delays in the establishment of proper diagnosis and introduction of surgical treatment ( table i ) . on october 19 , 2012 , he was admitted to the department of general surgery of a regional silesian hospital due to midepigastric pain which had been intensifying for the previous two days as well as dyspnea accompanied by dry cough . examinations performed on admission revealed the presence of peritoneal signs involving the whole abdominal cavity as well as asymmetry of the vesicular murmur ( r < an abdominal x - ray examination was performed with the patient in a standing position . subsequently , abdominal computed tomography ( ct ) was conducted ( october 20 , 2012 ) , demonstrating the presence of free air under the dome of the diaphragm and fluid in the right pleural cavity ( thickness : up to 18 mm ) . after a short preparation , the patient was qualified for exploratory laparotomy , which revealed a duodenal perforation and ulceration of the gastric cardia . due to the challenges associated with repairing the perforation , a gastric tube was introduced , and partial gastric resection was performed using the hofmeister - finsterer method . during the surgical procedure , a drop in the ventilation parameters was observed , and the vesicular murmur on the right side became less pronounced . therefore , after closure of the abdominal cavity , double drainage of the right pleural cavity was introduced , aspirating 2000 ml of brown fluid . chest ct performed on the 2 postoperative day visualized an esophageal fistula , massive contrast leakage into the pleural cavity , mediastinum , and abdominal cavity along the wall of the gastric stump , and fluid in both pleural cavities . diagnosed with esophageal perforation , the patient was transferred to the chair and clinic of general and thoracic surgery in zabrze for further treatment . after the performance of basic examinations , the pleural cavity was opened , and 1400 ml of brown , cloudy fluid was aspirated . during the procedure , gangrenous changes were found in the mediastinum , extending to the level of the left pulmonary artery . the perforation was located immediately above the diaphragm , on the posterior wall of the esophagus ; its size was estimated at 3 cm . despite the fact that more than 48 h had passed since the development of the perforation due to difficulties with accessing the injury site , the anterior wall was incised in order to uncover the perforation . the posterior wall was treated with single - layer repair , and the anterior wall was treated with double - layer repair . after the procedure , the patient was mechanically ventilated , received broad - spectrum antibiotics , and was fed parenterally ; on the second postoperative day , he underwent tracheotomy . control examinations demonstrated a clear elevation of inflammatory markers : procalcitonin ( pct ) 3.56 ng / ml , c - reactive protein ( crp ) 331 mg / l . two dye tests performed during the early postoperative period did not indicate the presence of an esophageal leak . on the 8 postoperative day , a follow - up chest ct showed contrast leakage from the lower segment of the esophagus , extending along the spine and into the right pleural cavity ( fig . after the procedure , the patient 's condition stabilized , which allowed him to be extubated ; on the 37 day of hospitalization , the tracheotomy tube was removed . the follow - up laboratory investigation showed stabilization of the inflammatory parameters ( pct < 0.01 ng / ml ) . the patient was discharged in good condition , remaining under the supervision of the thoracic surgery outpatient unit . during this time , he fed normally , eating solid foods . in january , the patient was again admitted to the clinic in order to undergo removal of the esophageal stent using gastrostomy access . control x - ray of the upper gastrointestinal tract revealed an esophageal stricture ( fig . the patient was qualified for a surgical procedure : the strictured segment of the esophagus ( approximately 4 cm in length ) was resected using a thick gastric tube , and an end - to - end anastomosis was performed . directly from the operating theater , the patient was transferred to the intensive care unit ( icu ) , where he stayed for three days . presently , the patient is fed orally , and his body mass has increased by several kilograms . visible fluid in both pleural cavities and contrast leakage outside the esophageal lumen x - ray of the upper gastrointestinal tract with contrast . esophageal stricture although the clinical signs of esophageal perforation have been described in a number of academic guidebooks and numerous scientific reports , diagnosing bs remains challenging in many cases . this stems from the topography of the esophagus , which passes through three different body regions ( neck , chest , and abdomen ) ; its perforation may , therefore , give diametrically different symptoms . the diagnostic difficulties are further compounded by the rarity of the disease . at present , the most recommended method for diagnosing bs is ct using an oral contrast agent ( water solution ) . typically , the ct examination reveals the presence of air in the mediastinum and/or pleural cavity , esophageal wall injury , an esophagopleural fistula , pleural effusion , and the presence of a mediastinal abscess connected to the lumen of the esophagus . we did not perform an endoscopic examination ( esophagoscopy ) to diagnose boerhaave syndrome as this method is not recommended by the literature as a first - line diagnostic tool ( in spite of its 100% sensitivity and 83% specificity ) because it is a risky procedure in patients in severe general condition . what is more , due to the necessity of air insufflation during esophagoscopy , there is a considerable risk of increasing the extent of the perforation by pumping air into the mediastinum and distributing the often limited purulent content across the whole pleural cavity . there are reports of patients in whom endoscopy of the upper gastrointestinal tract showed no esophageal perforation , and the presence of pathology was only confirmed after ct with contrast [ 1416 ] . the primary factor affecting selection of the treatment method is the time from the moment of perforation until establishment of the diagnosis . many authors believe that a delay in the start of treatment of more than 24 h increases the risk of death and complications by 50% , while primary surgical repair of the esophagus is associated with an over 20% risk of secondary leak development . in the available literature , we have not encountered any reports describing concomitant ulceration of the duodenum and complicated bs . acute abdominal signs with the presence of air under the diaphragm , observed by our colleagues from the regional hospital , clearly indicated the diagnosis of a perforated duodenal ulcer and largely obscured the signs of bs . it was only during the first procedure that the drop in ventilation parameters and the quieting of respiratory murmurs on the right side suggested the presence of pathology in the chest . the patient was brought to the clinic in a severe condition , with signs of sepsis . despite the substantial delay in diagnosis ( > 48 h ) and the patient 's condition after the partial resection of the stomach access to the perforation site ( posterior wall , immediately above the diaphragm ) was additionally impeded by the fact that we could not maneuver the esophagus due to concerns about tearing apart or damaging the already created gastrointestinal anastomosis hence the decision to access the perforation site through the anterior wall of the esophagus . the secondary anastomotic leak observed on the 8 postoperative day was successfully repaired with a self - expanding stent . the esophageal stricture after the removal of the stent was probably caused by the previously diagnosed ulceration of the cardia and , perhaps , the incision of the anterior wall . the aim of this report is to draw attention to the diagnostic and therapeutic challenges associated with bs .
legg - calve - perthes ( lcp ) is the avascular necrosis or idiopathic osteonecrosis of the epiphysis of the femur head1 ) . the main factor that plays a role in the etiology is the decreased blood flow to the epiphysis of the femur head . it is more frequently observed among male children between the ages of two and 12 years12 ) . hypopituitarism is a clinical syndrome that develops due to a partial or complete insufficiency of all , or one or more , of the hormones produced in the pituitary gland3 ) . growth hormone deficiency ( ghd ) is typically characterized by decreased growth hormone ( gh ) secretion despite spontaneous and pharmacological warnings when there are no other causes to explain significant growth retardation , slow growth rate , bone age lag , and short stature . causes of ghd include genetic ( e.g. , gh or gh - secreting hormone gene defects ) , anatomical or congenital ( e.g. , midline defects , septo - optic dysplasia , or vascular malformations ) , and acquired ( e.g. , craniopharyngioma , glioma , or histiocytosis ) hypothalamic and hypophyseal anomalies45 ) . the current article presents a patient with complaints of short stature , a diagnosis of multiple hypophyseal hormone deficiency , and a history of lcp and difficult birth - related pituitary stalk interruption syndrome . the case was presented for the following reasons : lcp and hypophyseal hormone deficiency might have developed secondary to difficult birth ; lcp might have developed secondary to insulin - like growth factor ( igf)-1 deficiency related to gh deficiency ; and this association has not been previously published . a male patient aged four years and 10 months was brought in with a complaint of short stature . it was indicated that although his development was normal until 12 months of age , it halted afterwards . it was found out that the patient was born at term in breech position through cesarean section with a normal apgar score and birth weight of 3,750 g , hospitalized postnatally for five days due to birth in breech position and was not noted to be hypothermic and hypoglycemic , was diagnosed with lcp at four years of age following detailed examination due to complaints of pain in the hips , and consequently underwent surgery ( fig . family history indicated that the patient 's parents were related to each other within the second degree of consanguinity . his first sibling was being followed up for tangier disease , and his second sibling was being followed up for hyperlipidemia by the department of pediatric metabolism . physical examination findings showed that the patient was prepubertal , and that he had a height of 96 cm ( standard deviation score [ sds ] , -3.2 ) , body weight of 14.6 kg ( sds , -1.9 ) , 2 cm shorter left leg compared to right leg , a surgical scar in the left inguinal region , and stretched penile length of 4.5 cm . other systemic examination findings were normal . while the patient 's chronological age was four years and 10 months , his bone age was consistent with a 2-year - old 's ( according to greulich and pyle ) . laboratory tests yielded the following results : white blood cell count , 11,300/mm ; hemoglobin , 12.1 g / dl ; thrombocyte , 320,000/mm ; urea , 7 mg / dl ; creatinine , 0.9 ml / dl ; serum sodium , 148 mmol / l ; potassium , 4.3 mmol / l ; chloride , 102 mmol / l ; calcium , 9.7 mg / dl ; phosphorus , 4.4 mg / dl ; normal liver function ; fasting blood glucose , 92 mg / dl ; total cholesterol , 130 mg / dl ; high - density lipoprotein , 41 mg / dl ; low density lipoprotein cholesterol , 69 mg / dl ; triglyceride , 50 mg / dl ; and urine density , 1,022 g / cm . detailed laboratory analyses showed thyroid function consistent with central hypothyroidism ( st4 , 0.49 ng / dl [ 0.8 - 1.9 ng / dl ] ; thyroid - stimulating hormone , 2.57 mu / l [ 0.5 - 5 mu / l ] ) . the patient was also determined to have hypocortisolemia ( basal serum cortisol level of 2.75 g / dl , stimulated serum cortisol level of 5.71 g / dl [ with low - dose adrenocorticotropic hormone ] ) and ghd ( peak gh values in glucagon and levodopa tests while the patient was euthyroid were 0.257 and 0.46 ng / ml , respectively ) . serum igf-1 level was 15 ng / ml ( sds , -3 ) and insulin - like growth factor binding protein-3 ( igfbp3 ) level was 900 ng / ml ( sds , -2.6 ) . an etiology - oriented hypophyseal magnetic resonance imaging was performed due to high prolactin levels ( 37.4 ng / ml [ normal range , 0 - 25 ng / ml ] ) , which revealed in the sellar cavity center that the adenohypophyseal gland height was 3 mm , neurohypophyseal localization was abnormal , and that there was no signal intensity . in addition , t1a hyperintense nodular appearance with dimensions of 3 mm2.5 mm showing diffused homogeneous contrast retention following injection of contrast agent was observed at the median eminence level ( ectopic neurohypophysis ) . it was deduced and evaluated as a pituitary stalk interruption syndrome due to difficult birth . the patient was sequentially started on hydrocortisone , l - thyroxin , and gh therapy . although several causes have been believed to be responsible for the development of avascular necrosis of the femur head , none of them , except trauma , have yet to completely explain the biological process that plays a role in the development of this pathological phenomenon . it is known that osteonecrosis can develop as a result of trauma even in the absence of a fracture12 ) . although igf-1 levels have been found to be lower in lcp patients compared to normal population in the literature6 ) , the present case with a complaint of short stature , a diagnosis of multiple hypophyseal hormone deficiency , and a history of lcp , was presented for the following reasons : lcp and hypophyseal hormone deficiency might have developed secondary to difficult birth ; lcp might have developed secondary to igf-1 deficiency related to gh deficiency ; and this association has not been previously published . lcp is most frequently observed in males ( male : female , 5:1 ) aged between two and 12 years7 ) . although increased rates of lcp incidence have been reported in the presence of various factors , such as low birth weight , low socioeconomic development , and exposure to cigarette smoke , the main factor that plays a role in the etiology of the disease is decreased blood flow to the epiphysis of the femur head12 ) . furthermore , many predisposing factors have been suggested for the etiology of the disease , most showing varying degrees of effects in every study1267 ) . the present case was a male within the age group that the disease is most frequently observed in . he was at normal birth weight for the gestational month , and had no prenatal exposure to cigarette smoke . he also had no history of trauma other than at birth , and his diagnosis was made based on clinical and radiological findings . it stimulates the synthesis and secretion of igf-1 in the liver , and the autocrine and paracrine effects of igf-1 increase its production . postnatal growth of cartilage tissue depends on hypophyseal gh , and igf-1 partly mediates this effect458 ) . while the primary agent is igf-1 in the postnatal growth of cartilage , it is igf-2 during the prenatal period . igf-1 is not only produced in the liver and kidneys , but also in the chondrocytes of the growing cartilage . igf-1 , produced in the chondrocytes , stimulates the growth of cartilage by binding to the igf-1 receptors in the chondrocytes with its autocrine effects48 ) . igf-1 has a direct effect on the differentiation of prechondrocytes to young chondrocytes and their clonal growth . studies in the literature showed that mice with excised hypophysis exhibited very little growth when igf-1 was administered locally to the bone epiphysis . this is because differentiated cells required for the binding of igf-1 appear only after the differentiation of precursor cells by gh in the germinal layer of the growth plate . in studies conducted on mice with excised hypophysis , both administering gh alone and igf-1 alone led to an increase in body weight , long bone growth , and cell division910 ) . following administration , gh stimulates igf-1 production in tissues , and consequently , growth through its autocrine and paracrine effects . when igf-1 is administered , it travels directly to the target tissues and exerts mitogenic effects1011 ) . many researchers have agreed that abnormal igf-1 levels have an effect on the pathogenesis of the disease in lcp patients691011 ) . we suggest that gh deficiency , and igf-1 deficiency secondary to gh deficiency , caused the short stature and osteonecrosis in the current case by affecting bone development through the abovementioned mechanisms . in the literature , even though igf-1 levels were determined to be lower in lcp patients than in normal population , there was no difference in igfbp3 levels . there was also no difference in hypophyseal hormone levels between lcp patients who underwent surgery and those who have not612 ) . in a different study where anterior hypophyseal hormones were evaluated in 139 pediatric lcp patients , hypophyseal hormone deficiency was not observed in any of the patients13 ) . in conclusion , even though igf-1 levels have been determined to be lower in lcp patients than in normal population in the literature , the present case study was in which lcp was identified in a patient with ghd as a part of multiple hypophyseal hormone deficiency . we propose that igf-1 deficiency secondary to gh deficiency can be an effective factor in the etiology of lcp , and that patients with gh deficiency should be monitored closely for lcp development both before treatment and during gh therapy .
interferons are well established agents for standard therapy in several malignancies , hepatitis c , idiopathic pulmonary fibrosis , and multiple sclerosis.1,2 despite this , adverse autoimmune effects associated with their use have been reported , including minimal change disease in the kidney,25 collapsing focal segmental glomerulosclerosis,6,7 membranous glomerulonephritis,8 acute renal failure,9 lupus nephritis,10,11 acute renal failure,12 and thrombotic microangiopathy.1318 these side effects are most often associated with interferon - alpha therapy , rather than interferon - beta . the mechanism for this is not clear . glomerular endothelial cells express and secrete adamts 13.17 the low activity of adamts 13 has been associated with the presence of an anti- adamts 13 igg antibody during treatment with interferon - alpha 2a,16 which could explain these adverse side effects , but no mechanism has been described to explain this with interferon - beta . in the following report , we describe a case of hemolytic uremic syndrome causing thrombotic microangiopathy and chorioretinitis after several months on treatment with interferon - beta which is much rarer . a 37-year - old woman was admitted to our hospital with acute renal failure , hypertension , subnephrotic proteinuria , nausea , and vomiting . she reported a 20-year history of multiple sclerosis , adequately controlled with steroids . she had been treated with interferon - beta due to a sensitive relapse affecting the spinal cord and both legs during the last five months . she reported a two - week history of mild fatigue and arthralgia in the left tarsus , treated with ibuprofen . on admission , she was well hydrated and , apart from pedal edema , physical examination was unremarkable . laboratory test results are shown in table 1 . a possible diagnosis of acute renal injury secondary to thrombotic microangiopathy associated with interferon - beta was suggested . chest x - ray was unremarkable and renal ultrasound showed kidneys of normal size with a normal echogenic cortex and no hydronephrosis . a kidney biopsy was performed and histological studies showed ischemic changes in 12 of 35 glomeruli studied ( figure 1a ) . some other glomeruli showed chronic glomerular microangiopathic lesions with duplication of the glomerular basement membrane ( figure 1b ) . the arterioles and intralobular arteries showed marked subintimal fibromucoid edema narrowing the lumen ( figure 1a arrows , figure 2a ) . an immunofluorescence study showed only fibrinogen deposits in the arterial wall ( figure 2b ) . the patient was finally diagnosed with thrombotic microangiopathy associated with interferon - beta , so the drug was withdrawn and immunosuppressive therapy was started with 1 mg / kg / day of prednisone because leg symptoms of multiple sclerosis had started immediately . doses of steroids were reduced and finally withdrawn over a period of one month , while glatiramer acetate was started . hematological abnormalities and serum lactate dehydrogenase levels returned to the normal range , and renal function slowly recovered a serum creatinine of 1.0 mg / dl . it is postulated that interferon - beta acts in this disease by inhibiting activation and proliferation of t cells.1,2 the drug is usually well tolerated , but constitutional side effects and autoimmune adverse events have been reported.19,20 the similarities between some manifestations of systemic lupus erythematosus , those of viral infections , and side effects of immunotherapy with recombinant interferons , such as fever , arthralgia , myalgia , and fatigue , are evident . in fact , interferon - alpha is a central mediator in systemic lupus erythematosus , and specific neutralizing antibodies are now in clinical trials for the treatment of this disease.21 our case can be immediately catalogued as a thrombotic microangiopathy - hemolytic uremic syndrome , which would explain the acute kidney injury . she had no symptoms or serological findings suggestive of systemic sclerosis , malignancy , malignant hypertension , or antiphospholipid syndrome . the presence of autoreactive antibodies , particularly antiphospholipid antibodies and antithyroid antibodies , are associated with an increased risk of interferon - beta antibodies in patients with multiple sclerosis on long - term therapy.22 however , we did not find any of these antibodies in our patient . kidney complications have not been directly attributed to multiple sclerosis.2 renal side effects including minimal change disease,25 collapsing focal segmental glomerulosclerosis,68 membranous nephropathy,9 lupus nephritis,10,11 acute renal failure,12 and thrombotic microangiopathy,1323 are most often associated with interferon therapy rather than with interferon - beta . the incidence of transient proteinuria during interferon - beta therapy is around 20% . to our knowledge , this is the fourth case of hemolytic uremic syndrome induced by interferon - beta . two of these patients were treated with corticosteroids and plasmapheresis , and another was only treated with supportive antihypertensive and antiproteinuric therapies . the fourth patient was diagnosed also with pseudo - sle and treated with immunosuppressants . in all of them the spectrum of kidney disease related to interferon - beta is shown in table 2 . the mechanism by which interferon could induce thrombotic microangiopathy lesions and nephrotic syndrome remains unclear.14 pleiotropic drugs such as interferon might disrupt complex pathways of complement regulation and play a role in endothelial damage.21 in recent years , mutation of complement system regulators ( factors h and i , and membrane cofactor protein ) have been directly implicated in the induction of atypical hemolytic uremic syndrome.24,25 however , we did not found any of these alterations in our case . furthermore , a recent publication described a case of low adamts 13 activity associated with the presence of an anti - adamts 13 igg antibody during treatment with interferon - alpha 2a.16 glomerular endothelial cells express and secrete adamts 13.17 it has been suggested that pre - eclampsia is also associated with decreased levels of adamts 13.18 the delayed appearance of thrombotic microangiopathy observed in our case and others suggest that the development of renal lesions may be the result of cumulative effects.14,22 in conclusion , thrombotic microangiopathy - hemolytic uremic syndrome induced by interferon - beta is an unusual side effect manifested as acute or subacute kidney injury . attempts should be made to detect it as soon as possible , and to clarify the mechanism of microangiopathy lesions .
the most common way to estimate rates of speciation is to use data from the fossil record . paleontological studies estimate how many new species formed over a given time interval ( the per lineage speciation rate ; e.g. , stanley 1979 ; van valen 1985 ; jablonski 1986 ; hulbert 1993 ; sepkoski 1993 ) . many paleontological estimates of speciation rates have been calculated from groups with reasonably complete fossil records like marine invertebrates ( e.g. , raup and sepkoski 1982 ; peters 2005 ) . for groups with incomplete fossil records , paleontological approaches for inferring speciation rates use direct information about species that lived in the past , whereas other methods ( discussed below ) must infer the past dynamics indirectly . the primary limitation of paleontological studies is the uncertainty that arises from gaps in the fossil record ( e.g. , limited specimen material , uneven sampling effort , and/or taphonomical biases ; raup 1979 ; sepkoski 1998 ; alroy et al . , paleontological studies generally rely on higher taxa ( valentine 2004 ) , and how these taxonomic groups are defined can have a profound influence on inferred speciation rates ( ezard et al . in press ) . most paleontological estimates of per - lineage speciation rates range from 0.01 to 10 speciation events per lineage per million years ( sepkoski 1998 ) ; sepkoski ( 1998 ) suggests a even though this rate varies tremendously both across taxa and through time ( sepkoski 1998 ) , it can serve as a rough but useful this approach requires phylogenies whose branch lengths have been scaled to time . the simplest way to calculate speciation rates from these trees is to compare species richness to clade age , which provides a minimum bound on the rate of speciation , assumed to be constant ( magallon and sanderson 2001 ; but see rabosky 2009 , 2010 who warns against this approach ) . another approach is to fit models ( i.e. , birth death models ) to phylogenetic branch lengths and estimate rates of speciation and potentially extinction ( reviewed in nee 2006 ) . using phylogenies of extant species takes advantage of the wealth of data from the tree of life ( hedges and kumar 2009 ) . however , phylogenetic trees are estimated with error , do not include direct information about extinct species , and suffer from a number of biases related to sampling , all of which can affect speciation rate estimates ( e.g. , revell et al . 2005 ; phillimore and price 2008 ; rabosky 2010 ; cusimano and renner 2010 ; brock et al . most phylogenetic studies of speciation rates recover estimates that are of the same order of magnitude as the fossil record . one meta - analysis of speciation rates estimated from phylogenies found rates that ranged from 0.01 to 10 speciation events per lineage per million years under a pure birth model ( mcpeek and brown 2007 ) . an approach that estimated both speciation and extinction rates simultaneously recovered speciation rates of the same order of magnitude ( e.g. , alfaro et al . 2009 ) . in contrast to the studies discussed above , many studies of young evolutionary radiations estimate very high rates of speciation . these estimates often come from adaptive radiations in insular habitats ( e.g. , islands and lakes ; see losos and ricklefs , 2010 , and chapters therein ) . for example , one of the best - known examples of rapid diversification is the cichlid fishes in lakes of the african rift valley ( seehausen 2006 ) . an extreme example occurs in lake victoria , where speciation rates might be as high as 400 speciation events per lineage per million years ( i.e. , the formation of ~450 species in ~15,000 years , johnson et al . 1996 ; genner et al . 2004 other studies suggest that similarly high speciation rates may occur in other systems [ e.g. , hawaiian drosophila ( coyne and orr 2004 ) , silverswords ( baldwin and sanderson 1998 ) ] . in fact , there are several well - known adaptive radiations that have occurred so quickly , it is extremely difficult to infer the true phylogenetic relationships among species [ e.g. , galapagos finches ( sato et al . ; burns et al . 2002 ; grant and grant 2007 ) , three - spined sticklebacks ( reviewed in schluter 2000 ) ] . concordant with studies of young evolutionary radiations , mathematical models of speciation also suggest that speciation rates can be quite high . mathematical models of speciation take a number of forms , including models of sympatric speciation ( e.g. , maynard - smith 1966 ; felsenstein 1981 ; dieckmann and doebeli 1999 ; doebeli and dieckmann 2003 ) , models of divergence with gene flow ( e.g. , wu 2001 ; hausdorf 2011 ) , models of speciation in allopatry ( e.g. , gavrilets 2003 ) , and metapopulation models of adaptive radiations ( e.g. , gavrilets and vose 2005 ) . modeling approaches allow for a mechanistic understanding of how specific parameters influence the process of speciation , but are obviously limited by their simplifying assumptions . for example , models of speciation make specific assumptions about population structure , genetic architecture , and the strength and type of selection . model assumptions and parameter values can have dramatic impacts on the dynamics of speciation models ( reviewed in gavrilets 2004 ) . mathematical models of speciation almost never focus on speciation rate per se , and instead generally ask whether or not speciation occurs and , if so , how long it takes from start to finish ( i.e. , the speciation transition time , transition times estimated from mathematical models are typically very fast ( e.g. , doebeli and dieckmann 2003 , but see orr and turelli 2001 ) . however , transition times are not directly related to speciation rates , which describe the time from one speciation event to the next ( i.e. , the speciation waiting time ) . one modeling study that focused explicitly on speciation rates found that when speciation occurred , the waiting time for speciation varied between 5,000 to more than 200,000 generations depending on model parameters ( gavrilets 2000 ) . for organisms with a generation time of 1 year , this corresponds to speciation rates of 2200 speciation events per lineage per million years . these rates are comparable to the highest speciation rates observed empirically in the young evolutionary radiations discussed above , and likely represent an upper bound for speciation rates . it is difficult to define a lower bound for speciation rates from mathematical model because these models typically do not focus on parameter values where speciation never happens . mathematical models of speciation and studies of young evolutionary radiations find that new species can form quickly and often . however , phylogenetic studies over longer time scales and paleontological studies find that new species usually form more slowly . what explains this apparent discrepancy in speciation rates across different types of studies ? here we call attention to an explanation that may help unify what we know about speciation rates from paleontological , phylogenetic , and mathematical studies : the ephemeral speciation model . it is possible that speciation is very common and very rapid , but that the new species produced almost never persist . therefore we suggest that some approaches ( e.g. , studies of speciation in action and mathematical models ) actually focus on the formation of ephemeral species while others ( e.g. , phylogenetic studies over longer time scales and paleontological studies ) focus on the persistence of these ephemeral species . instead of being a contradiction , these differences in speciation rates reflect two aspects of the same underlying model : the formation and the persistence of ephemeral species . the idea that many more incipient species form than persist traces back to mayr ( 1963 ) . stanley ( 1978 , 1985 ) also discussed this phenomenon , referring to these failed incipient species as aborted species levin ( 2000 , 2005 ) proposed a related idea for plants where many incipient species form and have differential survival ( i.e. , isolate selection ) . ( e.g. , geographic range expansion and ecological niche differentiation ( price 2008 ; rundell and price 2009 ) . hubbell s neutral theory of ecology also exhibits high turnover of young incipient species ( hubbell 2001 ; rosindell et al . our suggested namethe ephemeral speciation modeltakes inspiration from futuyma s ephemeral diversification model ( which focuses primarily on trait change : futuyma 1979 , 2010 ) . under an ephemeral speciation model , new incipient species are constantly forming at a high rate ( fig . 1 ) . recent research on mechanisms of speciation suggest that speciation can occur via a plurality of interacting mechanisms ( e.g. , geography , selection , genomic rearrangements ; see coyne and orr 2004 ; gavrilets 2004 ) . thus the conditions for some mode of speciation may often be met in natural populations . although speciation occurs frequently in the ephemeral speciation model , persistence of incipient species is rare . the lack of persistence could be due to extinction or reabsorption via hybridization of the incipient species ( seehausen et al . 2006 ; richmond and jockush 2007 ; behm et al . 2010 ) . for example suppose that in some clades speciation typically happens in small allopatric populations at the edge of a species range ( mayr 1963 ) . these new species will be very vulnerable to extinction and to changes in the conditions that maintain reproductive isolation ( mayr 1963 ) . new species are also likely to be fragile under other non - allopatric modes of speciation as well [ e.g. , new polyploid species have very small population sizes ( holloway et al . 2006 ) and new ecological species require continued divergent selection until other forms of reproductive isolation evolve ( nosil and sandoval 2008 ) ] . therefore failed speciation is common because speciation takes time to complete and because conditions change over time.fig . the model has three parameters : the incipient speciation rate , the incipient extinction rate , and the rate of formation of ( a ) a phylogenetic tree showing that species are composed of many incipient forms , most of which go extinct or are reabsorbed via hybridization ( inset ) . ( b ) a frequency distribution showing the uneven distribution of incipient forms within species . ( c ) a lineage through time plot showing an early burst of speciation due to the preferential survival of clades that form many new species by chance early in their history simulation of a hierarchical model of ephemeral speciation . the model has three parameters : the incipient speciation rate , the incipient extinction rate , and the rate of formation of ( a ) a phylogenetic tree showing that species are composed of many incipient forms , most of which go extinct or are reabsorbed via hybridization ( inset ) . ( b ) a frequency distribution showing the uneven distribution of incipient forms within species . ( c ) a lineage through time plot showing an early burst of speciation due to the preferential survival of clades that form many new species by chance early in their history the ephemeral speciation model is consistent with two key empirical observations . first , what taxonomists recognize as species are often comprised of many incipient forms . species typically show extensive genetic and phenotypic variation , and this variation is usually hierarchically structured ( avise 2000 ; bickford et al these incipient forms are recognized taxonomically by a variety of names ( e.g. , geographic races , subspecies , incipient species ; simpson 1944 ; mayr 1982 ) . here we follow the general lineage concept of species , which encompasses many different species concepts as points along a continuum and which is consistent with the idea that species themselves can be a collection of distinct lineages ( de queiroz 2005 ) . when incipient species form , they can go extinct or cause their parental forms to go extinct ( e.g. , hegde et al . 2006 ) . additionally , incipient species can collapse and be reabsorbed by their parental form ( mallet 2008 ) . one recent example is the collapse of a stickleback species pair in enos lake ( taylor et al . 2010 ) , but similar collapses of incipient species have been observed in cichlids ( seehausen 2006 ) ; trout ( bettles et al . 2005 ) , galapagos finches ( grant and grant 1993 , 2008 ) ; skinks ( richmond and jockusch 2007 ) and house spiders ( croucher et al . 2007 ) . one way to model ephemeral speciation over long time scales is to use a simple high turnover birth death model with high speciation and high extinction rates ( e.g. , alfaro et al . these high turnover models do a good job of predicting species diversity in clades of intermediate age [ e.g. , jawed vertebrates ( alfaro et al . 2009 ) and ray - finned fishes ( santini et al . 2009 ) ] . but high turnover birth death models are inconsistent with empirical data in three ways . first , high turnover models predict that species deep in the tree should accumulate exponentially through time ( i.e. , linear lineage through time plot , nee et al . 1992 ) , but this pattern is not commonly seen in phylogenetic trees ( rber et al . 2005 ; phillimore and price 2008 ) . second , high turnover models suggest that the persistence of incipient species should be random , but empirical work on persistence of populations across a species range suggests that population survival is not typically random with respect to both biotic and abiotic factors ( e.g. , levin 2000 , 2005 ; owens et al . third , in high turnover models , species fail to persist only because they go extinct , but incipient forms can fail to persist because they are reabsorbed . we suggest that ephemeral speciation models should be hierarchical ( gould 1980 ) where incipient forms are constantly forming and dissolving inside larger entities ( i.e. , species , fig . there are several existing models that consider a hierarchical process where incipient forms arise and go extinct at a higher rate than full species ( cadena et al . ( 2007 ) who find that the rate of bird subspeciation is between 30 and 40 times higher than the rate of speciation ( see also martin and tewksbury 2008 ) . there are several important consequences of hierarchical models of ephemeral speciation ( fig . 1 ) . first , species go extinct only when all incipient forms are lost . therefore the extinction rate is no longer a property of a species but depends on the number of incipient forms within the species and their extinction rate . second , like other hierarchical models ( e.g. , wakeley 2008 ; hubbell 2001 ) , the ephemeral speciation model predicts an uneven distribution of incipient form within species ( fig . 1b ) : a few species will contain many incipient forms while most species will contain few ( consistent with the observation that rare species are common ; lim et al . third , this uneven distribution will lead to differences in effective speciation and extinction rates across species . species with many incipient forms will have high speciation and low extinction rates compared to species with few incipient forms ( see also kisel and barraclough 2010 ) . the unevenness of speciation rates across taxa is consistent with the fact that phylogenetic trees tend to be more unbalanced than birth death models predict ( mooers and heard 1997 ) . finally , under the ephemeral speciation model it is very likely for entire clades of newly formed species to go extinct . the clades that survive to the present day are disproportionately likely to have undergone a burst of speciation early in their history ( phillimore and price 2008 ) , which could be an explanation for observed slowdowns in lineage accumulation through time ( see also pigot et al . finally , the conceptual link between the ephemeral speciation model and the ephemeral divergence model ( futuyma 1979 ; futuyma 2010 ) reflects a common pattern for rates of trait evolution and rates of speciation . similarly , traits are constantly changing in response to local selection pressures and/or drift . in both cases it is important to note that we are not arguing for a model of punctuated equilibrium ; trait change may or may not be associated with the formation of incipient species ( bokma 2008 ) . the important point is that most of the change that occurs over short time periods does not last . therefore a fundamental shift suggested by both ephemeral models is that evolutionary studies of diversity patterns should focus on not only the formation but also the persistence of new traits and species [ e.g. , why do some some species persist and others perish quickly ? ( levin 2000 , 2005 ; weir and schluter 2007 ; martin and tewksbury 2008 ; stanley 2008 ) ] . although some discussion of the fragile nature of species has occurred in the evolutionary biology literature over the last 50 years , we suggest that the idea of ephemeral speciation has not been deeply incorporated in the way scientists think about speciation . following hutchinson ( 1959 ) , evolutionary biologists have often referred to santa rosalia when asking why are there so many or so few species on earth ( e.g. , felsenstein 1981 ) . we suggest that the ephemeral speciation model provides a resolution to the goldilocks paradox of species diversity : the balance between rapid species formation and rare persistence can explain why the number of species on earth is just right .
diabetes type 1 is mainly results from auto - immune beta cell destruction , while viral infections and chemical agents seem to be the triggers of the disease . the well - documented effect of insulin is to mediate carbohydrates , proteins and lipids storage . therefore diabetes is considered as a defect of lipids , proteins and carbohydrates metabolism in which all the body systems and organs are affected ( williams and pickup 2000 ) . the effect of insulin deficiency mainly results in elevation of cholesterol , phospholipids and free fatty acid ( williams and pickup 2000 ) . natural toxins have been traditionally used to heal diseases and honeybee venom ( apitoxin ) is of a great importance in this regard . the venom is composed of varieties of peptides ( mellitin , apamin , secapin , tertiapin , ado lapin , and mcd peptid ) , enzymes ( phospholipase a2 , hyaluronidase , acid phosphomonoesterase , lysophospholipase ) , active amines ( histamine , dopamine , norepinephrine , serotonin ) and many other substances ( son et al . bee venom and bee sting had significant effect on quality of life ( wesselius et al . mellitin and phospholipase a2 are the most important ingredients and play a great role in irritation and allergic responses leading to anti - inflammatory and analgesic effects ( mazzanti et al . mellitin as the most important ingredient in bee venom is a strong phospholipase a2 activator which is composed of 26 amino acids and makes up 50% of the dried venom . those bee venom ingredients that contain a high molecular weight , like hyaluronidase and phospholipase a2 , cause the immune reactions ( bomalaski et al . apamin , mellitin and phospholipase a2 contained in bee venom , are strong immunoregulators but their effects on diabetes have not been investigated ( habermann 1972 , gauldie et al . bv apamin is known as a neurotoxin from central nervus system hyperexcitability through inhibition of the axonal potassium channels ( son et al . melittin , the major constituent of bee venom , has an anti - inflammatory effect through its functionality on the anterior pituitary gland , which in turns may result in stimulation of cortisol production from the adrenal gland ( son et al . part of anti - inflammatory effect of melittin may attribute to its interaction with cell surface moieties which seems to render the cytotoxic effects of melittin on cancer cells ( mirshafiey 2007 , son et al . lowering chemicals , achieving new therapeutic agents with low side effects seems to be necessary . nowadays diabetic s population is growing rapidly while encountering various life - threatening disease conditions that require researchers to evaluate related therapeutic , alleviative and preventive treatments . the main purpose of the study was to evaluate the effects of iranian honeybee venom ( apitoxin ) on blood levels of glucose and insulin in alloxan induced diabetic rat . bee venom samples were collected from beehives using an electric shocker , on february 2010 in khuzestan , iran . the electric shocker is composed of two components : one component as shocker and the other to collect the venom and concomitant material . the collecting unit is wooden and composed of a network of wires with small gaps between them . the collector panel was first located on bottom of the beehives and then on the top , to collect the desired amount of the bee venom . when the shocker was turned on , the honeybees stroked the wires and received a light electric shock and were stimulated them to sting and discharged their bee venom . the shocker was turned off after 25 minutes and the collecting panel was removed from the beehive , and the dried bee venom material scratched and transferred to a proper container . to evaluate the toxin production efficiency after collecting the samples healthy adult male lewwis rats weighting 20020 g were maintained in darou pakhsh pharmaceutical mfg co , tehran ( iran ) . the animal room was maintained under a constant 12-h light : 12-h dark cycle and temperature of 233 c and relative humidity of 7010% throughout the experimental period . for bees to be adapted to the new environment condition , all the experiments were carried out 10 days after their first residence . eighteen rats were placed randomly into three groups ( n= 6 ) : control group : normal saline 0/9% injected intraperitoneally . diabetic group : this group became diabetic by injection alloxan monohydrate at 150 mg / kg intraperitoneally . bee venom - treated group : at first received alloxan monohydrate to induce diabetes and iranian bee venom ( apitoxin ) at 0/5 mg / kg ( the best dose chosen after pretest ) after diabetes confirmation intraperitoneally at fasting condition every day for four consecutive weeks ( kim et al . 1999 , blood glucose level was measured using acua check ( germany ) . to measure the blood glucose level , a small incision was made on the animals tail using a lancet and a drop of fresh blood was extracted and used for glucometery . alloxan monohydrate ( sigma - aldrich germany ) was used to induce diabetes in rats . the drug was administrated intraperitoneally at the rate of 150 mg / kg ( viana et al . the method is preferred to induce diabetes in many other animals ( soto et al . a condition of hyperglycemia like diabetes type 1 appeared in rats ( byung - hyun and jin - woo 2001 ) . seventy two hours after the administration , blood glucose level was measured using acua check to determine diabetic condition . in this study , blood glucose level elevation over 280 mg / dl is supposed to reveal diabetic condition ( zhang and tan 2003 ) . usual signs of diabetes including polydipsia , polyuria and weight loss were observed 67 days following the administration ( nuraliev et al . the animal is kept between forefinger and thumb while the tube is inserted to the orbital foramen with a rotational movement . the capillaries are usually very sensitive and fragile here and burst following a light pressure . when a few almost large drops of blood were collected , the hematocrit tube is removed . the method is suitable for repetitive blood sampling especially for primary analysis following 2 weeks of administration . the animals were anesthetized with ether after 16 h of fasting to collect blood for analysis . forty eight hours after the last injection , blood samples were collected from the heart and centrifuged ( 3000 rpm for 15 min at 4 c ) for separating the serum . . then the amount of blood glucose , insulin , cholesterol and triglyceride in blood serum weredetermined.serum glucose level was measured by kinetic ( enzymatic ) and colorimetric methods using glucose estimation kit ( pars azmoon , iran ) . theserum insulin levels were assayed with an elisa , irma ( biosource europe sa ) , serum glucose , triglyceride and total cholesterol levels were determinedusingcommercial kits and enzymatic assays . the values were expressed as mean s.e.m statistical analyses were performed by one way analysis of variance ( anova ) followed by tukey multiple comparison test . bee venom samples were collected from beehives using an electric shocker , on february 2010 in khuzestan , iran . the electric shocker is composed of two components : one component as shocker and the other to collect the venom and concomitant material . the collecting unit is wooden and composed of a network of wires with small gaps between them . the collector panel was first located on bottom of the beehives and then on the top , to collect the desired amount of the bee venom . when the shocker was turned on , the honeybees stroked the wires and received a light electric shock and were stimulated them to sting and discharged their bee venom . the shocker was turned off after 25 minutes and the collecting panel was removed from the beehive , and the dried bee venom material scratched and transferred to a proper container . to evaluate the toxin production efficiency after collecting the samples healthy adult male lewwis rats weighting 20020 g were maintained in darou pakhsh pharmaceutical mfg co , tehran ( iran ) . the animal room was maintained under a constant 12-h light : 12-h dark cycle and temperature of 233 c and relative humidity of 7010% throughout the experimental period . for bees to be adapted to the new environment condition , all the experiments were carried out 10 days after their first residence . eighteen rats were placed randomly into three groups ( n= 6 ) : control group : normal saline 0/9% injected intraperitoneally . diabetic group : this group became diabetic by injection alloxan monohydrate at 150 mg / kg intraperitoneally . bee venom - treated group : at first received alloxan monohydrate to induce diabetes and iranian bee venom ( apitoxin ) at 0/5 mg / kg ( the best dose chosen after pretest ) after diabetes confirmation intraperitoneally at fasting condition every day for four consecutive weeks ( kim et al . blood glucose level was measured using acua check ( germany ) . to measure the blood glucose level , a small incision was made on the animals tail using a lancet and a drop of fresh blood was extracted and used for glucometery . alloxan monohydrate ( sigma - aldrich germany ) was used to induce diabetes in rats . the drug was administrated intraperitoneally at the rate of 150 mg / kg ( viana et al . 2004 , antia et al . 2005 ) . the method is preferred to induce diabetes in many other animals ( soto et al . 2001 ) . following the administration a condition of hyperglycemia like diabetes type 1 appeared in rats ( byung - hyun and jin - woo 2001 ) . seventy two hours after the administration , blood glucose level was measured using acua check to determine diabetic condition . in this study , blood glucose level elevation over 280 mg / dl is supposed to reveal diabetic condition ( zhang and tan 2003 ) . usual signs of diabetes including polydipsia , polyuria and weight loss were observed 67 days following the administration ( nuraliev et al . fasting blood samples were collected 2 weeks following bee venom administration using stone method . in this method , the animal is kept between forefinger and thumb while the tube is inserted to the orbital foramen with a rotational movement . the capillaries are usually very sensitive and fragile here and burst following a light pressure . when a few almost large drops of blood were collected , the hematocrit tube is removed . the method is suitable for repetitive blood sampling especially for primary analysis following 2 weeks of administration . the animals were anesthetized with ether after 16 h of fasting to collect blood for analysis . forty eight hours after the last injection , blood samples were collected from the heart and centrifuged ( 3000 rpm for 15 min at 4 c ) for separating the serum . then the amount of blood glucose , insulin , cholesterol and triglyceride in blood serum weredetermined.serum glucose level was measured by kinetic ( enzymatic ) and colorimetric methods using glucose estimation kit ( pars azmoon , iran ) . theserum insulin levels were assayed with an elisa , irma ( biosource europe sa ) , serum glucose , triglyceride and total cholesterol levels were determinedusingcommercial kits and enzymatic assays . the values were expressed as mean s.e.m statistical analyses were performed by one way analysis of variance ( anova ) followed by tukey multiple comparison test . bee venom preparation results : the bee venom samples were collected and panel scratched using a sterile blade and kept in clean dark vials . average amount of venom collected from three beehives ( each containing estimated 10000 bees ) was 147.7 mg . alloxan administration result showed a significant increase in blood glucose level in ( diabetic ) group compared with control group after 2 and 4 weeks ( p < 0.05 ) . in addition a significant increase was observed in blood glucose level in diabetic group after 4 weeks compared with 2-week period ( p < 0.05 ) . there was a significant decline in blood glucose level in bee venom- treated group compared with diabetic group ( p < 0.05 ) . also there was a significant decline in blood glucose level in diabetic treated group after 4 weeks compared with 2-week period ( p < 0.05 ) ( fig . the effect of bee venom administration on serum insulin level is illustrated in figure 2 . our results showed that serum insulin level significantly decreased in diabetic group compared with controls ( p < 0.05 ) . also there was a significant increase in bee venom - treated group compared with controls ( p < 0.05 ) ( fig . the effect of bee venom administration on serum triglyceride ( tg ) content is illustrated in figure 3 . there was a significant increase in serum tg content in diabetic group compared with controls ( p < 0.05 ) . a significant decline was observed in bee venom - treated group compared with diabetic animals ( p < 0.05 ) . but no significant difference was observed in serum tg content in bee venom - treated group compared with control ( fig . a significant increase was observed in serum total cholesterol in diabetic group compared with controls ( p < 0.05 ) . there was a significant decline in bee venom - treated group compared with diabetic animals ( p < 0.05 ) . but no significant difference was observed in cholesterol content in bee venom - treated group compared with control group ( p= 0.552 ) ( fig . in our study , blood glucose level increased following alloxan monohydrate administration which led to pancreas b - cells destruction ( byung - hyun and jin - woo 2001 ) . this may be contributed to substances like mellitin and phospholipase a2 contained in the venom . they may play a role in diminishing inflammation of islets of langerhans and thus elevating blood insulin level . with regard to the fact that insulin regulates blood glucose level , bee venom could decrease glucose content via increasing insulin secretion ( morgan and montague 1984 , fujimoto and metz 1987 , kim et al . 2000 ) . following alloxan administration in diabetic rats , blood glucose level and triglyceride ( tg ) content were elevated , which indicate insulin role in regulating lipid metabolism ( zhang and tan 2003 ) . insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides ( frayn 1993 ) . according to the obtained results one could explain the observed decline as follows : bee venom improves glycemic control and decreases blood glucose level . also glucose consumption is increased instead of lipids . acetyl coa derived from pyrovic acid enters krebs cycle which finally leads to glucose metabolism , however acetyl coa can enter tg synthesis pathway in usual condition ( zhang and tan 2003 ) . a decline was observed in cholesterol content of bee venom -treated group ( kim et al . probably cholesterol lowering effect is largely due to inhibition of its absorption in small intestine and promoting its hepatic release . the liver plays a critical role in discharging cholesterol via bile secretion ( reinner et al . alloxan administration led to destruction of islets of langerhans and diminished insulin secretion in diabetic rats . treating the rats with honeybee venom ( apitoxin ) increased insulin secretion up to control levels . according to the published reports , mellitin polypeptide and phospholipase a2 , which are two main component of the venom , promote insulin secretion . according to the literature , the observed effect is mediated by extracellular calcium and calcium channels . when these channels are opened large amounts of calcium enters the -cells and excite them to secret insulin ( morgan and montague 1984 , fujimoto and metz 1987 , kim et al . 1999 , simonson et al . 2000 ) . in a study on the effects of bee venom on islets of langerhans inflammation and onset of insulin dependent diabetes , kim et al . ( 1999 ) showed that intensity of inflammation and onset of diabetes declined following bee venom treatment . they also found that insulin , tg and cholesterol levels decreased in diabetic rats compared with non - diabetic animals ( kim et al . ( 1984 ) , mellitin polypeptide promotes insulin secretion from islets of langerhans in vitro . the obtained results suggest that mellitin as a valuable candidate for further studies on -cells plasma membrane role in regulating insulin secretion . the findings also indicate that mellitin can depolarize plasma membranes of -cells and acts as a calcium transporter in the cell , which in turn promotes insulin granules secretion . the effect of mellitin on insulin secretion depends on extracellular calcium ( morgan and montague 1984 ) . simonson et al . ( 2000 ) found that mellitin may promote insulin secretion via activating phospholipase a2 in islets of langerhans . their results indicate that phospholipase a2 activation plays a role in compensating insulin resistance response in islets of langerhans ( simonson et al . treatment with exogenous phospholipase a2 or mellitin promotes arachidonic acid and lysophospholipids production and insulin secretion . arachidonic acid produced by phospholipase a2 induction may act as a calcium transporter in to -cells and promote insulin secretion ( fujimoto and metz 1987 ) . in this study , our results indicate that honeybee venom ( apitoxin ) can be used as therapeutic option to lower blood glucose and lipids in diabetic rats , however further biochemical and pharmacological studies are necessary to provide more detailed understanding of the issue .
a pathology text describes choriocarcinoma ( cc ) as an epithelial malignancy of trophoblastic cells that lacks villous organization , with sites of origin being gestational as well as nongestational tissues . the former includes placental tissue ( whether intra or extrauterine ) whereas the latter comprises sites such as the ovary , testis , pineal gland , and ectopic rests in the mediastinum , retroperitoneum , stomach , esophagus , spleen , prostate , urinary bladder , and pancreas . tendency to metastasis is seen quite early with blood stream being the preferential route of spread . literature unfolds lungs to be the most common site of metastasis followed by the vagina , brain , liver , bone , intestine , and kidney . metastasis to uveal tissue is a rarity with the number of cases reported for gestational and nongestational cc , respectively , to be one and eighteen . additional sites of distant spread for nongestational cc include the spleen and small bowel . the metastases are typically hemorrhagic and hypervascular . in the present case , we describe various rare manifestations of gestational cc in a 30-year - old female patient . a 30-year - old female presented with generalized weakness , easy fatigability , high colored urine , icterus , and diminution of vision in both eyes since 2 months . the vision loss was painless and had gradually progressed to finger counting in the right eye and perception of light in the left . she also gave a history of amenorrhea since 2.5 months ; however , the urine pregnancy test was negative . prior to the amenorrhea , she had episodes of heavy intermenstrual bleed for a few months for which she had undergone dilatation and curettage which led to partial relief of her complaints . the patient was pale with a hemoglobin of 8.9 gm / dl ( normal 1215.8 g / dl ) and a peripheral blood smear negative for hemolysis . laboratory investigations revealed a total bilirubin of 3.57 mg / dl ( normal up to 1.2 mg / dl ) with direct fraction being 0.62 mg / dl ( normal up to 0.4 mg / dl ) , albumin 3.1 ( 2.33.5 g / l ) , serum glutamic - oxaloacetic transaminase ( aspartate aminotransferase ) 59 u / l ( normal 1238 u / l ) , serum glutamic - pyruvic transaminase ( alanine aminotransferase ) 70.1 ( normal sonography demonstrated coarse echotexture of the liver suggestive of liver parenchymal disease [ figure 1 ] . in addition , there were a few anechoic intraparenchymal spaces which on doppler interrogation had color signals with a velocity ranging between 55 to 79 cm / s and low impedance . pelvic sonography revealed bulky uterus with coarse echotexture [ figure 2 ] with numerous myometrial serpiginous vascular channels showing high blood flow of peak systolic velocity ( psv ) 113 cm / s . based on the available history of dilatation and curettage , a tentative diagnosis of iatrogenic uterine vascular malformation was made . ( a = anterior ; p = posterior ) endovaginal scan depicts a bulky uterus with anechoic spaces located in its anterior wall ( open arrow in a ) , which on doppler interrogation shows chaotic flow ( b ) . the block arrow points towards thinned endometrium , which at one place shows blurred interface with the myometrium on pelvic sonogram , the uterus reveals color aliasing in the region of the anechoic spaces [ figure 2 ] . the flow has high velocity ( 113 cm / s ) and low impedance in the next 2 days , she developed breathlessness and a computed tomography ( ct ) was performed . ct revealed hyperdense loculated collection in the anterior part of the right pleural cavity suggestive of hemothorax [ figure 4 ] . in addition , there was bilateral pleural effusion ( left > right ) with collapse of the left lung [ figure 4 ] . further , numerous haphazardly placed vascular spaces were present in anterior , superior , and posterior mediastinum , along left internal mammary and intercostal vessels , intraparenchymal in the right lung [ figure 5 ] , spleen [ figure 6 ] , liver [ figure 7 ] , and uterus [ figure 8 ] . a normal thrombophilia profile and multiplicity of sites of vascular channels made thrombosis a less likely etiology . further , there was a relative absence of stroma without local hypertrophy , as is seen typically in congenital vascular malformations . moreover , the patient did not have signs of cardiac failure despite numerous vascular channels suggesting short history of development of them . hence , based on clinical and imaging findings , uterine cc was suspected , and a b scan was done to rule out ocular metastasis from the same . b - scan revealed bilateral exudative retinal detachment with anechoic channels present in the subretinal space with high flow on b - mode and a peak systolic velocity of 61.6 cm / s and a low impedance [ figure 9 , videos 1 and 2 ] . serum hcg titres were sent which , however , was mildly raised to 183.15 miu / ml ( in nonpregnant females : < 5 miu / ml ) . other laboratory parameters included total bilirubin 4 mg / dl , direct bilirubin 1.2 mg / dl , prothrombin time ( pt ) 22 s ( normal 11.917 s ) , inr 1.9 ( normal < 1.41 ) , activated partial thromboplastin time ( aptt ) 46.9 s ( normal 25.337.9 s ) , d - dimer 6.8 mg / l ( normal 0.3 mg / l ) , creatinine 3.8 mg / dl ( normal 0.51.4 mg / dl ) . patient went into disseminated intravascular coagulation ( dic ) with acute hepatic failure and acute renal shut down and finally succumbed . non - contrast computed tomography of chest demonstrates a hyperdense lesion suggestive of an acute hemorrhage ( arrow ) . there is a collapse of the underlying left lung ( a - d ) contrast enhanced computed tomography of the chest in axial plane reveals multiple serpentine , dilated , and tortuous vessels along the left paraspinal space ( white arrow ) and internal mammary vessels ( red - bordered arrow ) . also noted are multiple intraparenchymal abnormal vessels within the right lung ( open arrows ) ( a - c ) arterial phase computed tomography axial view depicts multiple vascular spaces in spleen ( arrows ) ( a ) postcontrast axial computed tomography at the level of upper abdomen shows abnormal vascular spaces within the liver parenchyma ( arrows ) . besides , there are multiple ill - defined hypoechoic hepatic nodules ( more prominent in b , c ) sagittal reformation of venous phase computed tomography demonstrates multiple dilated vessels within the myometrium ; largest of which is located in the anterior wall ( open arrow ) ( also see figures 2 , 3 ) . also noted are vessels within the anterior ( white block arrow ) and posterior mediastinum ( black arrow ) doppler examination of the high flow in the subretinal space on right reveals chaotic flow with a peak systolic velocity of 61.6 cm / s and low impedance . molar gestation holds the biggest risk ; although a normal pregnancy also can culminate into tumorigenesis . sixty percent of women present with uterine enlargement . spotting and foul smelling discharge may be seen . one of the remarkable features of cc is extensive necrosis so much so that the primary site may sometimes be barely discernible . necrosis sometimes can give rise to clinically manifest hemorrhage at high tumor - volume metastatic sites , described by logothetis as choriocarcinoma syndrome , with diffuse alveolar hemorrhage as the most common manifestation . recognition of gestational cc is usually straightforward in the correct clinical setting of a recent molar pregnancy , rising titre of serum hcg , and a previously documented normal sonogram ; although a host of confounding factors may exist . these factors include cc following a normal or ectopic gestation or a spontaneous abortion . in addition , presentation with non - gynecologic problems such as respiratory compromise , hyperthyroidism , cerebral , gastrointestinal , or urologic hemorrhage add to the confusing clinical scenario . sonography plays an important role in detecting and staging cc and monitoring response to therapy . alternatively , the tumoral mass is composed of anechoic cystic spaces which either represent extensive necrosis or vascular channels ( arterio - venous shunts ) . this cystic appearance can mimic a mole , retained products of conception ( rpoc ) , or arteriovenous malformation . characteristic of cc is the presence of trophoblastic signals on spectral doppler ultrasound , which include a high psv with low impedance flow ( due to arteriovenous shunts ) , a low ri , and color aliasing . the psv is usually greater than 50 cm / s and is often more than 100 cm / s and an ri less than 0.5 . unfortunately , this flow pattern is not pathognomonic for cc ; various differentials include ectopic gestation , rpoc , and avm . an rpoc exhibits endometrial vascularity extending from myometrium but vascularity in uterine avm is isolated to myometrium . it reveals a bulky uterus with an ill - defined , inhomogenously enhancing mass with areas of hypervascularity . the metastases are also hypervascular like primary and often undergo hemorrhage and necrosis resulting in a variety of non - gynecologic problems . biochemical marker for detecting presence of cc or its response to therapy is human chorionic gonadotropin ( hcg ) . it is a heterodimer secreted by trophoblastic cells to facilitate corpus luteum to secrete the pregnancy hormone ( progesterone ) . once secreted , hcg undergoes multiple modifications to form different subtypes of hcg which may be secreted in urine and/or serum . for example , while beta core form of hcg is secreted only in urine , rising in concentration from 5 to 8 weeks of gestation and peaking in midtrimester , the hyperglycosylated form ( the only bioactive form other than the intact hcg ) , on the contrary , is secreted in excess soon after implantation and reaches undetectable levels toward the end of the first trimester . detection of hcg in urine or serum depends on the formation of a sandwich with hcg being the meat and two different antibodies ( binding to two different epitopes on the same hcg molecule ) being the bread . knowledge of this biochemistry is of prime importance since when to detect hcg ( timing after conception ) , which form to detect , and which commercially available kit to use ( since all kits do not measure all forms ) can avoid misinterpretation . it is a phenomenon wherein excess hcg saturates both the antibodies separately resulting in half - formed sandwiches ( so to speak ) and consequently falsely low titres ( the hook effect ) . similarly , variant hook effect too results in low detectable hcg levels . in variant hook effect , excess beta core variant in urine sample saturates one of the binding antibodies causing an incomplete reaction . both these phenomena of falsely negative tests can be surmounted by sample dilution especially in clinically suspected cases . yet another cause for low titre can be extensive necrosis of the tumor so much so that it becomes functionally inactive . spectacular results have been obtained with single drug ( methotrexate ) regimen for the former while the latter is usually fatal . for those of gestational type who fail to respond to single drug therapy , multi - agent regimen ( ema - co : etoposide , methotrexate and actinimycin d alternating with cyclophosphamide and vincristine ) comes to rescue . those with high hcg more than 40000 miu / ml , hepatic , or brain metastasis and presentation 4 or more months after pregnancy also benefit from this multiagent chemotherapy . presentation such as hepatits - like features , uveal metastases , cc syndrome , and the hook effect are rarely encountered ; simultaneous presence of these is unheard of . thus , if the clinical picture is confusing with a low hcg titre , radiological diagnosis should still be considered .
male accessory gland infection ( magi ) represents a potential cause of male infertility [ 1 , 2 ] , with a different prevalence in the previous published studies . magi may affect sperm parameters through three possible mechanisms : ( a ) secretory dysfunction of the accessory glands ( epididymis , prostate , and seminal vesicles ) [ 46 ] , ( b ) favoring an inflammatory microenvironment ( oxygen - free radicals , cytokines ) [ 712 ] , and ( c ) anatomical obstruction of the seminal tract [ 13 , 14 ] . the ultrasound examination of the epididymis and prostate - vesicular tract represents an important diagnostic tool for the clinical evaluation [ 9 , 15 ] , although it is not considered an essential step in the work - up . recently , a very comprehensive review on ultrasound physiopathology of male seminal tract has been published , confirming the importance of this diagnostic tool for the diagnosis of inflammatory diseases of the male genital tract . the ultrasound evaluation allows discriminating different diagnostic categories , in particular , uncomplicated form ( ultrasound criteria limited to the prostate : prostatitis ( p)),complicated form ( ultrasound criteria extended to seminal vesicles : prostatitis and vesiculitis ( pv ) , and the epididymis : prostatitis , vesiculitis , and epididymitis ( pve ) ) [ 9 , 15],unilateral form ( unilateral p - pv - pve ) [ 9 , 15 , 17],bilateral form ( bilateral p - pv - pve ) [ 9 , 15 , 17 ] : we have recently reported additional ultrasound criteria that allow the identification of two original ultrasound categories of magi , hypertrophic - congestive form ( fsuf),fibrosclerotic form ( hcuf ) . uncomplicated form ( ultrasound criteria limited to the prostate : prostatitis ( p ) ) , complicated form ( ultrasound criteria extended to seminal vesicles : prostatitis and vesiculitis ( pv ) , and the epididymis : prostatitis , vesiculitis , and epididymitis ( pve ) ) [ 9 , 15 ] , unilateral form ( unilateral p - pv - pve ) [ 9 , 15 , 17 ] , bilateral form ( bilateral p - pv - pve ) [ 9 , 15 , 17 ] : we have recently reported additional ultrasound criteria that allow the identification of two original ultrasound categories of magi , hypertrophic - congestive form ( fsuf ) , fibrosclerotic form ( hcuf ) . there are several risk factors that may favor the onset of magi , for example , lifestyle , diet , cigarette smoking , gastrointestinal diseases , and sexual promiscuity [ 19 , 20 ] . the role of low levels of testosterone ( t ) in promoting the onset of p in an experimental model was recently reported . in particular , a modulatory action of t on inflammatory response in the accessory glands has been demonstrated [ 22 , 23 ] . however , there is not evidence in the literature regarding the clinical characterization of hypogonadal patients with magi . on the basis of these premises , the aim of this study was to evaluate the different ultrasound characterization of fertile symptomatic patients with magi according to different serum concentrations of total t ( tt ) . out of a consecutive series of 2000 subjects , we selected 200 fertile ( spontaneous pregnancy during the previous 12 months ) symptomatic patients with magi evaluated during the period between january 2012 and january 2014 that were subjected to evaluation of serum concentrations of tt . the diagnosis of magi was carried out according to the criteria reported in table 1 and published in several previous studies of our group [ 13 , 15 ] . the diagnosis was further confirmed by the administration of a questionnaire dedicated to patients with magi , for the assessment of the severity of the associated symptoms , previously published by our group . this questionnaire ( si - magi : structured interview about magi ) is divided into 4 domains relative to urinary disorders , spontaneous and/or ejaculatory pain and/or discomfort , sexual disorders , and quality of life . all examined patients had ultrasound criteria suggestive of magi , as previously reported [ 15 , 17 , 18 ] . table 2 shows the ultrasound criteria used by our group and published in previous studies for the confirmation and greater characterization of the clinical diagnosis of magi [ 15 , 17 , 18 ] . other exclusion criteria were represented by hormone therapy with antiestrogens or gonadotropins or t. hormonal evaluations were performed by electrochemiluminescence with hitachi - roche equipment ( cobas 6000 , roche diagnostics , indianapolis , in , usa ) . the reference intervals were as follows : lh = 1.69.0 miu ml , fsh = 2.012.0 miu ml , and total testosterone = 2.88.0 ng ml . blood sampling was performed at 8:00 am , after at least 8 hours of sleep . we analyzed the ultrasound and hormonal data ( serum tt ) of 200 patients with magi aged between 24.0 and 67.0 years ( mean age = 36.0 14.0 years ) with bmi between 18.0 and 33.0 kg / m ( mean value = 25.0 7.0 kg / m ) . patients were divided into six groups according to the sextile distribution of total testosterone : group 1 : tt 2.7 ng ml , group 2 : tt > 2.7 and 3.6 ng ml , group 3 : tt > 3.6 and 4.4 ng ml , group 4 : tt > 4.4 and 5.3 ng ml , group 5 : tt > 5.3 and 6.6 ng ml , group 6 : tt > 6.6 ng ml . group 1 : tt 2.7 ng ml , group 2 : tt > 2.7 and 3.6 ng ml , group 3 : tt > 3.6 and 4.4 ng ml , group 4 : tt > 4.4 and 5.3 ng ml , group 5 : tt > 5.3 and 6.6 ng ml , group 6 : tt > 6.6 ng ml . the frequency of the following possible ultrasound conditions was evaluated for each group : p ( prostatitis alone ) , pv ( ultrasound criteria suggestive for prostatitis associated with criteria of vesiculitis ) , and pve ( criteria of prostatitis and vesiculitis associated with epididymitis ) , unilateral p , pv , and pve , bilateral p , pv , and pve , and hcuf or fsuf . the study was approved by the internal institutional board and all examined patients signed informed consent to the processing of personal data . accordingly , variables are presented as means standard deviations or median ( interquartile range ) and differences between groups were tested by student 's independent t - test or mann - whitney u test on the basis of their normal or not - normal distribution , respectively . pearson 's or spearman 's correlation coefficients were used in order to test the associations between the different variables . multivariate linear regression models adjusted for age and bmi were performed for factors significantly correlated at spearman 's analysis . multivariate logistic regression analysis was performed to assess significant predictors of magi after adjusting for age and bmi . the software spss 9.0 for windows was used for statistical evaluation ( spss inc . , the linear regression analysis showed inverse association between tt and duration of symptoms after adjusting for age and bmi ( r = 0.929 ; p < 0.01 ) . at multivariate logistic regression analysis adjusted for age and bmi , tt was an independent predictive factor of p ( or = 1.533 [ 95% ci : 1.2721.848 ] ; p < 0.01 ) , pv ( or = 0.818 [ 95% ci : 0.6750.992 ] ; p < 0.01 ) , pve ( or = 0.714 [ 95% ci : 0.5780.880 ] ; p < 0.01 ) , up ( or = 1.847 [ 95% ci : 1.4982.278 ] ; p < 0.01 ) , bp ( or = 0.615 [ 95% ci : 0.4930.766 ] ; p < 0.01 ) , bpv ( or = 0.505 [ 95% ci : 0.3210.796 ] ; p < 0.01 ) , bpve ( or = 0.610 [ 95% ci : 0.4580.812 ] ; p < 0.01 ) , hcuf ( or = 2.201 [ 95% ci : 1.7022.848 ] ; p < 0.01 ) , and fsuf ( or = 0.607 [ 95% ci : 0.4940.746 ] ; p < 0.01 ) . table 3 shows the differences between groups divided into sextiles according to different serum concentration of tt . the results of this study suggest that different serum concentrations of tt in patients with magi are associated with a different ultrasonographic characterization . in particular , the reduction of tt represents a predictive factor for ultrasonographic complicated form ( pv and pve ) , ultrasonographic bilateral form of p , pv , and pve , and ultrasonographic fibrosclerotic form . the increase in tt represents a predictive factor for ultrasonographic uncomplicated form ( p ) , ultrasonographic unilateral form of p , and ultrasonographic hypertrophic - congestive form . the comparison between groups showed a significantly lower frequency of pve ( ultrasonographic form with greater anatomic extension ) in group 6 ( tt > 6.6 ng ml ) . the same group showed a significantly higher frequency of the ultrasonographic hypertrophic - congestive form and a significantly lower frequency of the ultrasonographic fibrosclerotic form . finally , patients with tt 3.6 ng ml showed a significantly greater duration of symptoms of magi compared to the other groups . therefore , it is conceivable that , in clinical practice , the male hypogonadism could represent a risk factor for a complicated form of magi . there are no other studies in the literature that have examined the frequency and/or the clinical characteristics of patients with magi according to the different levels of tt ; however , it is known that magi represent a very important clinical problem for the symptoms and the reproductive consequences [ 3 , 24 , 25 ] . the low levels of tt could have an important significance from the diagnostic and therapeutic point of view . in fact , the results of this study suggest that male hypogonadism could be a risk factor for the development of magi and in particular for complicated form of magi ( ultrasound criteria suggestive for extension to the seminal vesicles and epididymis ) . furthermore , the hypogonadal patients showed a higher frequency of bilateral and fibrosclerotic ultrasound form of magi . ultrasound evaluation of the epididymis , prostate , and seminal vesicles represents an important tool for the clinical characterization of magi ; however , it is not a mandatory diagnostic step . in this field , the experience of our group showed that the progressive involvement of more accessory glands detectable by the ultrasound evaluation is associated with a poor quality of sperm parameters and severe symptoms [ 24 , 25 ] . moreover , the ultrasound examination allows discriminating the bilateral form of magi better than some semen parameters such as the fructose . we recently reported two other variants of magi , respectively , called hypertrophic - congestive and fibrosclerotic ultrasound form , associated with a different sperm quality after pharmacological treatment ( lower in patients with even more recently , two other categories of patients ( diabetes mellitus and irritable bowel syndrome ) have been characterized by ultrasound examination for the peculiar characteristics of the sex accessory glands . the evaluation of serum levels of tt is often required during an initial andrological consultation for various clinical problems ( sexual or reproductive pathology ) and therefore we wanted to examine retrospectively the frequency of ultrasound criteria suggestive of magi according to the different serum concentrations of tt . from the methodological point of view , there are no similar studies in the literature ; however , for the interpretation of the results , it is necessary to analyze some evidences that suggest a role of testosterone as a modulator of the inflammatory response in the prostatic tissue . several mechanisms have been proposed to explain the potential anti - inflammatory effects of t on the prostatic tissue . in particular , it has been suggested that t could reduce the tissue expression of proinflammatory cytokines and t - lymphocytes and antagonize macrophage activation and neutrophil in the prostatic tissue and the progression of the fibrosis . furthermore , t seems to positively modulate the expression of junctional proteins of the prostatic epithelium and negatively the activation of the immune response . finally , the antiestrogenic effect and the negative modulation of the expression of toll - like receptor 4 represent other mechanisms [ 2123 , 2830 ] . in detail , the study of vignozzi and colleagues showed that t reduced the expression of inflammatory prostatic markers in an experimental model of prostatic inflammation induced in rabbits by experiment fed with high - calorie diet , modulating the expression of il8 , il6 , il1 , and tnf ( proinflammatory cytokines ) , t lymphocytes , macrophages , markers of neutrophil activation , and prostatic fibrosis . the hypogonadism is associated with a reduced expression of claudina 4 and claudina 8 , the essential components in the formation of junctional structures of the prostatic epithelium , whose alteration induces inflammatory degeneration of the prostate tissue and the immune response ; instead , the t supplementation is associated with reexpression of these proteins . a reduction of t / estradiol ratio is associated with a higher frequency of prostatic inflammation in an experimental model . finally , in castrated rats , the expression of toll - like receptor 4 , functional complex involved in the activation of inflammatory and autoimmune response , is significantly increased . finally , the patients of group 1 have a condition of male hypogonadism probably related to the age , suggested by higher serum concentrations of lh and lower mean value of testicular volume . this aspect must be evaluated through further studies in order to understand a possible association between persistence of magi ( these patients have an increased duration of symptoms ) and the different serum concentrations of tt , as suggested in previous studies about a similar clinical model represented by luts ( lower urinary tract symptoms ) secondary to benign prostatic hyperplasia ( bph ) [ 31 , 32 ] . an important difference between these two clinical models is represented by the increase in prostate volume for the patients with luts - bph related that represent a potential contraindication to the hormonal treatment with t , while patients with magi have an inflammation of periurethral zone assessed by ultrasound evaluation associated with obstructive symptoms [ 11 , 15 , 17 , 18 ] , without a real increase in prostate volume . these patients may find advantages from hormone therapy with t when hypogonadism is associated ; however , further studies will need to evaluate this aspect . however , it should be noted that increasing evidence shows that prostate volume and activity are t dependent only in the overt hypogonadal range , for very low serum t concentrations [ 33 , 34 ] . in conclusion , the results of this study suggest evaluating the serum concentrations of total testosterone in patients with magi . in particular , male hypogonadism is associated with different clinical and ultrasound characterization of these patients .
conjugated organic structures have attracted significant recent attention due to their potential applications in single - molecule transistors and organic photovoltaics . in the quest for smaller and more efficient electronics , organic semiconductors serve as a promising alternative to their silicon counterparts because of their increased electronic efficiency and ease of chemical functionalization . in this context , oligoacenes which are composed of linearly fused benzene rings ( figure 1 ) have high application potential since they possess large charge - carrier mobilities and tunable electronic band gaps . most notably , pentacene is already utilized as an organic field - effect transistor due to its large hole mobility ( 5.5 cm / v s ) which exceeds that of amorphous silicon . in general , the linear acenes are especially important since they form the basic fundamental units of armchair graphene nanoribbons , which continue to garner enormous interest as novel nanoscale materials . molecular structure and atom labels for the linear acenes . the specific atom numbers depicted in this figure define an ordered basis for generating the transition density matrices discussed in section . in addition to their promising photovoltaic applications , the oligoacenes are also noteworthy as a unique system in which the successes and failures of time - dependent density functional theory ( tddft ) can be assessed and addressed . in 2003 , grimme and parac noted a dramatic failure ( > 0.5 ev error in excitation energies ) of tddft using standard hybrid functionals for the lowest - lying * states of the oligoacenes.(20 ) their findings were particularly unusual since these types of valence excitations are typically well described ( within 0.1 ev ) by hybrid tddft calculations . while it is well - known that long - range charge - transfer and rydberg excitations provide a significant challenge for tddft , these effects are not present in the acene systems since none of the valence excitations possess rydberg character or involve any long - range charge transfer ( both the ground- and excited - state dipole moments are exactly zero by molecular symmetry ) . as a result , the unexpected failure of tddft in these simple valence excitations is most unusual and somewhat surprising . first , we show that certain range - separated functionals , which incorporate both a position - dependent admixture and an asymptotically correct contribution of hartreefock ( hf ) exchange , yield substantial improvements over conventional hybrids for the various oligoacene excitations . numerical optimization of parameters in the range - separated and hybrid functionals is carried out to understand their effect on excitation energies and their overall trends . following the two - dimensional real - space analysis approach of tretiak et al . , we then examine excitonic effects for the various excitations and tddft methods . the transition densities and electron difference density maps enable us to understand why conventional hybrids fail and how range - separated functionals accurately reproduce excitation energies and quasiparticle energy gaps for each of the different transitions . we begin by briefly reviewing these two different formalisms and then compare their accuracy in predicting oligoacene excitation properties . recall that dft is an exact theory in which the only inaccuracies encountered in practice arise from approximations to the ( still unknown ) exchange - correlation functional . one of the most widely used dft schemes for the exchange - correlation energy is becke s three - parameter b3lyp method,(51 ) which has a relatively simple formulation given by in this expression , ex , hf is the hf exchange energy based on kohnsham orbitals , ex , slater is the uniform electron gas exchange - correlation energy,(52 ) ex , becke88 is becke s 1998 generalized gradient approximation ( gga ) for exchange,(53)ec , vwn is the voskowilknusair 1980 correlation functional,(54 ) and ec , lyp is the leeyangparr correlation functional.(55 ) depending on the choice of gga , there are numerous other hybrid functionals in the literature which combine different gga treatments of exchange and correlation with varying coefficients . in these global hybrid functionals , the fraction of nonlocal hf exchange , a0 , is held constant in space and fixed to a gga - specific value ( the b3lyp functional , for example , is parametrized with a0 = 0.20 ) . in contrast to conventional hybrids which incorporate a constant fraction of hf exchange , the long - range - corrected ( abbreviated as lc or lrc in the literature ) formalism mixes hf exchange densities nonuniformly by partitioning the electron repulsion operator as the erf term denotes the standard error function . r12 = \|r1 r2\| is the interelectronic distance between electrons at coordinates r1 and r2 , and is the range - separation parameter in units of bohr . the first term in eq 2 is a short - range interaction which decays rapidly on a length scale of 2/ , and the second term is the long - range part of the coulomb potential . for a general gga or hybrid functional , the corresponding exchange - correlation energy according to the lc formalism is in this expression , ec , dft is the original , unmodified dft correlation contribution , ex , dftsr and ex , hfsr are the respective dft and hf contributions computed with the short - range part of the coulomb operator ( first term in eq 2 ) , and ex , hflr is the hf exchange contribution evaluated using the long - range part of the coulomb potential(56 ) ( second term in eq 2 ) . the ahf parameter is the coefficient of hf exchange present in the original hybrid functional ( ahf = 0 if the original functional is a pure density functional , i.e. , blyp , bop , or pbe ) . it is important to mention at this point that there are also several other range - separation techniques and functionals in the literature , and that the prescription given in eqs 2 and 3 is only one of many lc forms . for example , the range - separation technique has been further modified by handy et al . with their cam - b3lyp ( coulomb - attenuating methodb3lyp ) methods . similarly , the scuseria group has also developed several new range - separated functionals based on a semilocal exchange - hole approach . these exchange - hole models have been further refined by the herbert group to design new functionals which accurately describe both ground and excited states . in terms of chemical applications , have also presented benchmarks for several families of excitations including the electronic spectra of anthroquinone dyes,(57)n * transitions in nitroso and thiocarbonyl dyes,(58 ) and * excitations in organic chromophores.(59 ) very recently , there has also been pioneering work by the baer group and the kronik group in constructing range - separation functionals tuned entirely from first principles . the key to their success is the choice of a range - separation parameter , , which minimizes the difference between the ionization energy ( ie ) and the negative of the highest - occupied molecular orbital ( homo ) energy , ehomo , of the molecule . since the ionization energy is rigorously equal to ehomo for an exact functional , the formalism by baer and co - workers is entirely self - consistent and does not require any experimental input or high - level benchmark calculations . in all of these various range - separated methods , the key improvement in their accuracy is the smooth separation of dft and nonlocal hf exchange interactions through the parameter . specifically , the exchange - correlation potentials of conventional density functionals exhibit the wrong asymptotic behavior , but the lc scheme ensures that the exchange potential smoothly recovers the exact 1/r dependence at large interelectronic distances . it is important to point out that the length - scale partitioning in the lc formalism is essential for obtaining accurate tddft results . more precisely , a 100% global hf exchange fraction without range separation can corrupt the delicate balance between exchange and correlation contributions , resulting in large errors in excitation energies . for extended charge - transfer processes , the long - range exchange corrections are also particularly vital since these types of excitations are especially sensitive to the asymptotic part of the nonlocal exchange - correlation potential . for the linear acenes in this work , we compared the performance of global hybrid functionals against range - separated and wave function - based calculations . in order to investigate the role of different hf exchange schemes in the various tddft methods , we explored the effect of changing the hf exchange fraction , a0 , in the global hybrid model and the result of varying the range - separation parameter within the lc formalism . for the parametric study on global hybrids , we kept the same functional form in becke s three - parameter model ( eq 1 ) and computed vertical singlet excitation energies as a function of a0 ranging from 0.0 to 1.0 in increments of 0.05 . in these calculations , we fixed ax = 1 a0 in eq 1 but kept the correlation contribution with ac = 0.81 unchanged . the ax = 1 a0 convention is a common choice used in many hybrid functionals such as becke s b1 convention(61 ) ( in a previous study on large oligothiophenes,(31 ) we had carried out calculations with ax fixed to the original 0.72 value recommended by becke and found that all of the excitation energies were nearly identical compared to the ax = 1 a0 convention ) . to explore the effect of range - separated exchange on the optoelectronic properties of the acenes , we computed vertical singlet excitation energies as a function of ranging from 0 to 0.90 bohr ( in increments of 0.05 bohr ) while keeping the correlation contribution ec , dft in the lc - blyp functional unchanged . in our study , we utilized several range - separated functionals including cam - b3lyp , lc - bop , lc - pbe , lc-pbe , and lc - blyp but found that all of the full - hf - exchange lc functionals gave similar results for the linear acenes . it is very important to note that the original cam - b3lyp functional is defined(39 ) with a coulomb - attenuating parameter of + = 0.65 and , therefore , exhibits a 0.65/r dependence for the exchange potential . as a result , the cam - b3lyp functional is particularly different than the other lc functionals considered in this work since it does not incorporate a full 100% hf exchange at large interelectronic distances . the very similar results obtained from the other full - exchange lc functionals imply that the excitation energies are not very sensitive to the specific dft correlation contribution used , and that the systematic error observed previously by grimme and parac(20 ) is largely due to the hf exchange component for the acene systems . in light of these similarities , much of our parametric study focuses on the lc - blyp results since the other full - hf - exchange lc methods give very similar energies as a function of . we should also note that a direct comparison between lc - blyp , cam - b3lyp , and the global hybrid model in eq 1 allows a very fair and consistent evaluation since all of these methods have similar correlation contributions . as benchmarks for assessing the quality of the various tddft methods , we calculated cc2/cc - pvtz excitation energies for the linear acenes ranging from n = 2 to 7 benzene rings ( we stop at n = 7 since our cc2/cc - pvtz calculations indicate a very abrupt and large multireference / diradical character for n = 8) . we use the cc2 excitation energies as reference values since eom - ccsd and caspt2 calculations with the cc - pvtz basis set were out of reach for larger acenes containing five or more benzene rings . furthermore , we consider the cc2 results as reliable reference values since they accurately reproduce solvent - corrected experimental excitation energies(20 ) ( see table 1 ) and are close to cc3 benchmark calculations for the smaller acenes.(64 ) as an additional check on the quality of the cc2 calculations , we found that none of the acene systems required a multireference treatment of electron correlation ( d1 diagnostic values were in the 0.040.06 range ) , and contributions from single excitations were always greater than 90% . the mean absolute errors ( mae ) relative to solvent - corrected experimental results are listed below each of the various methods . excitation energies were computed with the cc - pvtz basis with the same reference geometry for all of the different methods . in order to maintain a consistent comparison across the b3lyp , cam - b3lyp , lc - bop , lc - pbe , lc-pbe , lc - blyp , and cc2 levels of theory , these reference geometries were optimized at the b3lyp / cc - pvtz level of theory and are available in the supporting information . for all of the tddft excitation energies , we used a cc - pvtz basis set and a high - accuracy lebedev grid consisting of 96 radial and 302 angular quadrature points . all tddft calculations were performed with a locally modified version of gamess,(65 ) and the cc2 calculations were carried out with the turbomole package.(66 ) we focus on two different valence excitations in the linear acenes , commonly labeled in the literature as la ( lowest excited state of b2u symmetry ) and lb ( b3u symmetry ) . the la excited state results from a homo lumo transition with polarization along the molecular short axis , and the lb state is characterized by a nearly equal mixture of homo1 lumo and homo lumo+1 excitations with a total polarization along the long axis.(68 ) using the cc2 excitations as reference values , we performed a total root - mean - square error ( rmse ) analysis for all 12 energies ( six la and six lb transitions ) as a function of and a0 . as seen in figure 2a , the rmse curve for lc - blyp has a minimum at = 0.29 bohr with a rms error of 0.10 ev . perhaps , surprisingly , this rmse - optimized value of is nearly identical to the 0.31 bohr value recommended for simultaneously describing excitation and fluorescence energies in large oligothiophenes.(31 ) the rmse in figure 2b for the b3lyp - like global hybrid functional has a minimum at a0 = 0.50 , with a larger error of 0.20 ev . it is worth noting that our rmse - minimization with a0 = 0.50 and ax = 1 a0 yields a functional very similar to the bhhlyp functional ( originally defined with ac = 0 ) with the exception that our choice has an extra correlation contribution due to the ec , lyp term in eq 1 . we denote this reoptimized hybrid functional with a0 = 0.50 as b3lypopt in the remainder of this work . unless otherwise noted , all further lc - tddft calculations indicate a range - separation parameter of = 0.29 bohr . total root - mean - square errors ( rmse ) as a function of ( a ) the range - separation parameter in the lc - blyp functional and ( b ) the hf exchange fraction a0 in a b3lyp - like hybrid functional . part a shows the rmse curve having a minimum at = 0.29 bohr , and part b shows the rmse curve having a minimum at a0 = 0.50 . table 1 compares the la and lb excited - state energies between b3lyp , b3lypopt , cam - b3lyp , lc - bop , lc - pbe , lc-pbe , lc - blyp , and cc2 , and figure 3a , b depicts in more detail the general trends in transition energies ( expressed in wavelength units ) between the various tddft and cc2 results . it is most important to note in these figures that the energetic ordering of the two electronic states is different , depending on the size of the acene . specifically , both cc2 and experimental studies(69 ) indicate that a curve crossing between the la and lb states occurs slightly before n = 3 benzene rings ( anthracene ) . for all of the other larger acenes , the la state lies energetically below the lb state . examining table 1 and figure 3b , we find that the full - exchange lc - tddft calculations are unique in that they show excellent agreement with cc2 energies for both the la and lb excitations . moreover , all of the lc - tddft methods preserve the correct ordering of electronic states between n = 2 and n = 3 benzene rings . in the case of the cam - b3lyp functional though ( which only has 65% hf exchange at long - range ) , there are still some systematic discrepancies for the la excitations which are still somewhat underestimated . although the energetic ordering of the la and lb states is correctly predicted by cam - b3lyp , the energy differences are almost negligible , with only a 0.05 ev difference separating the la and lb states of naphthalene ( compared to a 0.2 ev difference with the full - exchange lc functionals ) . these observations strongly indicate that a range - separated partitioning of exchange alone , without 100% asymptotic hf exchange , is not sufficient , and a full asymptotic contribution of exchange is essential for accurately describing both the la and lb excitations . turning now to the global hybrids , figure 3a shows that the b3lyp functional severely underestimates excitation energies ( i.e. , overestimates absorption wavelengths ) for the la electronic state . the situation is somewhat improved upon using the rmse - optimized a0 = 0.50 value in b3lypopt ; however , this procedure results in lb excitations which are now overestimated and la excitations which are still quite underestimated . the crossing between la and lb curves occurs much too early in both functionals , and the electronic symmetries in naphthalene have the wrong order . in general , the accuracy in excitation energies and trends is significantly improved with the lc scheme , while conventional hybrids are unable to reproduce the qualitative behavior in excitations even if the fraction of hf exchange is optimized . comparison between tddft and cc2 excitation energies ( in wavelength units ) for ( a ) conventional global hybrid and ( b ) range - separated lc - blyp functionals . the b3lypopt functional denotes a modified b3lyp functional with a rmse - optimized exchange fraction of a0 = 0.50 , as discussed in the main text . from these results , it is interesting to note that long - range charge transfer is not responsible for the unexpected failure of b3lyp in these highly symmetrical systems . in a recent benchmark study , peach et al.(26 ) introduced a diagnostic test which quantifies the spatial overlap , , between the occupied and virtual orbitals involved in an excitation . this diagnostic metric is typically used to postprocess a converged tddft calculation and has an intuitive form given by in this expression , xia and yia are the virtualoccupied and occupiedvirtual transition amplitudes , respectively , and oia is the spatial overlap integral of the moduli of the two orbitals , oia = \|i(r)\|\|a(r)\| dr . by construction , the diagnostic metric is bounded between 0 and 1 , with small values signifying a long - range excitation and large values indicating a localized , short - range transition . on the basis of their extensive benchmarks , if is less than 0.3 , indicating little overlap and significant long - range charge transfer character , hybrid functionals are predicted to yield inaccurate results . in table 2 , we computed the diagnostic for both the la and lb states and found that all values were well above the 0.3 threshold ( some of them even approaching 0.9 ) , indicating a substantial overlap and no long - range charge transfer in these systems ( it is rather interesting though that the diagnostic incorrectly predicts the la excited state to be more accurately described than the lb state in both the b3lyp and lc - blyp functionals ) . thus , instead of long - range charge transfer from one end of the molecule to the other , we do find that the la excitation involves a sizable local rearrangement of electron density . in support of this assertion , figure 4 depicts the electron density difference map ( excited ground ) for the la and lb excited states in pentacene computed at the cc2 level of theory ( difference maps for the other acenes can be found in the supporting information ) . the electron density difference map gives a dynamic visualization of electronic rearrangement for a transition , with red regions ( positively valued ) denoting an accumulation of density and blue regions ( negatively valued ) representing a depletion of density upon excitation . as depicted in figure 4 , the la state involves significantly more local charge redistribution than the higher - energy lb state . in contrast , the lb excited - state density is very similar to the ground state , as evidenced by the very small and sparsely distributed isosurface regions . these cc2 difference densities confirm the long - held valence - bond viewpoint that the la state possesses an notice also that the length scale of charge redistribution is on the order of the carboncarbon bond length ( 1.4 ) , which is comparable to the length scale at which lc - blyp predicts long - range hf exchange to dominate short - range dft correlation ( 1/ 1.8 ) . even though none of these transitions have long - range charge transfer character , our findings do support the physical interpretation that a range - separated contribution of full hf exchange on the length scale of the molecule is still necessary for accurately describing these local charge rearrangements . electron density difference maps ( excited ground ) for the la and lb excited states of anthracene computed at the cc2 level of theory . red regions denote a positive density difference ( accumulation of density upon electronic excitation ) , and blue regions represent a negative density difference ( depletion of density upon excitation ) . both densities are plotted using the same isosurface contour value . in order to provide further insight into these optoelectronic trends , we carried out an investigation of excitonic effects by analyzing electronhole transition density matrices for the various excitations and tddft methods . , one can construct coordinate qv and momentum pv matrices with elements given by where g and v are ground and excited states , respectively . the fermi operators ci and ci represent the creation and annihilation of an electron in the ith basis set orbital in . for the acene systems analyzed in this work , the qv and pv matrices each form a two - dimensional xy grid over all of the carbon sites along the x and y axes . the specific ordering of the carbon sites used in this work is shown in figure 1 . the ( qv)mn coordinate matrix gives a measure of exciton delocalization between sites m and n , and the ( pv)mn momentum matrix represents the probability amplitude of an electronhole pair oscillation between carbon sites m and n , respectively . each of these matrices provides a complementary view of exciton delocalization and electronhole coherence for optical transitions within the acene systems . figure 5 displays the absolute value of the coordinate density matrix elements , \|(qv)mn\| , for the la and lb excitation energies computed at the lc - blyp level of theory . the x and y axes in this figure represent the benzene repeat units in the molecule , and the individual matrix elements are depicted by the various colors . on the basis of its construction , off - diagonal elements with large intensities represent widely separated electronhole pairs between different atoms . as shown in figure 5 , the la density matrix has more off - diagonal elements than the corresponding lb excitation , whose matrix elements are primarily confined along the diagonal . these figures reflect the more delocalized nature of the la state , in agreement with the electron density difference maps discussed previously . it is also important to note that all the transition density plots are symmetric along the counterdiagonal , verifying that no long - range charge transfer occurs in these systems ( an asymmetric transition density along the counterdiagonal implies more electrons than holes are localized on one side of the molecule ) . contour plots of coordinate density matrices ( q ) for the la and lb excited states computed at the lc - blyp level of theory . the x- and y - axis labels represent the number of benzene repeat units in the molecule . the elements of the coordinate matrix , qmn , give a measure of exciton delocalization between sites m ( x axis ) and n ( y axis ) . the coherence size , which characterizes the distance between an electron and a hole , is given by the width of the momentum density matrix , pv . to compare excitonic effects between global and range - separated hybrids , we plot the absolute value of the momentum density matrix elements , \|(pv)mn\| , for both the b3lyp and lc - blyp functionals in figure 6 ( transition density plots for all of the different functionals and excited states can be found in the supporting information ) . these figures show that the b3lyp functional gives a more delocalized density - matrix pattern and a larger coherence size compared to the lc - blyp functional . furthermore , the coherence size as predicted by the b3lyp functional is larger by nearly one repeat unit in comparison to the lc - blyp results . these findings are consistent with the b3lyp formalism which only incorporates a global fraction of 20% hf exchange and , therefore , exhibits a 0.2/r dependence for the exchange potential . as a result , the asymptotically incorrect b3lyp exchange potential is not attractive enough , leading to an over - delocalized electronhole pair and , therefore , an overestimated coherence size in the acene systems . contour plots of momentum density matrices ( p ) for the lb excited state computed at the b3lyp and lc - blyp levels of theory . the x- and y - axis labels represent the number of benzene repeat units in the molecule . the elements of the momentum matrix , pmn , represent the probability amplitude of an electronhole pair oscillation between sites m ( x axis ) and n ( y axis ) . finally , it is interesting to compare quasiparticle energy gaps predicted by both global hybrid and range - separated functionals in the acene systems . within kohnsham theory,(73 ) the quasiparticle gap can be approximated by the difference between the lowest unoccupied and highest unoccupied molecular orbital energies , elumo ehomo . table 3 compares elumo , ehomo , and the experimental ionization energies ( ie ) for the linear acenes computed at the b3lyp , cam - b3lyp , and lc - blyp levels of theory . from kohnsham theory , it is well - known that an ( if one had access to such a functional ) , would yield an ionization energy exactly equal to ehomo . for the pentacene molecule , as a specific example , the b3lyp functional provides a ehomo value of 4.78 ev which significantly underestimates the experimental ionization energy(74 ) of 6.61 ev . the ehomo values predicted by cam - b3lyp are an improvement over the b3lyp energies , but the average deviation of 0.70 ev from the experimental ies is still quite large . in contrast , the lc formalism , which incorporates a correct asymptotic behavior of the exchange potential by construction , gives ehomo values in exceptional agreement with all of the experimental ies , resulting in an impressive average deviation of 0.07 ev . these results complement our previous discussion of la and lb excitation energies by further demonstrating that a full 100% asymptotic contribution of hf exchange is necessary to provide a consistent description of electronic properties in these systems . furthermore , these findings demonstrate that the range - separated formalism with full asymptotic hf exchange is very self - consistent both the excitation energies and quasiparticle properties in these systems are predicted accurately while simultaneously satisfying the energy constraints as required by kohnsham theory . the average deviation of ehomo relative to the experimental ie is listed below each of the various methods . in conclusion , the present study clearly indicates that both a range - separated partitioning as well as an asymptotically correct contribution of exchange play a vital role in predicting optoelectronic properties in the linear acenes . even though none of the excitations involve extended long - range charge transfer , we find that a range - separated contribution of full exchange is still necessary to accurately describe both the valence excitation energies and the la lb curve crossing in these simple systems . the results of our observations also strongly indicate that a range - separated partitioning of exchange by itself , without 100% asymptotic hf exchange ( i.e. , cam - b3lyp ) , is not sufficient to accurately describe both the la and lb states . conversely , reoptimization of functional parameters toward 100% full exchange without range separation in a global hybrid does not improve the situation either ; in fact , this reparameterization results in a corruption between exchange and correlation errors with trends in la and lb excitations being even more poorly described . in particular , we find that global hybrid functionals overdelocalize excitons , underestimate quasiparticle energies , and are unable to reproduce general trends in both la and lb , even if the fraction of hf exchange is optimized . the most important results of our observations indicate that a simultaneous use of range - separated partitioning as well as a full contribution of exchange at large interelectronic distances is essential for accurately describing both the la and lb states in these systems . as acenes form the basis of nanoribbons and other polycyclic aromatic hydrocarbons,(75 ) this study serves an important role in determining which tddft methods are most appropriate for these systems , especially since wave function - based calculations on carbon nanostructures are still prohibitively demanding . looking forward , it would be extremely interesting to see if the range - separated formalism also provides a similar accuracy for describing triplet states in acenes and other chromophores . while this study focused on only singlet excitations , further work is still needed to understand triplet excitations since exciton fission to low - lying triplet states ultimately control the electronic efficiencies in photovoltaic systems.(76 ) we are currently investigating these triplet states , with further calculations on extended organic light - harvesting systems,(9 ) to help predict the efficiencies of these materials . with this in mind , we anticipate that the lc - tddft technique will play a significant role in understanding and accurately predicting the optoelectronic properties in these novel nanostructures .
cavernous hemangiomas ( ch ) are well - defined lesions composed of closely packed sinusoidal vascular channels with structurally incomplete vessel walls and no intervening neuronal parenchyma . although cavernous malformations are thought to arise sporadically in many individuals , a substantial number of patients have been characterized with a heritable form of this disease , which is transmitted in an autosomal - dominant mode , with variable expression and incomplete penetrance , often presenting as multiple intracranial lesions on magnetic resonance imaging ( mri ) . the de novo appearance of ch has been firmly established , most notably after radiation therapy . the lesions usually appear 810 years later within brain tissue that was included in radiation fields . in contrast to the infrequency of ch , medulloblastomas are the most common malignant brain tumors of the posterior cranial fossa in children , being generally associated with several familial cancer predisposition syndromes.[57 ] although caf - au - lait lesions occur with variable frequency in a number of disorders , such as neurofibromatosis types 1 ( nf1 ) and 2 , albright syndrome , among others , to the best of our knowledge this phenotype has never been reported before in association with a non - radiation - induced brain ch and medulloblastoma . a 13-month - old boy , with consanguineous parents , was referred to the pediatric emergency department of our hospital with partial - complex seizure . prominent caf - au - lait skin lesions were observed on the patient 's neck and midchest [ figure 1a - c ] . his father and brother had similar dermatologic signs , the latter having died at the age of 6 years due to t - cell acute lymphocytic leukemia . he also had an older brother who died owing to congenital heart disease [ figure 1d ] . mri of the brain showed an isolated lesion in the left mesial - temporal lobe [ figure 2a ] . ( b , c ) caf - au - lait lesions associated with multiple ephelides . ( d ) pedigree of the family . squares , male ; circles , female ; symbols with a diagonal , deceased individuals . age at diagnosis is indicated on the right bottom of the symbols . solid symbols , affected individuals ; shaded symbol with a diagonal , congenital heart disease ( a ) axial t2-weighted magnetic resonance imaging ( mri ) showing the left mesial temporal cavernous hemangioma . ( b ) axial t2-weighted mri showing a left - cerebellar posterior fossa tumor with brainstem displacement . ( c ) fluid - attenuated inversion - recovery image showing an asymptomatic right frontal radiationinduced cavernous hemangioma 60 months after radiation terapy ; ( d ) axial t1-weighted mri enhanced with gadolinium showing recurrence of the medulloblastoma in the cerebellar vermis . ( e , f ) axial and sagittal t1-weighted image enhanced with gadolinium two months later showing massive brain stem infiltration at the age of 4 years he presented with a 7-day history of progressively worsening headache , vomiting , and gait ataxia . mri scans showed supratentorial hydrocephalus associated with a posterior fossa tumor in the left cerebellar hemisphere displacing the brainstem [ figure 2b ] . five years later , control mri scans confirmed no recurrence of the lesion , however showed multiple and asymptomatic radiation - induced cavernous hemangiomas [ figure 2c ] . at the age of 10 a massive recurrence of the medulloblastoma was observed in the posterior fossa associated with severe hydrocephalus [ figure 2d ] . a surgical resection was attempted with the aim of reducing the lesion . in spite of aggressive adjuvant treatment , the child died 3 months later due to infiltration of adjacent brain stem and clinical complications [ figure 2e , f ] . caf - au - lait skin lesions are more commonly associated with neurofibromatosis type 1 ( nf1 ) , or von recklinghausen syndrome . the presence of six or more large skin lesions with diameters above 1.5 cm is considered to be virtually diagnostic of nf1 . our patient fulfilled this criterion , with more than a dozen large caf - au - lait skin lesions in addition to numerous ephelides on the neck , midchest and armpits . although various skin lesions have been reported in association with familial ch , documented cases of caf - au - lait lesions are scarce . venous restrictive disease has been postulated to be a consequence of impaired venous flow caused by radiation change . a cumulative increase of radiation - induced ch occurs over time in children who receive cranial radiation therapy . in our case , posterior fossa tumors associated with nf1 are very infrequent , differently from those tumors located in other regions , such as optic pathway tumors and brain stem tumors , which are common and histologically benign . therefore , medulloblastoma is a typically malignant cerebellar tumor of infancy rarely associated with nf1 clinical features . in view of these facts , our case has been extensively analyzed in order to define a syndrome that could explain the presence of nf1 clinical features associated with non - radiation - induced brain ch and posterior fossa medulloblastoma in a child with consanguineous parents . therefore , the department of genetics of our university has investigated whether the patient 's presentation was related to constitutional mismatch repair - deficiency ( cmmr - d ) . the mismatch repair ( mmr ) machinery contributes to genome integrity , and the mlh1 , msh2 , msh6 and pms2 genes play a crucial role in this process . mmr corrects single base - pair mismatches and small insertion - deletion loops that arise during replication . moreover , the mmr system is involved in the cellular response to a variety of agents that damage dna , as well as in immunoglobulin class switch recombination . heterozygous germline mutations may cause lynch syndrome , an autosomal dominant cancer syndrome associated with hereditary nonpolyposis colorectal cancer , endometrial carcinoma , and other malignancies , generally occurring in the fourth and fifth decade of life . in contrast , rare cases of biallelic deleterious germline mutations in mmr genes leading to cmmr - d have been recognized since 1999 . this cancer syndrome is characterized by a wide spectrum of early - onset malignancies such as hematological , brain or gastrointestinal cancers and a phenotype that resembles nf1 . interestingly , several reports stress that caf - au - lait macules differ from typical nf1-associated lesions in that they have variable degree of pigmentation , irregular borders , and , usually , a segmental distribution pattern . other features of nf1 found in cmmr - d patients include lisch nodules , neurofibromas , skinfold freckling , and tibial pseudarthrosis . hence , it is not surprising that a number of cmmr - d cases were initially diagnosed as nf1 . it has been speculated that nf1-like clinical features in cmmr - d patients result from germline mosaicism arising early during embryonic development . there are data supporting the hypothesis that the nf1 gene is a mutational target of mmr deficiency . the use of such analysis may facilitate identification and surveillance of heterozygous and homozygous individuals in the wider family , in addition to allowing for informed decision - making about prenatal genetic diagnosis .
in the history of treating night blindness , the ancient egyptian , the babylonians , the greeks , and many other cultures after them , used animal liver as treatment , like what we did in rak . in the egyptian papyrus ebers ( 1500 b.c . ) the recommended cure was : roasted ox liver , pressed , applied ( topical to the eye ) . another ancient egyptian papyrus , kahun 1 ( 1825 b.c . ) , a gynecological treatise , mentions instructions for a woman , can not see , to eat raw liver of an ass . the assyrian medical texts ( 700 b.c . ) they thought it was caused by rays of the moon and cured by application of ass 's liver to the eyes . they did not put the liver itself in the eye but used the extracted oil and probably enjoyed eating the cooked liver . it is very likely that the ancient egyptian ritual treatment also ended with the patient eating the liver . the greeks shifted the recommendation from topical application to frank eating raw beef liver , soaked in honey , to be taken once or twice ( daily ) by mouth . galen ( 130 ad-210 ad ) , recommends : continuous eating of liver of goats . it is clear that the choice of the animal is influenced by its availability in the community . the chinese sun - szu - mo ( 7 century ad ) in his 1000 golden remedies describes a cure by administration of pig 's liver . the juice of lamb liver was applied topically to the eyes of night - blind children . the procedure was exactly as described by the ancient egyptians , except for one small addition : rather than discarding the remaining organ , the ( practitioners ) fed it to the affected child this was never considered part of the therapy itself . similarly , in 1928 aykroyd wrote : i have been told of a custom of steaming the eyes over cooking liver , which is then eaten , as a remedy for night blindness in the canadian newfoundland island . with respect to arab civilization i found that the abbasid physician hunayn ibn ishaq ( 809873 ) recommended in his book seven articles on the eye the same procedure of rubbing the liver oil in the eye , but also added two more suggestions : get the smoke from the cooking liver goes into the eye plus eat the liver . now we know that the cause of night blindness is deficiency in vitamin a and the real therapeutic benefit comes from eating the liver . animal liver , including that of fish , is very rich with vitamin a , because the liver is the store house of vitamin a in the body . some ancient medical practitioners prescribed rubbing the liver oil in the eye , but patients could not resist eating the tasty barbequed liver . hunayn ibn ishaq took the idea from them because he is credited for translating several greek medical books to arabic . in ancient time , they must have realized that there was a nutritional deficiency as the cause of that disease . arabic poetry written by hunayn 's contemporary poet ibn duraid ( 837933 ) who lived in the same city of bagdad , said that night blindness ( asha ) was caused by dinner ( asha ) , that is , lack of dinner : the two words in arabic not only rhyme together but actually sound the same , hence the poetic effect . in the later middle ages , the dutch physician jacob van maerlandt ( 12351299 ) wrote the following poem recommending eating the liver : so , from the above historical review , it was not surprising that such ancient treatment of night blindness was transmitted over centuries to the arabs and reached rak for me to witness . in the late 1920s , the fat - soluble compound in liver was isolated by karrer a swiss scientist and his colleagues , and termed vitamin a. there are two main forms of vitamin a : provitamin a and preformed vitamin a. provitamin a carotenoids are found in plants . there are many forms of provitamin a , but beta - carotene is the only one that is metabolized by mammals into vitamin a. so , after ingestion , the body converts the provitamin into vitamin . preformed vitamin a ( retinol , retinal , retinoic acid , and retinyl esters ) is the most active form of vitamin a ; it is mostly found in animal sources of food , e.g. , liver , and toxicity can occur if excessive quantities are ingested . the liver is rich with vitamin a because about 5085% of the total body retinol is stored in the liver . the top food sources of vitamin a in our area in the gulf are dairy products , liver , and fish . the top sources of provitamin a are carrots , dates , cantaloupe , and squash . dates are very rich with carotenoid . because people in our region consume a lot of dates , night blindness is not very common . in the eye , vitamin a has two major roles : prevention of xerophthalmia ( abnormalities in corneal and conjunctival development ) and photo - transduction ( transforming light into electrical signals for the brain ) . the cone cells are responsible for the absorption of light and color vision in bright light . vitamin a deficiency is rarely seen in the rich communities and industrialized countries . however , it is still the third most common nutritional deficiency in the world . in a large part of the third world ( southern and southeast asia , africa , and south america ) , night blindness , complete blindness , and advanced stages of xerophthalmia occur in many malnourished children and adults . according to the world health organization , 190 million preschool - aged children and 19.1 million pregnant women around the world have a serum retinol concentration below 0.70 mol / l . in our area , the arabian gulf , citizens rarely get night blindness , not only during these days of wealth , but also during the preoil days . both are rich with vitamin a and provitamin a. so , why did my father develop night blindness while the rest of the family , eating the same food , did not ? the answer is that , at that time , he was ill with gallbladder disease . fat soluble vitamins like vitamin a needs to be mixed with bile salts to form small aggregates before it could be absorbed by the gut . people with insufficient bile flow are usually deficient in vitamin a and hence the night blindness . in populations where dietary intake of vitamin a is adequate , there is no evidence that supplementation of vitamin a as an antioxidant is helpful for preventing cardiovascular disease . randomized trials of vitamin a and beta - carotene have shown no benefit for primary or secondary prevention of coronary heart disease . such supplementation may even have harmful effects on cardiovascular mortality , cancer , and bone health . the lesson from the above story of the ancients and how they cured night blindness with food , tells us to avoid unnecessary vitamin supplements and get our nutritional requirement through balanced and healthy food .

the increased life expectancy of brazilians , due to scientific and technical advances has contributed to the changing epidemiological profile . in this sense , the model of health care focused on infectious diseases has been gradually replaced by a model directed at chronic diseases . stroke is classified as chronic degenerative disease and is characterized by an abrupt episode of vascular origin , which results in focal or generalized disturbance of brain function that persists for more than 24 hours1 . after a stroke , an individual may present neurological alterations related to motor functions , speech and cognition , as well as impairment of independence in daily activities of life2 . hemiplegia is characterized by an abnormal physical state of the hemibody , accompanied by total loss of strength , alteration of the senses , and mental , language and perceptual disorders , while hemiparesis results in partial loss of strength and the other symptoms mentioned above , with hemiplegia / hemiparesis3 being the most commonly found symptom in patients after stroke4 . a functional recovery strategy for limitations resulting from motor impairment , the use of motor imagery ( mi ) stands out especially with regard to daily activities . mi is defined as the mental process dynamic in which the subject simulates a motor task that occurs without the movement of the segments associated with this task , and can it be presented in two modalities : visual and kinesthetic . these differents modalities of mi activate distinct brain regions , and these activities can be investigated through electroencephalograms ( eeg)5 . besides mi , another therapeutic strategy is the mechanism of mirror neurons . these are visualmotor neurons that are triggered both when an individual performs a certain gesture as when volunteer observes another individual performing the same movement . the presence of mirror neurons in humans has been demonstrated in studies of the reactivity of eeg brain rhythms during the observation of movements6 . furthermore , the utility of eeg as a tool for the analysis of brain function can be observed in protocols that use actual execution of motor tasks , like mi7 . the present study used electroencephalography to compare the values of absolute power of the alpha , beta and gamma frequency bands in the central cortical areas during performance of the motor gestures of feeding , and the drive system of mirror neurons and imagery between a right hemiparetic volunteer ( rhv ) and a healthy volunteer ( hv ) . this study was conducted at the laboratory of brain mapping and functionality ( lamcef ) at the federal university of piau ( ufpi ) and presents the report of two cases , one healthy volunteer and one right hemiparetic ; both were male and right - handed . to check the handedness then , they were evaluated using to the modified ashworth scale to identify hypertonia degrees . the mini - mental state examination ( mmse ) was conducted as a cognitive screening instrument . in order to verify the overall functionality of the subjects , the barthel index was assessed as well . the ability of imagining a motor gesture in the first or third person was investigated through the scale of motor imagery ( the kinesthetic and visual imagery questionnaire ; kviq ) . the subjects were duly informed about the objectives , the experimental procedure involved , and the methodology of this study , and voluntarily signed a consent form . the study was approved by the ethics committee on human research of ufpi , under registration number 246.283/2013 . electrodes were placed according to the 10 - 20 international system , and the reference electrodes were placed on the earlobes ( bi - atrial ) . the eeg signal frequencies fluctuated between 0.01 and 50 hz . to ensure a more judicious selection , parts contaminated by muscle artifacts were removed using an automatic rejection algorithm to reject values exceeding a threshold ( 100 microvolts ) . the first eeg measure was performed with the subject at rest for 4 minutes with his eyes open . immediately after , the subject watched a 6-second video showing the motor gestures of picking up a cup , bringing it to the mouth and putting it back on the table in front of the subject . it is noteworthy that the individual in the video sat in a comfortable position , under the same conditions as the subjects of our experiment . after watching the video the subjects performed three randomized tasks . 1 ) practice the subjects performed the motion of withdrawing the right hand that was resting on the table , picking up a glass , bringing it to the mouth and return it to the initial site marked on the table , and replacing the hand in the starting position . 2 ) imagery the subjects imagined the motor gesture described above in the first person for 4 minutes . 3 ) mirror neurons the subjects watched the video of the motor gesture described . immediately after the experimental tasks the data were analyzed used matlab r2011a , converted by microsoft excel 2010 , and transformed into log 10 values . the data in relation to age , and cognitive functional capacity , as well as the scale of imagery are presented in table 1table 1 . all values of the magnitude of absolute power in the alpha band of hv were lower than those of rhv ( fig . comparison of the absolute power of the alpha band across the five tasks ) . regarding the imagery data scale , despite the individuals finding it easy to imagine , the electroencephalographic results were not the same in hv . the analysis verified higher values at cz , c4 and t4 , corresponding to the right hemisphere . in the beta band , in the practice task , an increase in the magnitude of power at t3 of hv ( fig . comparison of the absolute power of the beta band across the five tasks ) was observed . in contrast , in rhv , there was no increase in absolute power values in practice compared to rest , but at t4 greater activation was observed . comparison of the absolute power of the alpha band across the five tasks comparison of the absolute power of the beta band across the five tasks the analysis of the gamma band showed there was increased activity in rhv during the imagery task , specifically at t4 ( fig . comparison of the absolute power of the gamma band across the five tasks ) . during all other tasks , we observed a reduction in the magnitude of the gamma band power . during initial rest hv showed high power at c3 that corresponded to representation of the hand . in general , hv showed lower cortical activation during all tasks at all measurement sites ; however , at c3 , there was an increase in the magnitude of the absolute power during the following tasks : initial rest , practice , imagery and final rest . the hv also showed increased power at t4 during tasks of initial rest , practice , and imagery , and reduced activity during rest and final video . hv showed higher magnitude at c4 during initial rest , but in the other tasks only reductions were observed . hv showed reduced cortical activation at all the other measurement sites during all of the tasks . the present study aimed to compare between rhv and hv , the values of absolute electroencephalographic in the alpha , beta and gamma frequency bands of the central cortical areas , resulting from the execution of the motor gesture of feeding , the activation of the mirror neuron system , and imagery . for this study , the central electrode sites of c3 , cz , c4 , t3 and t4 were chosen in order to represent the motor areas related to the planning and execution of movement , and the sensory areas corresponding to the areas of the body activated whenever any sense or sensory receptor is specifically stimulated8 . the alpha band is inversely related to cortical activation7 , 9 , cognitive processes and wakefulness required during task execution10 , 11 . changes in alpha band power seem to be associated with cognitive tasks of different levels of complexity13 . alpha band power is also associated with highly specific perception , attention and memory processes10 . the increase in the absolute power of the alpha band observed in this study appears to be related to the mental consolidation of the task9 . alpha rhythm activation is inversely related to the proportion of the population of cortical neurons activated during perception , motor and cognitive processes14 . thus , we suggest that hv responded adequately to the demands of the tasks , while rhv showed lower mental activity in all tasks , possibly because of brain injury . the lower values of the absolute power verified at c3 , demonstrate that hv showed greater mental activity during imagery and video than rhv . the results of hv showed higher activities at cz , c4 and t4 , corresponding to the right hemisphere . this shows that due to lesion in the left hemisphere , rhv had cortical activation suggestive of prior motion inexperience . it has been reported that cortical activation occurs in the contralateral hemisphere during the execution and learning of movement13 . the beta frequency band spans 12 to 30 hz . from a functional perspective , beta frequencies are commonly associated with motor activities11 , 12 . when this band is presented in cortical activity , suggests mental activity and sharp attention9 . the beta rhythm seems to be linked to movement precisely in the frontal and central areas of the cortex that are related to the planning and execution of voluntary movements , and it can be influenced by exercise13 , 15 . the beta band is related to motor activities , especially in the frontal and central cortical areas which are , responsible for activating conditions of planning and execution of voluntary movements12 , 13 . the magnitude of the absolute power at t3 increased during the practice by hv ( fig . 2 ) , possibly because t3 is representative of secondary motor areas which work as planners and organizers of the sequence of movements performed by the hands of the subject . in contrast , in rhv , there was no increase in power values during rest , but at t4 greater activation was observed , presumably to assist the work of t3 during practice12 . this would indicate synchronization between the same associated regions of the left and right sides of the brain contralateral to the motor task requested . beta waves appear in association with maximum attention of the dominant hemisphere , which possibly lends itself to motor planning strategy , as shown predominantly at t38 . the gamma band is related to memory , integrating factors and integrating spatial / temporal proprioception9 . however , uhlhaas demonstrated frequencies from 60 to 200 hz , which were correlated to specific cognitive functions8 . these frequencies were associated with pace , waking as a preparatory state to movement , and processing of diffuse cortical information before the motor event happen10 . the increase in the absolute power values of rhv at t4 , which is located in the right hemisphere and corresponds to the area responsible for motor control of the hand , during imagery , this shows preservation of the ability to imagine the tasks in rhv , and use of the hemisphere opposite to the brain injury to assist in achieving the movement6 , 10 . hv during initial rest showed high power at c3 corresponding to representation of the hand . probably this was due to prior knowledge of the task ; however , during practice , the increase was not significant compared to the other conditions . the magnitude of power of the gamma band is related to the state of preparation for movement and memory ; however , the results of this study indicate that there was increased activation in this frequency in rhv10 , 17 , possibly because neural activity was lowered by the presence of brain injury . the analysis of absolute power of the alpha , beta and gamma bands allows observation of changes in patterns of consolidation in mental tasks , motor planning , and evocation of memory , which may be associated with brain injury in hemiparetic subjects . studies indicate that the applicability of motor imagery associated with other physiotherapy resources enhances treatment targeting motor functionalization of patients with stroke18 . future studies should investigate experimental models to complement the present findings , associating different tasks and retention periods , in order to obtain more information about the patterns of cortical activation .
excessive gestational weight gain ( gwg ) , defined as that above the 2009 institute of medicine ( iom ) recommendations,1 is associated with a greater likelihood of having a large for gestational age neonate2 with increased adiposity and insulin resistance.3 similarly , excess gwg is also implicated in adverse maternal outcomes , including gestational diabetes,4 hypertension , and preeclampsia,5 as well as greater postpartum weight retention ( ppwr).6 the insulin - like growth factor ( igf ) axis plays a vital role in growth and development and regulates cellular differentiation , hypertrophy , and glycemic homeostasis.7 igfs elicit their intracellular effects by interacting with the igf type 1 receptor ( igf-1r ) or igf type 2 receptor ( igf-2r ) and insulin igf receptor hybrids.8 initial reports identified igf binding protein ( igfbp)-3 as an insulin - antagonizing peptide , binding with high affinity to free igf(s ) and governing bioactivity . recently , igfbp-3 has been shown to act independent of the traditional receptors via nuclear translocation and is thereby capable of directly interrupting transcriptional regulation.8 in respect of excessive gwg , igfbp-3 has been shown to inhibit adipocyte differentiation9 and induce insulin resistance in vivo and in vitro.10 furthermore , igf axis protein expression is compromised in pregnancies complicated by obesity.11 however , the influence that excessive gwg has on igf and igfbp expression during human pregnancy is unknown . understanding differences in weight accretion during critical periods of maternal fetal growth and development may lead to more efficacious obesity management strategies , as maternal insulin sensitivity and ppwr are known contributors to the intergenerational obesity cycle.12 consequently , the igf system may be a candidate pathway involved in maternal substrate storage and nutrient flux through the placenta during pregnancy where excessive weight is gained . positive energy balance and aberrant hormone responses may contribute to alterations in maternal fetal body composition . for instance , fetal adipogenesis peaks in late gestation,13 with more than 90% of fetal body fat accrued in the third trimester.14 in obese women , bioactive igf - i is directly correlated with igfbp-3 concentrations in blood,15 and igf - i stimulates the differentiation of preadipocytes through modulation of the igfbps.16 at term , the pregnancy - associated rise in maternal igfbp-3 serum level correlates positively with birth weight,17,18 whereas the increase in free igf - i during pregnancy is thought to be mediated by igfbp-3 proteolysis.19 traditionally , igfbp-1 acts to inhibit igf function and is inversely related to insulin levels . instances of chronic weight gain over time ( ie , obesity ) display low serum concentrations of igfbp-1 that may regulate in vivo igf - i bioavailability based on fuel supply.15 lastly , although the exact function of igfbp-4 is not yet understood , it may play a role in stem cell differentiation into muscle and fat and be modified in both pregnancy and weight gain.20 thus , we aimed to examine protein expression of regulatory components of the igf system in controls ( adhere group : ie , those who gained in accordance with iom gwg recommendations ) and mothers and their term neonates who exceeded iom recommendations ( exceed group : ie , those who exceeded iom gwg recommendations ) . specifically , we determined whether igf - i , igf - ii , and igfbp-1 , -3 , and -4 expression differed between groups of mother - infant dyads . given that mothers who gain excessively during pregnancy generally give birth to larger infants , we hypothesized that ( 1 ) the growth - enhancing components of the igf system would be expressed to a greater extent , and ( 2 ) the growth - inhibiting proteins downregulated in pregnancies with excessive gwg patients were recruited from the ottawa hospital general campus ( a tertiary care center ) , maternal fetal medicine , and obstetrics clinics after the study was approved by the ottawa hospital research ethics board . women who smoked and those with type 1 , type 2 , or gestational diabetes or any other medical complication such as hypertension , cardiac disease , vascular disease , autoimmune disease , and thrombophilias and a past history of fetal loss , fetal growth restriction , or hypertensive diseases of pregnancy were excluded from either group . all women were classified as glucose tolerant following their prenatal glucose challenge test , according to canadian guidelines . fasting blood samples were obtained via peripheral venipuncture from 21 gravidas ( eight who met and 13 who exceeded iom gwg recommendations relative to prepregnancy body mass index [ bmi ] ) immediately prior to term elective cesarean section , to avoid any potential influence of labor . corresponding venous serum the placenta was then weighed to the nearest gram using a calibrated electronic scale ( olympic smart scale ; natus medical inc , seattle , wa , usa ) . following blood draws , the samples remained at room temperature for 30 minutes to allow for clotting , and were subsequently spun at 1,700 g for 15 minutes at 4c . the supernatant was removed aliquoted into separate cryovials , and frozen at 80c for batch analysis . the expression of igf - ii in human circulation was determined as previously described by qiu et al.21 briefly , aliquots of 0.5 l of sera were diluted with 1x sds nonreducing sample buffer ( 62.5 mmtris - hcl , ph 6.8 , 2% sodium dodecyl sulfate [ sds ] , 10% glycerol , 0.01% bromophenol blue ) , and subjected to electrophoresis with 10% tricine sds - polyacrylamide gel electrophoresis ( page ) . the separated proteins were blotted on to a nitrocellulose membrane , treated with an antibody extender solution ( pierce , rockford , il , usa ) , blocked with 5% dehydrated nonfat milk in tris - buffered saline ( ph 7 with 0.3% tween 20 ) , and subsequently probed with mouse anti - igf - ii(167 ) monoclonal antibody ( clone s1f2 ; upstate biotechnology , lake placid , ny , usa ) and horseradish peroxidase ( hrp)-conjugated antimouse immunoglobulin g. bands of igf - ii variants were visualized with enhanced chemiluminescence , and their relative contents quantified using densitometry with alpha ease fctm software ( alpha innotech , san leandro , ca , usa ) . this assay can detect a quantity of recombinant human igf - ii as low as 6.25 pg.21 the presence of igf - i and igfbp-4 in human and cord serum was determined as described previously , with the following modifications . aliquots of serum ( 0.5 l ) were resolved by 15% glycine sds - page under nonreducing conditions and electrotransferred to nitrocellulose membranes . the membranes were blocked for 1 hour in 5% milk , immunoblotted with highly specific antibodies for igf - i ( # ab9572 ; abcam , ca , usa ) and igfbp-4 ( # sc6005 ; santa cruz biotech , ca , usa ) , and quantified as described previously . igfbps were determined using aliquots of 0.5 l of sera subjected to electrophoresis , transfer , antibody extend solution treatment , and blocking agents as described previously , for the determination of igf - i and -ii . the membranes were subsequently probed with biotinylated igf - ii ( gropep ltd , adelaide , sa , australia ) ( 50 ng / ml prepared with tris - buffered saline and tween 20 ) at 4c overnight , followed by antibody highly specific to igfbp-1 ( # sc55474 ; santa cruz biotech ) and igfbp-3 ( # sc135947 ; santa cruz biotech ) and finally by streptavidin - hrp ( ge healthcare , buckinghamshire , uk ) for 1 hour at room temperature . bands of igfbps were visualized after being independently probed on separate days with highly specific antibodies targeting solely the protein of interest , with enhanced chemiluminescence , and their relative contents were quantified via densitometry with alpha ease fctm software as described previously . glucose concentrations were measured by oxidase enzymatic methodology using the cholestech ldx analyzer as described by the manufacturer ( hayward , ca , usa ) . circulating levels of insulin and leptin were analyzed in duplicate by enzyme - linked immunosorbent assay using luminex xmap technology for the human metabolic hormone magnetic bead panel assay as described by the manufacturer ( millipore corporation , billerica , ma , usa ) . the intra- and interassay coefficients of variation were 3% and 6% for insulin and 3% and 4% for leptin . gwg was calculated from anthropometric values obtained from the medical record using the difference of directly measured maternal weight at last prenatal visit minus directly measured prepregnancy weight obtained by the patient s general practitioner . percentage ( % ) exceeding gwg was calculated as the quotient of total gwg divided by the upper limit of recommended gain using the 2009 iom guidelines relative to pregravid bmi , then multiplied by 100.1 this allowed us to categorize women into adhere ( 100% recommended gwg ) or exceed ( > 100% recommended gwg ) groups independent of pregravid bmi , and to assess the effect of relative excessive gwg based on evidence - informed guidelines for prepregnancy bmi . homeostasis model of assessment for insulin resistance ( homa - ir ) was calculated as fasting glucose ( mmol / l ) fasting insulin ( pmol / l)/22.5 and subsequently log - transformed.22 fetal placental weight ratio was calculated as infant birth weight ( grams [ g])/placenta weight ( g ) . spss statistics software version 19 was used for outcome analysis ( ibm corporation , armonk , ny , usa ) . protein band optical densities were log - transformed and presented as the mean standard deviation percentage above internal control . the internal control used was a pooled sample consisting of equal volumes of maternal and fetal sera taken from each subject in the study . all other outcome variables are presented as the mean standard deviation unless otherwise specified . a nonparametric two - tailed independent samples kruskal wallis test was used to make conservative comparisons between groups for all patient characteristics as well as maternal spearman correlations were used to examine relationships between maternal and fetal characteristics and outcomes of interest . patients were recruited from the ottawa hospital general campus ( a tertiary care center ) , maternal fetal medicine , and obstetrics clinics after the study was approved by the ottawa hospital research ethics board . women who smoked and those with type 1 , type 2 , or gestational diabetes or any other medical complication such as hypertension , cardiac disease , vascular disease , autoimmune disease , and thrombophilias and a past history of fetal loss , fetal growth restriction , or hypertensive diseases of pregnancy were excluded from either group . all women were classified as glucose tolerant following their prenatal glucose challenge test , according to canadian guidelines . fasting blood samples were obtained via peripheral venipuncture from 21 gravidas ( eight who met and 13 who exceeded iom gwg recommendations relative to prepregnancy body mass index [ bmi ] ) immediately prior to term elective cesarean section , to avoid any potential influence of labor . corresponding venous serum was obtained from the umbilical cord following removal of the placenta as clinically indicated . the placenta was then weighed to the nearest gram using a calibrated electronic scale ( olympic smart scale ; natus medical inc , seattle , wa , usa ) . following blood draws , the samples remained at room temperature for 30 minutes to allow for clotting , and were subsequently spun at 1,700 g for 15 minutes at 4c . the supernatant was removed aliquoted into separate cryovials , and frozen at 80c for batch analysis . the expression of igf - ii in human circulation was determined as previously described by qiu et al.21 briefly , aliquots of 0.5 l of sera were diluted with 1x sds nonreducing sample buffer ( 62.5 mmtris - hcl , ph 6.8 , 2% sodium dodecyl sulfate [ sds ] , 10% glycerol , 0.01% bromophenol blue ) , and subjected to electrophoresis with 10% tricine sds - polyacrylamide gel electrophoresis ( page ) . the separated proteins were blotted on to a nitrocellulose membrane , treated with an antibody extender solution ( pierce , rockford , il , usa ) , blocked with 5% dehydrated nonfat milk in tris - buffered saline ( ph 7 with 0.3% tween 20 ) , and subsequently probed with mouse anti - igf - ii(167 ) monoclonal antibody ( clone s1f2 ; upstate biotechnology , lake placid , ny , usa ) and horseradish peroxidase ( hrp)-conjugated antimouse immunoglobulin g. bands of igf - ii variants were visualized with enhanced chemiluminescence , and their relative contents quantified using densitometry with alpha ease fctm software ( alpha innotech , san leandro , ca , usa ) . this assay can detect a quantity of recombinant human igf - ii as low as 6.25 pg.21 the presence of igf - i and igfbp-4 in human and cord serum was determined as described previously , with the following modifications . aliquots of serum ( 0.5 l ) were resolved by 15% glycine sds - page under nonreducing conditions and electrotransferred to nitrocellulose membranes . the membranes were blocked for 1 hour in 5% milk , immunoblotted with highly specific antibodies for igf - i ( # ab9572 ; abcam , ca , usa ) and igfbp-4 ( # sc6005 ; santa cruz biotech , ca , usa ) , and quantified as described previously . igfbps were determined using aliquots of 0.5 l of sera subjected to electrophoresis , transfer , antibody extend solution treatment , and blocking agents as described previously , for the determination of igf - i and -ii . the membranes were subsequently probed with biotinylated igf - ii ( gropep ltd , adelaide , sa , australia ) ( 50 ng / ml prepared with tris - buffered saline and tween 20 ) at 4c overnight , followed by antibody highly specific to igfbp-1 ( # sc55474 ; santa cruz biotech ) and igfbp-3 ( # sc135947 ; santa cruz biotech ) and finally by streptavidin - hrp ( ge healthcare , buckinghamshire , uk ) for 1 hour at room temperature . bands of igfbps were visualized after being independently probed on separate days with highly specific antibodies targeting solely the protein of interest , with enhanced chemiluminescence , and their relative contents were quantified via densitometry with alpha ease fctm software as described previously . glucose concentrations were measured by oxidase enzymatic methodology using the cholestech ldx analyzer as described by the manufacturer ( hayward , ca , usa ) . circulating levels of insulin and leptin were analyzed in duplicate by enzyme - linked immunosorbent assay using luminex xmap technology for the human metabolic hormone magnetic bead panel assay as described by the manufacturer ( millipore corporation , billerica , ma , usa ) . the intra- and interassay coefficients of variation were 3% and 6% for insulin and 3% and 4% for leptin . gwg was calculated from anthropometric values obtained from the medical record using the difference of directly measured maternal weight at last prenatal visit minus directly measured prepregnancy weight obtained by the patient s general practitioner . percentage ( % ) exceeding gwg was calculated as the quotient of total gwg divided by the upper limit of recommended gain using the 2009 iom guidelines relative to pregravid bmi , then multiplied by 100.1 this allowed us to categorize women into adhere ( 100% recommended gwg ) or exceed ( > 100% recommended gwg ) groups independent of pregravid bmi , and to assess the effect of relative excessive gwg based on evidence - informed guidelines for prepregnancy bmi . homeostasis model of assessment for insulin resistance ( homa - ir ) was calculated as fasting glucose ( mmol / l ) fasting insulin ( pmol / l)/22.5 and subsequently log - transformed.22 fetal placental weight ratio was calculated as infant birth weight ( grams [ g])/placenta weight ( g ) . spss statistics software version 19 was used for outcome analysis ( ibm corporation , armonk , ny , usa ) . protein band optical densities were log - transformed and presented as the mean standard deviation percentage above internal control . the internal control used was a pooled sample consisting of equal volumes of maternal and fetal sera taken from each subject in the study . all other outcome variables are presented as the mean standard deviation unless otherwise specified . a nonparametric two - tailed independent samples kruskal wallis test was used to make conservative comparisons between groups for all patient characteristics as well as maternal fetal levels of glucose , insulin , and leptin . spearman correlations were used to examine relationships between maternal and fetal characteristics and outcomes of interest . eight women who met and 13 who exceeded 2009 iom gwg recommendations , of comparable age and height , were studied . to assess relative gwg , the mean percentage of women exceeding gwg guidelines based on prepregnancy bmi differed between the groups , as designed . mean prepregnancy bmi and weight were greater in the exceed group , as expected , given the narrower range of acceptable gain as pregravid bmi increases ( table 1 ) . in the adhere group , seven had pregravid bmis classified as normal weight , whereas one was classified as obese . in the exceed group , four and nine women were categorized as normal weight and obese , respectively . however , birth weight , gestational age at delivery , prenatal glucose screen , placenta weight , and fetal placental weight ratio did not differ between groups . maternal protein expression of igf - i , igf - ii , igfbp-1 , and igfbp-4 and cord blood protein expression of igf - i , igf - ii , igfbp-1 , igfbp-3 , and igfbp-4 were similar between groups ( p > 0.05 ) . protein expression of igfbp-3 was increased in maternal serum from the exceed group when compared with the adhere group ( p 0.05 ) ( figure 1 ) . maternal glucose , insulin , and cord concentrations of insulin , glucose , and leptin did not differ between groups ( table 2 ) . however , maternal leptin levels were elevated in the exceed group ( figure 2 ) and displayed a strong direct relationship with excess gwg and homa - ir ( figure 3 and table 3 ) . following bonferroni adjustment , nonsignificant trends ( ie , where p < 0.05 prior to adjustment and p = 0.010.05 postadjustment ) were observed between excessive gwg and infant birth weight , maternal igfbp-4 expression , and homa - ir ( table 3 ) . eight women who met and 13 who exceeded 2009 iom gwg recommendations , of comparable age and height , were studied . to assess relative gwg , the mean percentage of women exceeding gwg guidelines based on prepregnancy bmi differed between the groups , as designed . mean prepregnancy bmi and weight were greater in the exceed group , as expected , given the narrower range of acceptable gain as pregravid bmi increases ( table 1 ) . in the adhere group , seven had pregravid bmis classified as normal weight , whereas one was classified as obese . in the exceed group , four and nine women were categorized as normal weight and obese , respectively . however , birth weight , gestational age at delivery , prenatal glucose screen , placenta weight , and fetal placental weight ratio did not differ between groups . maternal protein expression of igf - i , igf - ii , igfbp-1 , and igfbp-4 and cord blood protein expression of igf - i , igf - ii , igfbp-1 , igfbp-3 , and igfbp-4 were similar between groups ( p > 0.05 ) . protein expression of igfbp-3 was increased in maternal serum from the exceed group when compared with the adhere group ( p 0.05 ) ( figure 1 ) . maternal glucose , insulin , and cord concentrations of insulin , glucose , and leptin did not differ between groups ( table 2 ) . however , maternal leptin levels were elevated in the exceed group ( figure 2 ) and displayed a strong direct relationship with excess gwg and homa - ir ( figure 3 and table 3 ) . following bonferroni adjustment , nonsignificant trends ( ie , where p < 0.05 prior to adjustment and p = 0.010.05 postadjustment ) were observed between excessive gwg and infant birth weight , maternal igfbp-4 expression , and homa - ir ( table 3 ) . the present study illustrates a preliminary examination of the igf axis in otherwise healthy mothers and newborns who adhere to and exceed 2009 iom gwg guidelines . the fact that participants suffered no medical or obstetrical complications ( ie , no gestational diabetes mellitus [ gdm ] or hypertension ) provided us with a unique opportunity to examine the influence of excessive gwg without the potential effects of the commonly associated complications or comorbidities . this investigation reveals a small , albeit significant , difference in the igf axis in the exceed group versus the adhere group : specifically , augmented expression of igfbp-3 in maternal blood from normoglycemic pregnancies where gwg is excessive . given our design , we are unable to assess causality . however , the metabolic dysregulation of maternal growth - controlling factors in excessive gainers aligns with previous observations , demonstrating that igfbp-3 induces insulin resistance in adipocytes10 via inhibition of adipocyte differentiation,9 thus modifying metabolic homeostasis . although induced during adipocyte differentiation , igfbp-3 can paradoxically inhibit this process.23 as adipogenesis proceeds , increasing adipose tissue igfbp-3 levels may signal a feedback mechanism , limiting further differentiation of preadipocytes.9 in fact , the dual regulatory role of igfbp-3 in differentiation has been identified in other cell types , including myoblasts,24 keratinocytes,25 and chondrocytes.26 as such , it is possible that augmented protein expression of maternal igfbp-3 may be , in part , due to a feedback response to excessive weight gain , whereby igfbp-3 sequesters free igfs , preventing further differentiation of adipocytes and antagonizing igf - induced glucose uptake and insulin - like activity . previous investigations have demonstrated that igfbp-310,27 inhibits insulin - mediated glucose uptake in adipocytes , and have suggested that the increase in free igf - i during pregnancy is mediated by igfbp-3 proteolysis.19 in fact , chan et al,9 using 3t3-l1 adipocytes , demonstrated that igfbp-3 inhibits the peroxisome proliferator activated receptor--dependent process of adipocyte differentiation by inhibiting the dimerization of retinoid x receptor- and peroxisome proliferator activated receptor-. taken together , these findings suggest that igfbp-3 has the potential to disrupt glycemic control and substrate uptake in adipose tissue . for instance , kim et al10 convincingly demonstrate that igfbp-3 induces insulin resistance in adipocytes in vitro and in vivo . furthermore , nguyen et al28 show that mice overexpressing human igfbp-3 in vivo have delayed insulin clearance and reduced glucose - stimulated insulin secretion in pancreatic islets acting by both igf - dependent and igf - independent mechanisms . although several physiological factors are suspected to regulate igfbp-3-induced alteration of glycemic control , we can not preclude the possibility that augmented igfbp-3 with excessive weight gain and obesity may preferentially elicit a hypertrophic response in existing adipocytes at the expense of attenuated adipogenesis . this may predispose women to preferentially store substrate in existing adipocytes , a fuel partitioning strategy that is metabolically disadvantageous when adipocyte proliferation rates are low.29,30 if excessive gwg with obesity compromises functional components of the igf axis , as demonstrated in the present investigation , this suggests that the igf axis may be implicated in maternal fat accretion , leptin metabolism , and insulin resistance . it is known that serum levels of leptin correlate with total body fat and insulin resistance31 in pregnant32 and nonpregnant women.3133 in a group of healthy pregnant women in the first trimester,34 as well as those with gestational diabetes,35 serum leptin positively relates with insulin resistance , a finding we substantiate at term in pregnancies with excessive gwg and obesity . given that serum leptin concentrations decrease following delivery , it has been argued36 that factors other than fat mass alone cause increased leptin production throughout pregnancy , including placental contributions . however , eriksson et al37 showed that serum leptin was elevated in gestational week 8 when increases in fat mass are minimal and when the placenta is not fully established , providing support that increased leptin levels are not only due to increased fat mass but influenced by other unknown factors . these findings parallel with our observation in women who gain excessively , as leptin levels have been reported to be commensurate with adipose stores in pregnant women at term.32,37 although we acknowledge the possibility that the observed differences in maternal leptin at the end of pregnancy may simply reflect between - group differences in pregravid bmi , shaarawy and el - mallah,38 comparing nonpregnant and pregnant women matched for pregravid bmi , determined that elevated serum leptin was directly associated with maternal adiposity of pregnancy . furthermore , in observations from a randomized controlled trial with and without gwg restriction in bmi - matched obese pregnant women , wolff et al39 showed that when patients in the intervention group successfully limited food intake and restricted gwg , a significant 20% reduction in both insulin and leptin was observed . lastly , in our adhere group , only a single woman was categorized as obese . collectively , these results suggest that appropriate gwg may attenuate adipose accretion and the commensurate leptin response , whereas excessive gwg may exacerbate maternal fat accretion , thus raising leptin levels . further , we present a strong direct relationship between maternal leptin levels and homa - ir , supporting previous work identifying pregnancy as both an insulin- and leptin - resistant state,32 partly mediated by increasing adiposity during pregnancy.37 indeed , eriksson et al37 showed that total body fat percentage was significantly correlated with homa - ir and with serum leptin before and during pregnancy , whereas leptin correlated with homa - ir , identifying a complex interactive role of maternal leptin , adiposity , and insulin sensitivity . although we can not make conclusive inferences about the composition of excessive gwg based on the results of our study , previous work suggests that high maternal fat content predominates and stimulates fetal growth , as evidenced by data showing that maternal total body fat40 and excessive gwg2 positively relate to birth weight , and that neonates born to mothers who gain in excess have higher body fat.41 furthermore , the pregnancy - enhancing effect on the relationship between maternal body fatness and insulin resistance37 may represent a physiological mechanism predisposing pregnant women to preferentially store body fat as weight is gained . it is now widely accepted that maternal body size affects fetal growth and metabolic homeostasis.3,42 our findings , in addition to others findings,43 suggest that maternal body size and weight gain are important factors determining the amount of dietary energy required by the developing child . excessive gwg with obesity may present a mechanism by which offspring size and body composition are regulated via alterations in the igf axis in response to energy availability in the maternal milieu . although we were unable to assess maternal or fetal body composition , we can not exclude the hypothesis that gwg - induced perturbations in igf axis function influence maternal fetal body composition a phenomenon thoroughly documented in mother infant dyads who gain excessively and exacerbated as pregravid bmi increases , leading to greater relative fat mass accretion.6 therefore , future longitudinal investigations should examine the influence of physical activity energy expenditure and dietary intake on igf axis dynamics and maternal fetal body composition to confirm our findings in a larger sample . the limitations of our study include the cross - sectional design , relatively small sample size , and inability to control for maternal dietary intake and physical activity patterns . however , we present a novel finding identified with validated and highly specific methodology for the outcomes of interest , while controlling for the acute effects of postprandial metabolism , as all women were fasted for at least 12 hours . through this design , we also attempted to control for diurnal variation in igf metabolism , given that all blood was drawn at the same time of day ( ie , the morning upon arrival to hospital admittance and fasting ) . further , our study population was homogenous in nature , as all women were normoglycemic as defined by clinical standards . in an attempt to avoid a confounding effect of mode of delivery on cord blood igf profile , lastly , we used the most recent 2009 evidence - based recommendations for gwg as outlined by the iom.1 overall , this study provides further support for the clinical value and use of the 2009 iom gwg recommendations in primary care , as women and providers can track and monitor weight status throughout pregnancy to reduce not only the likelihood of impairing maternal fetal metabolic signals but also the incidence of fetal overgrowth.2 specifically , if altering weight prepregnancy is not a feasible option , being aware that gwg is a modifiable risk factor may motivate women to gain within the limits in order to optimize maternal fetal health , chiefly ppwr and infant birth weight , known contributors to the intergenerational obesity cycle . our results align with epidemiological data suggesting that excessive gwg and obesity influence maternal growth signals during pregnancy . although it appears that igfbp-3 attempts to sequester free igfs and prevent undesired increases in igf bioactivity , the potential induction of impaired glycemic control renders excessive gain harmful . therefore , this study suggests that small deviations in igfbp - regulated igf bioavailability arising from excessive weight gain and obesity may influence adipocyte differentiation and glycemic control .
aloe vera is a common ingredient in cosmetics , skin care products , and increasingly , beverages and food products . recent consumer interest in aloe beverages may stem from the association of aloe juice with a variety of both anecdotal and experimental research - supported health benefits including the prevention or treatment of various tumors [ 2 , 3 ] , arthritis , diabetes , enhanced immunity , and decreased cholesterol levels . aloe juice is approximately 99% water and the remainder consists of minerals , vitamins , polysaccharides , lipids , phenolic compounds , and organic acids . according to the international aloe scientific council , the aloe leaf can be processed into two types of juices for commercial use : inner leaf gel juice and decolorized whole leaf juice . decolorized whole leaf juice is produced by grinding the leaf followed by treatment of extracted juice with activated charcoal to remove aloe charcoal treatment is necessary since the latex , which exists as a separate liquid between the outer rind and inner fillet gel , contains bitter phenolic molecules including anthraquinone c- and o - glycosides , anthrones , and some free anthraquinones . the major c - glycoside , aloin a , is the major anthraquinone in aloe latex and when oxidized , yields aloe - emodin , a free anthraquinone . anthraquinones are associated with diarrhea in vivo [ 12 , 13 ] , weight loss , gall bladder lesions , renal tubule pigmentation , and renal tubule hyaline droplets . in vitro , anthraquinones cause mutations in mouse lymphoma and salmonella assays . aloe beverages , which follow iasc standards contain less than 10 ppm aloin a and its isomer , aloin b . toxicology of various aloe products has been examined in vivo , and these studies collectively suggest that the potential for toxicity depends on the plant sections used in juice production . oral consumption of an ethanol extract of whole leaf aloe over 3 months by mice resulted in reproductive toxicity and increased mortality . however , subchronic administration of aloe taken from the inner leaf gel resulted in no evidence of oral toxicity to rats . in 2010 , the national toxicology program released a technical report on aloe vera that concluded that there was clear evidence of carcinogenic activity of a nondecolorized whole leaf extract of aloe vera in male and female f344/n rats based upon increased incidences of adenomas and carcinomas of the large intestine . the results of this report have been referred to on various health - related websites suggesting caution in consumption of aloe products [ 1922 ] and have generated substantial interest and concern by aloe producers , because the study results were based on a nondecolorized whole leaf extract that is not commercially available . the ntp study was designed to deliver nondecolorized whole leaf , freeze - dried juice powder to test animals through their drinking water at levels up to 3.0% ( wt / wt ) , and the study examined potential toxicologic effects of aloe in both b6c3f1 mice and f344 rats over time periods of 14 days , 13 weeks , and 2 years . while an increased incidence of tumorigenesis was observed in the large intestine of rats in the 2-year study , no lesions were observed in mice or rats in acute studies ; however , there was decreased water consumption in both rodent species at the highest aloe levels . during a subchronic period ( 13 weeks ) , mice administered aloe were similar to controls in most parameters with the exception of increased incidences of mucosal hyperplasia with goblet cell hyperplasia in the cecum and large intestine . rats exposed for two years also were found to have mucosal hyperplasia in the colon , particularly in the cecum and ascending portions . large intestine mucosal / goblet cell hyperplasias were also observed in mice exposed for two years ; however , these mice did not show increased neoplasms and had similar survival and body weights as control groups . the nondecolorized whole leaf powder used by the ntp contained the bitter latex of the aloe plant and therefore , the addition of this aloe directly to drinking water could have contributed to the study 's outcomes . palatability can influence water consumption and conceivably could have also contributed to the weight loss and mortality in the two - year study . commercial aloe beverages that contain little or no latex anthraquinones may have yielded different outcomes in the subchronic or chronic exposure tests . the ntp also assessed the acute effects of aloe gel only and decolorized ( activated charcoal treated ) whole leaf aloe , and in these studies , mice and rat body weights , water and feed consumptions , organ weights , hematology , clinical chemistry , and urine chemistry were generally similar to controls . although the reported incidence of large intestinal tumors has garnered the most attention from the ntp report , the incidences of intestinal mucosal hyperplasia were the most consistent observations across species exposed both subchronically and chronically . this mucosal hyperplasia suggests a toxicological response to one or more whole leaf aloe components . in the rat , hyperplasia of both the mucosa and goblet cells may indicate cellular damage and could be prognostic of later tumor development since control rats showed little or no hyperplasia at 13 weeks or adenoma / carcinomas of the large intestine at 2 years . to date , no evaluation of the de - colorized whole leaf aloe vera juice has been reported . in the present study , we report the first toxicological evaluation of a commercially available , decolorized , whole leaf ( dcwl ) aloe vera beverage , lily of the desert filtered whole leaf aloe vera juice . this particular product can be considered as being well characterized with regard to high - molecular - weight polysaccharide content and immunomodulating activity [ 25 , 26 ] . testing included assessment of potential genotoxicity in vitro and acute toxicity in vivo using a b6c3f1 mouse model . further testing included a 13-week study utilizing f344 rats in which the dcwl juice was administered through the rat 's chow in order to impart a minimal effect on palatability . examinations included large intestinal histology to understand if aloe beverages derived from dcwl aloe vera juice produced toxicologic signs associated with nondecolorized whole leaf juice . aloe vera leaves were harvested and maintained under cold storage ( 8c ) until processing . the harvested leaves were washed , disinfected , and macerated to yield an intermediate raw material representative of a nondecolorized whole leaf extract as was used in the aforementioned ntp study . the nondecolorized extract was then filtered using carbon / diatomaceous earth to yield a dcwl extract . to this extract , a proprietary isolate of high - molecular - weight aloe vera polysaccharide , aloesorb , the resulting extract was flash - pasteurized to yield the commercial product ( lily of the desert filtered whole leaf aloe vera juice with aloesorb ) . nmr analysis of the resulting extract established ( 1 ) the presence of aloe vera ( by confirming the presence of aloe vera polysaccharide , malic acid , and glucose ) , ( 2 ) the presence of whole leaf markers ( isocitrate and isocitrate - lactone ) , and ( 3 ) the absence of adulterants / stabilizers in the extract . hplc analysis confirmed the presence of low levels of anthraquinones ( aloin a at 0.868 ppm , aloin b at 1.335 ppm , and aloe - emodin at 0.200 ppm ) . prior to testing , the aloe juice was lyophilized and determined to be 10.65 0.11% nonaqueous . prior to analysis in genotoxicity assays , the aloe juice was filtered using positive pressure to render it sterile and the ph adjusted to 7.5 to accommodate salmonella bacterial assays that are ph - sensitive . in order to maximize the level of aloe administered to test mice , the 7x juice was further concentrated by lyophilization to a final concentration of 35x in a labconco lyophilizer over a 6-hour period . this concentrated juice was administered in gavage studies . a single lot ( # 021412 - 4 ) was used throughout the studies . an assay for mutagenesis was used , which is based on the ames test utilizing salmonella typhimurium strain ta100 but modified for liquid culture and a 96-well plate scale . the second assay detected potential dna damage utilizing an e. coli strain containing a transgene for beta - galactosidase downstream of the sos - dna repair promoter system . both assays were purchased in a commercial format from ebpi bio - detection products ( mississauga , on , canada ) , referred to the as muta - chromo plate and sos - chromo test assays , respectively . to test for potential metabolic generation of mutagens , dcwl aloe vera juice was also tested in the presence of s9 liver extract . for testing with salmonella , the juice was combined with a reaction mix containing growth substances and a ph indictor . the juice was administered at 21x , 14x , and 7x concentrations . as a positive control for direct acting mutagenesis ( i.e. , independent of metabolic conversion ) , either sodium azide ( 0.38 m final concentration ) or 2-nitrofluorene ( 7.1 m final concentration ) was included in one plate . 2-aminoanthracene ( 2.60 m final concentration ) was used as a positive control for mutagenesis requiring metabolism . plates containing no aloe juice were used to measure spontaneous mutations over the incubation periods , and these blanks were compared to all aloe - containing plates . the number of positive mutated wells was scored on days 3 , 4 , and 5 of growth . these numbers were then compared statistically to blanks . for the dna damage assay in e. coli , the dcwl juice was tested at levels of 21x , 14x , 7x , 3.5x , 1.75x , 0.88x , 0.44x , 0.22x , 0.11x , and 0.055x . when diluted , the juice was suspended in sterile 10% dimethyl sulfoxide ( dmso ) in sterile 0.85% saline . a positive control for dna damage independent of metabolism was included ( 4-nitroquinoline oxide [ 4nqo ] ) and used at levels of 10.0 g / ml ( 52.6 m ) , 5.0 , 2.5 , 1.25 , 0.625 , and 0.3125 g / ml . a positive control for mutagenesis requiring metabolism was included in another plate ( 2-aminoanthracene ) and used at 100.0 ( 2.6 m ) , 50.0 , 25.0 , 12.5 , 6.25 , and 3.125 g / ml . all mice were purchased from jackson laboratories , inc . and were ordered between 4 and 6 weeks of age . after a two - week period of quarantine , the mice were numbered for identification by ear tag . upon initiation of studies , mice were weighed , sorted into boxes of 5 mice each , and then randomly assigned to control or aloe juice groups . mice were housed within the louisiana state university division of laboratory medicine 's aalac - approved vivarium in super mouse micro isolation 750 boxes that are racked in an enviro - gard - b microisolation control cage rack ( lab products , seaford , de ) . mouse boxes were stocked with clean / autoclaved bio - serv ( frenchtown , n.j . ) mouse igloo and fast - trac spinning enrichment devices and nestlets pads ( ancare , bellmore , ny ) . all animal procedures followed the national research council 's guide for the care and use of laboratory animals and were first reviewed and approved by the lsu iacuc . f344 rats were purchased from charles river laboratories and arrived at 7 weeks of age . after a two - week period of quarantine , rats were identified by permanent marker on the tail . cage boxes were changed with fresh litter and fresh water weekly , and boxes were stocked with clean / autoclaved crawl balls enrichment devices ( bio - serv , frenchtown , n.j . ) . dcwl aloe juice was administered by gavage with a 20-gauge curved stainless steel gavage needle ( popper & sons , new hyde park , ny ) to male and female b6c3f1 mice twice over a 24-hour period at 1.0% of body weight . seven mice per test group were gavaged either concentrated juice or water then observed daily for abnormal behaviors including hypoactivity , isolation from littermates , ataxia , dyspnea , or prostration . in addition , mice were monitored for a weight loss of 10% or greater , decreased feed consumption of 10% or greater , and death . through the oral gavage , female mice consumed an estimated amount of aloe vera juice equal to 32.1 times the amount a person would normally drink per 24-hour period . these estimates were determined by assuming the recommended daily amount of aloe juice a person drinks to be 8.0 ounces ( 236.6 ml ) . to scale this quantity to a mouse , we used body surface area ratio comparing mice groups with a 5 feet , 10 inches person weighing 70 kg . at the conclusion of the 3- and 14-day periods , gross necropsies were performed on all animals , and final body weights plus weights of the kidney ( right ) , heart , lung , liver , and testes ( males ) were recorded . this analysis included rbc , hb , hct , rdw , mcv , mch , mchc , platelets , mpv , and total wbcs . finding of pathologic anomalies would trigger further microscopic evaluation of all organs collected from mice . body weight , organ weights and all hematologic and clinical chemistry parameters were analyzed within each sex by student 's t - test , with significance as p 0.05 . control groups received aim-93 g chow ( teklab , madison , wi ) ad libitum while aloe - treated rats consumed chow formulated with concentrated aloe juice in place of water ( teklab , madison , wi ) for 13 weeks . each week , weights of individual rats and feed consumed were recorded . animals were assessed daily for general behavior , health , and appearance as well as appearance of stools . at the conclusion of the 13-week study period , rats were anesthetized with isoflurane for blood collection and then humanely euthanized . at necropsy , abdominal , pelvic , and thoracic organs were observed in situ , then the liver , kidneys , testes ( in males ) , heart , and lungs were removed , weighed , rinsed with phosphate - buffered saline ( pbs ) , and placed into 10% neutral buffered formalin ( nbf ) . the intestinal tract was examined from the stomach distally , and then the tract from the cecum distally was labeled using colored thread for cecum , ascending , transverse , and descending colon and rectum . the large intestines were then removed , placed in pbs , and sectioned according to anatomic region . intestinal contents were gently flushed with pbs , and using a blunt needle , the cecum was opened along the greater curvature and flushed and examined for gross lesions . the cecal - colic junction was opened and grossly examined . fixed tissues were embedded in paraffin and sectioned at 7 um onto glass slides and stained with hematoxylin and eosin . histological preparations and microscopic evaluations were made of the liver , cecum , and colon tissues ( ascending , transverse , descending ) and rectum . each large intestine anatomic region was evaluated and scored using a semiquantitative scale in increments of 0.1 in which 00.9 indicated hypoplastic mucosa , 1.01.9 indicated mucosal architecture and cellular populations within expected ranges , 2.02.9 indicated moderate hyperplasia of the mucosal epithelia and increased lymphocytic infiltrate and/or goblet cell hyperplasia and secretion , and 3.03.9 indicated pronounced hyperplasia of mucosa and goblet cells , lymphoid aggregates with enlarged germinal centers , and inflammatory infiltrate . mucosa of rats was independently scored by three reviewers , and the average scores for each large intestinal region were computed for aloe treatment and controls of each sex . feed consumption and body weight changes during the 13-week period were compared using two - way repeated measures anova with bonferroni post - tests . organ weights were compared using an unpaired t - test with significance set a p 0.05 . hematologic tests included rbc , hgb , hct , rdw , mcv , mch , mchc , platelets , mpv , plasma protein , pcv , wbcs , neutrophils , lymphocytes , monocytes , and eosinophils . clinical chemistry assays included glucose , ast , alt , alp , ck , t protein , albumin , globulin , cholesterol , bun , creatinine , calcium , phosphorus , sodium , potassium , chloride , bicarbonate , and anion gap . samples were compared with gender - matched controls using an unpaired t - test ( p 0.05 ) . large intestinal mucosal thickness scores were compared using mann - whitney u ranking to obtain a p value set as p 0.05 . to verify dcwl aloe juice levels in the compounded feed , one juice constituent , malic acid , was determined in samples of 9.3x concentrated juice prior to compounding and in samples of the final aloe - containing chow . a malate assay kit ( biovision , milpitas , california ) was used to measure l(- ) malate . the 9.3x aloe juice contained a measured 362.0 18.0 mm malate ; therefore , the chow was expected to contain a malate of 36.2 mm . the final measured malate concentration extracted from the finished chow was 37.8 1.29 mm , or 104.4 3.4% of the expected . male rats in this study consumed an average of 14.3 g aloe chow / day or 540.5 mol malate / day . female rats consumed an average of 9.13 g of aloe chow / day or 344.0 mol malate / day . these malate levels indicate that the male rats exceeded a recommended 8.0 ounce human consumption by 5.9-fold and the female rats exceeded the human consumption of aloe juice by 5.1-fold based on body surface area . dcwl aloe vera juice extract when tested at up to a 21x concentration was not significantly mutagenic to test salmonella spp . the blank plate without s9 showed a similar number of revertant wells in aloe plates of 21x , 14x , or 7x ( table 1(a ) ) . similarly , the blank plate with s9 did not show any significant differences from revertant wells in aloe + s9 plates of 21x , 14x , or 7x ( table 1(b ) ) . the p value was 0.05 for all aloe juice concentrations , without and with s9 . a bacteria - free plate remained without growth indicating that there was no external bacterial contamination in the assay and positive controls for both sodium azide and 2-aminoanthracene plus s9 extract verified assay function ( data not shown ) . sos dna repair in e. coli was not activated by dcwl aloe vera juice extract in concentrations ranging from 21-fold concentrated to approximately 20-fold diluted ( figure 1(a ) ) . the juice product also did not show significant concentration - related dna damage / repair in the presence of metabolically active liver extract ( figure 1(b ) ) . these data indicate that juice does not contain significant levels of active or latent genotoxic compounds . sos dna repair was activated in the positive control 4-nitroquinoline oxide ( 4nqo ) resulting in a concentration - dependent increase in dna repair gene activity ( figure 1(c ) ) . bacteria also showed a concentration - dependent increase in dna damage repair gene activity when exposed to the positive control chemical 2-aminoanthracene ( 2-aa ) in the presence of metabolic extract from rat liver ( figure 1(d ) ) . male and female mice after both 3 and 14 days postadministration of aloe had body and organ weights similar to control mice ( table 2 ) . hematologic values from blood sampled from mice at 3 days and 14 days postexposure to the aloe vera juice were generally similar to control group mice ( table 3 ) . the few values that appeared to be different between groups had very small variances within groups ; thus , small differences between means were statistically calculated as different . however , in all cases the differences between means were 10% or less . clinical chemistry values from blood sampled at these time periods were also similar to values for control group mice ( table 4 ) . the architecture of the tissues and cellular morphology were considered typical of mice at their age ( data not shown ) . for instance , male livers from both control and mice treated with the test extract showed moderate cytoplasmic vacuolation , instances of binucleate hepatocytes , and extramedullary hematopoiesis ( emh ) . the female mice treated with the test extract as well as controls showed multifocal emh , some cytoplasmic vacuolation , and binucleate hepatocytes , but did not display the occasional mild hepatocellular necrosis seen in males . no pathologic features were determined to be exclusively or predominantly associated with test groups . during the course of study dcwl aloe vera - fed rats were equivalent to control rats in behavior . both body weight gains and feed consumption of rats fed the test extract were equivalent to the same parameters in the control males and females ( figures 2 and 3 ) . one female rat died during transport from the vendor , and thus the control group of females contained 6 rats , while all other groups contained 7 . the rat was treated with an antibiotic ( enrofloxacin ) and nonsteroidal anti - inflammatory ( meloxicam ) for a one - week period . this female responded well to therapy and was included in final data analyses . at the study 's conclusion , weights of organs were not significantly different from control in either sex ( table 5 ) , and gross necropsy was otherwise unremarkable . hematologic analyses showed that no parameter assessed in rats fed with the dcwl aloe vera differed significantly from the respective control group ( table 6 ) . clinical chemistry values from blood were also generally not different from control group mice ( table 7 ) . there were no differences in males ; however , in females , values for albumin and cholesterol were lower in rats fed the test extract versus control rats . cholesterol was reduced in males fed the test extract , but this difference was not statistically significant . the cecum ( including the cecal - colic junction ) , ascending , transverse , and descending colon and rectum were opened and examined for gross abnormalities ( i.e. , masses or ulcerative lesions ) . in this study , we sought to determine if oral administration of concentrated levels of a commercially available dcwl aloe juice , lily of the desert filtered whole leaf aloe vera juice with aloesorb , produced genotoxicity in vitro , acute / subacute toxicity in mice or subchronic toxicity in rats . in agreement with the results of others who have tested aloe vera from the inner gel fillet previously reported in vivo toxicity tests , aloe derivatives without the anthraquinone - containing latex are not associated with adverse effects . tested dried powder from the inner leaf fillet in a feed study with fischer 344 rats and found no toxicity . more recently , tanaka et al . reported no mortalities , no abnormalities at necropsy , and no differences in body weight gain after 14 days in a rat study . this study tested an aloe vera gel extracted with supercritical carbon dioxide administered as a single oral dose of 150 mg / kg . in our in vivo assays , we also found no evidence of aloe toxicity . in mice , our acute studies demonstrated no mortalities , no changes in behavior , body weight , or organ weights . hematology and clinical chemistry values were generally not significantly different compared to controls although in some instances , a measured value for one parameter was different from the control at p 0.05 . if the mouse showed no other subjective / objective change suggesting toxicity , we considered these values to be the occasional outliers , which would be anticipated given the large number of individual parameters quantitated , and we did not feel that these were biologically relevant differences between aloe - treated and untreated groups . necropsy evaluations , hematology , and clinical chemistries were generally similar to the control groups . the difference in plasma albumin was not felt to be a toxic effect since we found no differences in total protein , globulin , or serum protein in females and because albumin in males fed with the test extract was not different ( p = 0.94 ) . cholesterol has been reported to be decreased in rats that were administered aloe vera previously [ 30 , 31 ] ; thus , this result in our analysis is not surprising . huseini et al . have reported that aloe gel reduced cholesterol in humans in a clinical trial ; however , these clinical results have not been verified in other studies to date . this was a relatively short - termed study with young , healthy animals , and this effect may have been more pronounced and/or seen in males over a longer term or in aged animals . studies employing innately hypercholesterol rat models have shown a greater cholesterol - reducing effect of aloe . therefore , although this study was not one of efficacy determination , it is a notable observation . of particular interest , in our 13-week rat study , there was an analysis of the large intestine for signs of mucosal pathology . these pathologies were reported in f344 rats with water - administered , nondecolorized whole - leaf aloe vera extract and featured a significantly increased incidence and severity of mucosal hyperplasia with goblet cell hyperplasia that was more pronounced in male rats . our data obtained using a dcwl aloe vera juice contrast with these previous results . no gross or microscopic evidence of intestinal pathologies was observed . rat intestinal sections from both control and rats fed with the test extract displayed typical mucosal phenotypes including sections with variable levels of focal lymphoid cellularity in the lamina propria and occasional variations in the degree of fibrous tissue between crypts ; however , crypts were uniform in length and depth and goblet cells were evenly distributed and not over productive with mucous secretions . the differences in findings between our present study and that of the ntp are most likely due to the different aloe vera juices tested . we have not identified specific components in nondecolorized whole - leaf aloe extract that are reduced or absent in purified whole leaf beverages and which may have led to the mucosal response observed by the ntp . however , attractive candidates are the anthraquinones associated with the latex in whole leaf juice but largely absent from activated charcoal - treated aloe . free anthraquinones , released in animals after intestinal microbial oxidation of the glycosidic bonds , have been associated with in vitro mutagenesis , although aloe - emodin , the predominant anthraquinone released by microbial action on aloin glycosides , has been repeatedly found to be negative in a variety of in vivo assays ( reviewed by ) . longer termed animal studies , particularly with a sensitive species , could reveal toxicologic or carcinogenic effects not seen with acute assays , and the rat model seems well suited to potentiate toxic and carcinogenic effects of aloe 's anthraquinones . rats ( as well as mice ) have substantial quantities of microbial flora throughout their upper as well as lower gastrointestinal tracts including those such as eubacterium spp . which are capable of oxidizing the barbaloin c - glycosidic bond [ 35 , 36 ] . in contrast , humans have a relatively sterile upper gi tract , and anthroid glycosides pass intact to the colon before limited c - glycosidase activity occurs . other aloe vera components , such as polysaccharides that serve as growth substrates for eubacterium , could alter intestinal flora in favor of glycosidase - capable bacteria . the rat may therefore be an ideal sentinel for intestinal toxicities associated with the phenolics in aloe ; however , extrapolation of carcinogenic outcomes should account for the degree of relative human exposure to the free anthraquinones and the levels of anthraquinone glycosides in the aloe juice extract tested . the levels of aloe vera consumed by rats in the present study are similar to a concentration consumed by rats in the 2-year rat ntp study ( see table 8) . the ntp study used dried nondecolorized whole leaf aloe juice powder at levels of 0 , 0.5 , 1.0 , and 1.5% in drinking water . at the 1.0% level , male rats in the ntp study drank an average of 28.80 mls / day at approximately the 4-week time point . males and females consumed approximately 232 mol malic acid / day/100 g weight . these data indicate that the rats in our study consumed an amount of aloe juice equivalent to the 1.0% aloe drinking water in the ntp study by malic acid equivalency . in summary , after assessing a dcwl aloe beverage for both genetic and in vivo toxicity , we found no adverse effects associated with high intake of aloe at acute or subchronic periods . importantly , we did not find hyperplastic mucosal changes that were so notable in a recent report using nondecolorized whole - leaf aloe . therefore , it is reasonable to believe that components in the untreated leaf extract are responsible for the large intestinal hyperplastic reactions at 13 weeks .
overweight and obesity are a global concern in both developed and developing countries and in school age children , the prevalence continues to remain high . australian childhood overweight and obesity rates have been shown to be 25.3% for boys and girls aged 517 years , comprised of 17.7% overweight and 7.6% obese . there is a clear need for effective prevention efforts to address the high prevalence of childhood and adult obesity without which obesity will become the primary cause of preventative deaths worldwide . management of this epidemic requires action at a number of levels including broad - based community interventions that focus on environmental change to support individual behaviours [ 46 ] . governments need to contribute to less obesogenic environments through political , physical , sociocultural and structural change , and prevention programs [ 2 , 7 , 8 ] . despite a large body of the literature pertaining to the management of childhood obesity , there are a limited but increasing number of community - wide prevention projects . to effectively target childhood obesity , existing and new programs need to be systematically evaluated to determine the efficacy of the implemented strategies and such evaluations should be of high quality in order to contribute to the evidence for addressing childhood obesity . however , these evaluations are limited by a lack of setting specific tools which allow evaluation specific to a particular setting , such as a school or home environment , and further limited by a lack of reliable tools suitable for evaluation purposes in these settings . the eat well be active ( ewba ) community programs were implemented in south australia from 2005 to 2010 , focusing on prevention of obesity through environmental change using a community development approach . briefly , the program aimed to increase the proportion of 018 year old children within the healthy weight range by effecting environmental change which in turn would influence healthy eating and physical activity behaviours . results from the evaluation of ewba have been reported elsewhere [ 1215 ] . due to the lack of relevant tools to evaluate the impact of the intervention , a number of program - specific questionnaires were developed to assess behaviours , knowledge , attitudes , and environments relevant to the goals of the program of increasing healthy eating and activity . four of these tools were completed by children aged 9 to 11 years , their parents , and teachers . assessment of these tool properties is important to ensure that accurate and appropriate assessments can be conducted . the aim of this paper is to report the reliability of the parent and teacher questionnaires , tools that assess the diet and physical activity environments of children in the home and school , respectively . these questionnaires can provide relevant insight into the domains which influence nutrition and physical activity behaviours in children , and may be used to evaluate obesity prevention interventions . ethics approval for this study was obtained from the flinders university social and behavioural research ethics committee , the south australian health ethics committee , and the department of education and children 's services ethics committee . school 1 was a reception - year 12 ( 518 years ) government school in the north of adelaide and school 2 a reception - year 12 catholic school in a regional centre in south australia . school 2 was part of the wider ewba evaluation , but school 1 was not . all parents of children in years 57 at school 1 were invited to participate in the test - retest reliability study , and all teachers were invited at a staff meeting , in october 2008 . at school 2 , the test - retest reliability study was conducted in conjunction with the ewba follow - up evaluation . parents who participated in this ewba evaluation follow - up ( sept nov 2009 ) were invited by letter to complete the questionnaire on a second occasion , and all teachers were asked at a staff meeting to complete the survey a second time . at school 1 , test 1 was administered to all teachers at a staff meeting . at school 2 , test 1 was administered to teachers who agreed to participate whose classes were participating in the wider ewba evaluation . at school 1 , the parent questionnaires were sent home by the school to all parents of students in school years five , six , and seven . an introductory letter , an information sheet , and reply - paid envelope to allow return by post accompanied the questionnaire . at school 2 , the parent questionnaires were administered as part of the wider ewba evaluation , as reported in . two weeks later at both schools , teachers completed the questionnaire again at a staff meeting , and parents who completed the first questionnaire were mailed the second questionnaire with a reply - paid envelope . reminder letters were sent two weeks later by the school to parents who had not returned the second questionnaire . the parent and teacher questionnaires were part of a suite of questionnaires developed for evaluation of the ewba community programs . the questionnaires were developed by the program evaluation committee which included academics with expertise in childhood obesity , nutrition , physical activity , and community development . these were developed in response to a lack of valid and relevant tools being available in the published or the grey literature . the items included in the program - specific questionnaires were specifically selected to evaluate each of the program 's relevant objectives . thus , these questionnaires were likely to be more sensitive to the programs ' goals and objectives than any existing questionnaires which were more general and did not include the breadth of the programs ' inquiry . the parent questionnaire contains 25 questions requiring 67 responses covering the following domains : demographics ; obesogenicity of the home environment ; parental knowledge and attitudes towards healthy eating and physical activity ; child physical activity and healthy eating behaviours . two additional questions ask about food security and difficulty of breastfeeding in public places ( table 1 ) . the teacher questionnaire consists of 15 questions requiring 44 responses covering teaching practices around healthy eating and physical activity inclusion in the school curriculum ; training / experience in healthy eating and physical activity ; teacher knowledge and attitudes towards healthy eating and physical activity ( table 2 ) . to produce more meaningful and reliable results this is one way of interpreting information obtained from dietary indexes and has been used previously [ 10 , 1719 ] . in order to make the analyses specific to ewba and to be able to measure the impact of the intervention directly by relating to the specific project goals , items were grouped into scores based on the goals of the ewba community programs . for example , goals related to diet included decreasing child intake of sweetened beverages and noncore foods and increasing intake of water and fruit and vegetables ; hence , these were used as categories for the basis of developing scores related to child intake . similarly , goals related to physical activity were increasing active pastimes and decreasing sedentary activity ; hence , these were used as the basis for classifying items into scores demonstrating child physical activity . generally , ewba sought to influence at multiple levels including attitudes , behaviours , and environments ; therefore , scores assessed one of these three domains in the context of the diet or activity factors mentioned previously ( non - core food , sweetened beverages , water , fruit and vegetables , active pastimes , and sedentary pastimes ) . items were reverse coded where necessary , so a higher score represented a more positive attitude or behaviour . the created scores allow better assessment of health themes , enablers , and barriers and can be used to compare baseline data against a target score . thus , there are 14 outcomes ( seven scores and seven single items ) from the parent questionnaire and 12 outcomes ( six scores and six single items ) from the teacher questionnaire . a target score which identifies a healthy attitude or environment was determined for each score ( tables 3 and 4 ) . targets for assessment of intake were largely based on the australian guide to healthy eating ( aghe ) . assessment of physical activity was based on the physical activity guidelines for five to 12 year olds and 12 to 18 year olds . the 14 parent and 12 teacher outcomes were assessed for test - retest reliability using the intraclass correlation coefficient ( icc ) . internal consistency of the teacher and parent scores ( outcomes with multiple items ; parent questionnaire seven scores ; teacher questionnaire 6 scores ) at time 1 was assessed using cronbach 's alpha . sixty parents ( school 1 : 22 , school 2 : 38 ) completed the questionnaire on two occasions one to two weeks apart . all but one respondent was female , 46 ( 77% ) were married or in a defacto relationship , half ( n = 33 , 55% ) were from two children households , 13 ( 22% ) had a university degree , and two ( 3% ) had year 10 schooling or less . forty - six ( 77% ) were working full - or part - time , and 20 ( 33% ) held a government health care card . it is not possible to calculate response rates for parents at both schools as questionnaires were posted to parents by the school and exact numbers posted are unknown . table 3 shows the outcomes for each questionnaire and the icc ( 95% confidence interval ) for each score . all iccs were statistically significant and ranged from 0.37 to 0.92 with eight of the fourteen greater than 0.7 . cronbach 's alpha varied from 0.13 to 0.78 for the scores from the parent questionnaire , with three of seven scores having alpha values greater than 0.5 . twenty - eight teachers ( school 1 : 24 , school 2 : 4 ) completed the teacher questionnaire twice . this is a response rate of 100% at school 1 and 50% at school 2 . most ( n = 23 ) were female , and a majority taught across multiple year levels with half the respondents teaching in each of reception to year 5 and approximately one third teaching year 6 . thirteen respondents had been at their current school for four years or longer , and for eight , it was their first year at the school . table 4 shows the outcomes for each completion of the questionnaire and the icc ( 95% confidence interval ) for each score . all iccs were statistically significant and ranged from 0.42 to 0.86 with five of the twelve greater than 0.7 . cronbach 's alpha varied from 0.11 to 0.91 for the scores from the teacher questionnaire , with four of six scores having alpha values greater than 0.5 . the purpose of this study was to determine the reliability of the parent and teacher questionnaires developed to evaluate the ewba community programs , two questionnaires that assess the healthy eating and physical activity environments of children . the parent questionnaire assesses child dietary intakes , parent knowledge of health - related recommendations , and attitudes about healthy behaviours . the teacher questionnaire assesses the degree to which teachers incorporate healthy eating and activity facets in their daily teaching regime and their skills , attitudes , and knowledge around healthy eating and physical activity . overall the surveys showed good test - retest reliability with only one outcome in each of the parent and teacher surveys having an icc of less than 0.5 ( parent , money , and teacher , knowledge ( fruit ) ) . the test - retest reliability of knowledge questions in both the parent and teacher surveys was generally lower than the other items , with iccs between 0.4 and 0.6 . the commonly promoted message in south australia is go for 2&5 which are the adult recommendations for fruit and vegetable intake , respectively , and a message that is actively used in schools . however , the aghe recommends a minimum of one and three serves of fruit and vegetables , respectively , for 811 year olds and two and four serves , respectively , for 1218 year olds . the aghe includes additional options according to varying eating patterns with serves above these minimums . these different messages are a potential source of confusion which may mean that respondents are guessing the correct option and this in turn would be a source of retest error . the internal consistency of the scores in the parent and teacher questionnaires was poor to moderate . only one score from the parent questionnaire ( non - core food ) and three from the teacher questionnaire were in line with this recommendation ; however , the value is affected by the number of items in the scale , and it is common to find low cronbach 's alpha values with scales with less than ten items . four scores from the parent questionnaire had between five and ten items and three scores had less than five items . in the teacher questionnaire , four scores had between five and nine items and two scores had less than five items . hence , the low number of items in the scores could be a reason for the poor to moderate internal consistency observed . furthermore , the parent and teacher scores were created by summing items that measure the goals of the ewba program , specifically reducing intake of noncore foods and sweetened beverages , reducing screen time , and increasing fruit and vegetable intake , water intake , and physical activity . unlike other scales in the literature , we would not necessarily expect all items to correlate . for example children with a high intake of crisps would not necessarily have a high intake of lollies ( both single items in the noncore food score ) . this could explain the lower than ideal cronbach 's alpha values for some of the scores . four parent questionnaires were identified in the literature that had similar ranges ( or slightly better ) of internal consistency and test - retest reliability observed in this study . the home environment survey is completed by parents and measures how supportive the home environment is of healthy eating and physical activity . cronbach 's alpha for four subscales ranged from 0.66 to 0.84- and test - retest reliability was high with icc more than 0.75 for all scales . the children 's dietary questionnaire , measuring parent report of child eating patterns , had four subscales with cronbach 's alpha for fruit and vegetable and noncore food subscales ranging from 0.62 to 0.76 with icc for test - retest reliability ranging from 0.51 to 0.90 which was concluded to be satisfactory . the meals in our household questionnaire measured parent report of six domains , including family meal structure and mealtime behaviours . test - retest reliability was assessed using the spearman correlation for the six domains , and this ranged from 0.80 to 0.95 , while cronbach 's alpha ranged from 0.39 to 0.93 across the six domains . similarly , a questionnaire measuring constructs believed to predict fruit and vegetable consumption ( in children , completed by parents ) had pearson correlation ranging from 0.61 to 0.84 and cronbach 's alpha from 0.31 to 0.91 . a strength of this study is the report on two questionnaires with multiple scores / indexes that simultaneously measure diet and physical activity environments of children . many evaluation tools measure healthy eating or physical activity , while these tools measure the two simultaneously . additionally , these tools are unique because they focus on behaviours , environments and attitudes , all of which have been demonstrated as factors contributing to the obesity epidemic . hence these two tools have the potential to be used in the evaluation of obesity prevention programs and consequently contribute to the evidence about obesity prevention . the indexes also cover a broad range of domains relevant to healthy eating and activity across the home and school environment and use scores as a way of interpreting information and gaining an overall picture . tools which simultaneously evaluate environments that children are exposed to at home and school , as well as the attitudes of parents and teachers and diet and activity behaviours of children , are lacking in the literature . the use of an internal consistency analysis in addition to testing test - retest reliability is a strength , as is the participation of parents and teachers from both metropolitan and regional schools . a notable limitation of the study is the sample size . as previously mentioned , the recommendation for a test - retest reliability study is a sample size of 100 . the sample size for the teacher questionnaire fell well short of this ( n = 28 ) , and the sample size for the parent questionnaire was also below this ( n = 60 ) . ( n = 4 ) because only teachers of classes participating in the ewba evaluation were asked to participate ( n = 8) . the low sample size has implications for interpreting the results of the study , in particular those for the teacher questionnaire , because a larger sample size results in a smaller confidence interval which means we can be more certain that the true reliability coefficient is close to that which has been calculated . hence , the reliability of the teacher questionnaires in particular should be interpreted with caution . despite the recommendation of 100 as the sample size for a test - retest reliability study , the sample size for similar studies varies considerably in the literature and the sample size for the parent questionnaire falls within this range . forty - four parents were used to assess reliability of the meals in our household questionnaire and 38 childcare directors completed the nutrition and physical activity self - assessment for child care in a test - retest reliability study . other studies had a larger sample size to test test - retest reliability , including gattshall et al . who tested reliability of the home environment survey with 156 parents and randall - simpson et al . who had 140 parents participate to assess reliability of the nutrition screening tool for every preschooler in addition , while creation of scores can provide more meaningful and reliable results , important information may be missed or results misinterpreted without some secondary investigation of reliability of individual items such as in the parent attitudes to healthy eating and physical activity scores . a possibility for future research is to test the internal validity of the parent and teacher questionnaires and to retest the reliability of the teacher questionnaire with a larger sample size . the parent and teacher questionnaires for the ewba community programs are a moderately reliable method for assessing child intakes , environments , attitudes , and knowledge associated with healthy eating and physical activity . these questionnaires assess relevant information and the scores present this information in a meaningful manner , suggesting that they may be useful in similar settings to evaluate similar obesity prevention programs .
although copper is a trace mineral and accounts for only 0.01% of total body weight , it plays an important role in electron transport , neurotransmitter synthesis , collagen cross - linkage , and melatonin production and is an important factor in the coagulation cascade . toxicity associated with abnormal metabolism , as seen in wilson 's disease , can result in excessive copper accumulation and deposition in many tissues . this can lead to cardiac dysfunction , liver cirrhosis , pancreatic dysfunction and neurological abnormalities . pregnancy induces little change in the metabolism of this trace metal , only retention in the amounts needed for fetal growth . abnormal copper metabolism may be associated with intrauterine fetal growth restriction , preeclampsia , and neurological sequelae . we present a case of unexplained abnormally elevated copper levels outside of wilson 's disease only during pregnancy . the patient is a 32-year - old gravida 4 para 2012 who presented for routine obstetrical care at 12 weeks gestation . evaluation for wilson 's disease , in both the patient and her children , was negative . her two sons , from different fathers , had elevated copper levels at birth . her first child was diagnosed with autism and had three myocardial infarctions . her second child was diagnosed with autism and his copper levels are now normal . during those pregnancies , the patient declined treatment for elevations in her copper . during the current pregnancy , copper levels were again elevated . she was followed in the high - risk obstetrical clinic . in the first trimester , her copper level was 154 g / dl , the high end of normal , with normal copper levels ranging from 70155 g / dl . as her pregnancy progressed , the levels rose as high as 260 g / dl with zinc levels decreasing to 61 g / dl ( normal levels of zinc range from 70150 g / dl ) . the remainder of her blood work , including liver function tests , urine drug screens , and complete blood counts were within normal limits . she repeatedly refused treatment with chelation therapy , but instead she opted to increase her intake of zinc . following zinc supplementation of 100 mg daily , her serum zinc levels normalized to 140 g / dl . this led to a decreased , but still elevated , copper level of 245 g / dl . at 36 weeks she delivered a liveborn male weighing 2700 grams with apgar scores were 9 and 9 at 1 and 5 minutes , respectively . he showed no evidence of infection ; all of his labs and physical examinations were normal . while thyroid function tests were normal , the baby did not have evidence of heart disease during cardiac and nicu evaluation and workup . majority of copper is absorbed in the enterocytes of the duodenum and proximal small intestine and incorporated in the liver into apoceruloplasmin , forming ceruloplasmin . copper participates in multiple enzymatic reactions with varied physiological roles from melanin production to wound healing to electron transport . it stimulates the absorption of iron and is required for the synthesis and function of hemoglobin . it is also involved in the production of elastin and collagen which contribute to the structural stability of bone , cartilage , and tendons . wilson 's disease is an autosomal recessive disorder of copper metabolism with a worldwide prevalence of 1 : 30,000 . the cause of copper elevations is due to an inherited genetic defect in the copper transport which reduces biliary excretion . mutation in the allele for this gene leads to absence or diminished function , resulting in decrease copper excretion . once the capacity of the liver is exceeded , copper diffuses into the bloodstream and deposits into other organs , causing damage specifically to the brain , eyes , and kidneys . recent studies have shown that during pregnancy , atp7b plays a role in transporting copper from the placenta to maternal circulation , thus preventing fetal overload . if dysfunctional , excess copper remains in the fetus and placenta leading to oxidative damage resulting in fetal loss or damage . while our patient did not have wilson 's disease , abnormal copper metabolism is a rare condition . workup for other causes of abnormal elevated copper metabolism were negative in this patient ; nevertheless , her treatment and management were the same . excess copper levels can be associated with preeclampsia secondary to excess buildup in the liver , and the fetus can become growth restricted and have neurological sequelae because of oxidative damage caused by copper accumulation in the placenta and fetal tissue [ 4 , 5 ] . there are few case reports in the literature about successful pregnancies in women with wilson 's disease , probably due to hormonal changes secondary to chronic liver disease , endocrine disorders along with infertility , and recurrent miscarriages due to excess copper accumulation in the uterus [ 3 , 4 ] . however , patients who receive proper treatment can conceive and have a favorable outcome in pregnancy . penicillamine , trientine , tetrathiomolybdate , and zinc are drugs used for the treatment of wilson 's disease . penicillamine reduces copper levels by chelation and forms a soluble complex that is excreted in the urine . it has been observed in more than 100 pregnancies for a variety of medical conditions including rheumatoid arthritis . while the majority of those pregnancies resulted in healthy newborns , some defects were observed such as vein fragility , impaired wound healing , cutis laxa , low - set ears , micrognathia , and hyperflexion of hips and joints . counseling and close monitoring is essential to prevent defects in newborn and maintain effective therapy for mother [ 3 , 6 ] . the alternative , zinc , is usually reserved for maintenance therapy ; nevertheless , it appears to be equally effective as penicillamine and easily tolerated . the effects of these treatments suggest that it is relatively safe during pregnancy . however , there are too few exposed infants to be certain . to our knowledge , case reports of abnormal elevated copper metabolism in pregnancy outside of wilson 's disease are nonexistent . nonetheless , we treated this patient the same , because the consequences of untreated wilson 's disease in pregnancy can lead to preeclampsia , fetal loss or fetal growth restriction , and neurological damage . in the limited case reports on successful pregnancies complicated by wilson 's disease , penicillamine was the drug of choice and is the mainstay of therapy , and very few case reports used zinc as the only therapeutic option during pregnancy . in our patient , zinc therapy seems to have limited the effects of excess copper to the fetus . and while it is still too early to know whether or not this child will develop autism or any other neurological sequelae , zinc regimen seems to have achieved its purpose , which is a safe maternal and neonatal outcome . additionally , counseling , along with treatment options and timely delivery , can greatly improve neonatal and maternal outcome .
xylene ( aromatic hydrocarbon ) has been widely used as a de - alcoholization agent of choice , in spite of its toxicity to laboratory personnel and the danger it poses to the environment . the toxic effects of xylene include acute neurotoxicity , cardiac and kidney injury , cancer , blood dyscrasias , skin diseases , gastrointestinal disturbances , musculoskeletal system disorders , fetotoxicity and so on . on account of the occupational safety and health administration ( osha ) regulations , various xylene substitutes , such as , limonene reagents , aliphatic hydrocarbons , vegetable oils and mineral oils were tried in the past to avoid xylene in the laboratory . it is non - toxic , heat stable , slow to oxidize and has highest resistance to rancidity . in the present study , we have tried to compare the efficacy of coconut oil with that of xylene , as a clearant , as it is readily available , less expensive and a safer alternative to xylene . the first half of the tissue bit was processed in xylene and the other half simultaneously in coconut oil [ figure 1 ] . the duration of clearing was constant for both the solutions ( one hour each : two changes ) . after de - alcoholization , the specimens were also tested for gross changes after clearing . all the sections were stained with hematoxylin and eosin ( h and e ) to permit evaluation of the histological details . few of the sections ( salivary gland specimens ) were subjected to periodic acid schiff ( pas ) also , in order to see whether coconut oil was interfering in this routinely used special staining procedure . the sophisticated technique of computer - assisted morphometry was performed , to observe the morphological features like the mean cell area , to see if any consistent change existed between the study groups . each equal half of every tissue cleared in parallel solutions , either in xylene / coconut oil specimens for this study were selected from the anatomical structures in the head and neck region , such as , skin , buccal mucosa , salivary gland , tendon , muscle and lymph node . the inclusion criteria were as follows : only soft tissue was considered for this study . the specimen size was 0.5 1 cm or greater and a thickness of 3 - 5 mm was taken for processing ( for better penetration of the processing fluids ) . the tissue was then divided into equal halves : during clearing , one was processed in coconut oil and the other in xylene . gross tissue specimen : after clearing in two different solvents , the gross tissue features , such as , translucency ( surface translucency when viewed for reflected light ) , rigidity ( palpation with two fingers ) , change after impregnation ( change in the rigidity because of infiltration of wax ) and ease in section cutting , were noted down for each specimen separately , for co - s and xy - s . scoring was done while comparing the parameters for both the agents : the finding of co - s that was inferior to xy - s was considered as score 0 , similar to xy - s as score 1 and superior to xy - s as score 2 [ table 1 ] . the tissue bits were measured just after clearing , to compare the gross - shrinkage for the two solvents [ figure 2 ] . comparison of gross features of co - s with respect to xy - s measurements for gross tissue shrinkage cellular architecture : ( a ) for cellular details , distinct architecture and good nuclear - cytoplasmic contrast is considered as score 1 and indistinct / blurred nuclear - cytoplasmic contrast as score 0 . ( b ) for nuclear details , distinct chromatin condensation , prominent nuclear membrane and crisp staining of the nucleus is considered as score 1 and indistinct smudging and pyknosis of the nuclei as score 0 . quality of staining : the staining of tissues was evaluated as poor , satisfactory and good . poor indicated that the tissue failed to take up the stain adequately , stained unevenly ( score = 0 ) . satisfactory pointed toward details like not visualized up to the mark ( score = 1 ) . good designated good contrast between the nucleus and cytoplasm and visibility of details , along with brilliance of staining ( score = 2 ) . the images were captured using a three - chip ccd camera attached to a trinocular research microscope with a 100x objective . the final image captured on the monitor had a magnification of 1000x . for each specimen , five most representative fields were selected . the selected fields included representative cells where distinct cellular and nuclear outlines were seen , avoiding overlapping . a total of 100 cells ( 20 cells in five different high - power fields ) were randomly selected and measured for any difference in the xy - s specimens and co - s specimens . histologically identifiable acini , adipocytes and epithelial cells in the para basal layer were subjected for measurement . the actual measurements of the morphometric parameters were done using the image analyzer software image - proexpress ( media cybernetics , silver spring , md , usa ) . the cell area ( ca ) was measured in square microns when the perimeter was traced ; the software automatically calculated the ca ( number of pixels detected , converted to micrometers ) [ figure 3 ] . morphometrical analysis of the mean area of an individual cell to assess shrinkage at the cellular level as most of the evaluative criteria were subjective , the scoring and assessment was carried out by three different observers and the mean scoring was considered , which would prevent interobserver and intraobserver bias . the obtained data was subjected to statistical analysis using the wilcoxon matched pair test and the mann - whitney u test . specimens for this study were selected from the anatomical structures in the head and neck region , such as , skin , buccal mucosa , salivary gland , tendon , muscle and lymph node . the inclusion criteria were as follows : only soft tissue was considered for this study . the specimen size was 0.5 1 cm or greater and a thickness of 3 - 5 mm was taken for processing ( for better penetration of the processing fluids ) . the tissue was then divided into equal halves : during clearing , one was processed in coconut oil and the other in xylene . gross tissue specimen : after clearing in two different solvents , the gross tissue features , such as , translucency ( surface translucency when viewed for reflected light ) , rigidity ( palpation with two fingers ) , change after impregnation ( change in the rigidity because of infiltration of wax ) and ease in section cutting , were noted down for each specimen separately , for co - s and xy - s . scoring was done while comparing the parameters for both the agents : the finding of co - s that was inferior to xy - s was considered as score 0 , similar to xy - s as score 1 and superior to xy - s as score 2 [ table 1 ] . the tissue bits were measured just after clearing , to compare the gross - shrinkage for the two solvents [ figure 2 ] . comparison of gross features of co - s with respect to xy - s measurements for gross tissue shrinkage cellular architecture : ( a ) for cellular details , distinct architecture and good nuclear - cytoplasmic contrast is considered as score 1 and indistinct / blurred nuclear - cytoplasmic contrast as score 0 . ( b ) for nuclear details , distinct chromatin condensation , prominent nuclear membrane and crisp staining of the nucleus is considered as score 1 and indistinct smudging and pyknosis of the nuclei as score 0 . quality of staining : the staining of tissues was evaluated as poor , satisfactory and good . poor indicated that the tissue failed to take up the stain adequately , stained unevenly ( score = 0 ) . satisfactory pointed toward details like not visualized up to the mark ( score = 1 ) . good designated good contrast between the nucleus and cytoplasm and visibility of details , along with brilliance of staining ( score = 2 ) . the images were captured using a three - chip ccd camera attached to a trinocular research microscope with a 100x objective . the final image captured on the monitor had a magnification of 1000x . for each specimen , the selected fields included representative cells where distinct cellular and nuclear outlines were seen , avoiding overlapping . a total of 100 cells ( 20 cells in five different high - power fields ) were randomly selected and measured for any difference in the xy - s specimens and co - s specimens . histologically identifiable acini , adipocytes and epithelial cells in the para basal layer were subjected for measurement . the actual measurements of the morphometric parameters were done using the image analyzer software image - proexpress ( media cybernetics , silver spring , md , usa ) . the cell area ( ca ) was measured in square microns when the perimeter was traced ; the software automatically calculated the ca ( number of pixels detected , converted to micrometers ) [ figure 3 ] . morphometrical analysis of the mean area of an individual cell to assess shrinkage at the cellular level as most of the evaluative criteria were subjective , the scoring and assessment was carried out by three different observers and the mean scoring was considered , which would prevent interobserver and intraobserver bias . the obtained data was subjected to statistical analysis using the wilcoxon matched pair test and the mann - whitney u test . most of the specimens ( 73% ) were more rigid in xy - s when compared with co - s . although in 16 specimens , the rigidity was same in both the groups [ table 1 ] . translucency was visibly better in all co - s than xy - s [ table 1 ] . however , there was no difference observed in the tissue bits as far as rigidity after impregnation and ease of sectioning was concerned , in both the groups [ table 1 ] . there was no significant shrinkage in the tissue bits after clearing in coconut oil ( p = 1.000 ) . however , with respect to xy - s , the specimen shrank significantly , when compared with the measurements taken before clearing ( p = 0.0117 ) [ table 2 and figure 2 ] . there was no difference in staining quality and tissue architecture in both kinds of specimens [ table 3 and figure 4 and 5a ] . co - s , when stained with periodic acid - schiff ( pas ) , showed similar details as seen in xy - s [ figure 5b ] . morphometrically , there was a significant decrease in the mean area of the individual cells in xy - s , compared to co - s ( p = 0.0006 ) , [ table 4 and figure 3 ] . comparison of gross shrinkage after clearing comparison of staining quality a , b , c hematoxylin and eosin stained tissue sections of xylene - treated specimen . a1 , b1,c1 hematoxylin and eosin stained tissue sections of coconut oil treated specimen . [ a and a1 skin tissue , b and b1 salivary gland tissue , c and c1 lymph node tissue ] a , b xylene treated specimen . [ a and a1 oral epithelium ; b and b1 salivary gland stained with pas ] morphometric analysis of mean area of individual cell considering the toxicity of xylene and its hazards , various substitutes , including vegetable oils and mineral oils , have been tried in the past . however , most of them showed an inconsistent outcome , which motivated us to take up this study . coconut oil was selected , as it is , profusely available in the tropical world , especially in south asia , it is less expensive and non - hazardous . when compared with xylene , it is not harmful to the environment . the results of the present study showed that co - s , after clearing , was apparently more translucent compared to xy - s . although less rigid in contrast to xy - s , it did not adversely affect impregnation and section cutting . morphometrically , the shrinkage was relatively less in co - s when compared with xy - s , which was noted as a statistically significant difference in the mean cell area of individual cells between sections ( p = 0.0006 ) . however , there was no change in cellular , nuclear and cytoplasmic staining , when both groups were compared . buesa used a mixture of ethanol , isopropyl alcohol and mineral oil as an alternative for xylene and found the mixture to be as efficient as xylene in de - alcoholization . instead , we considered the environment - friendly , readily - available alternative , coconut oil , as we wanted to avoid chemicals such as ethanol and isopropyl alcohol , which were also hazardous . a mixture of coconut oil and olive oil was tried by rasmussen et al . and they noted incomplete impregnation , leading to problems in the cutting sections and therefore , they concluded that this mixture was ineffective as a clearing agent . in contrast to their observation , we found that co - s , when used alone , was as effective as xylene , without interfering with further impregnation and cutting . this difference could be because of the olive oil in the mixture , which would have adversely affected the procedure , counteracting with the favorable properties of coconut oil . a study by andre et al . substituted xylene with a mixture of peanut oil , soyabean oil , coconut oil and cotton oil and concluded that it was a poor alternative , as the quality of sections with respect to xy - s were better . even the special staining procedure showed good results , proving no interference by coconut oil with the tissue composition and it just acted as a transient media . as the result of our study showed less shrinkage in co - s , compared to xy - s , we would suggest that this would be a preferred procedure , where morphometric studies have to be carried out . the only drawback associated with coconut oil , is its tendency to get solidified at a lower temperature . however , this can be overcome by performing the clearing procedure in an incubator , maintaining the required temperature . this research study is unique , as we have tried to assess the efficacy of two clearing agents at different stages of the histopathological procedure , such as , processing , impregnation , sectioning , staining and microscopic evaluation , including morphometry . the results of the present study infer that coconut oil is an efficient substitute for xylene , as it is non - hazardous , less expensive and causes less shrinkage of the tissue . it can be used as a de - alcoholization agent in the histopathological laboratory , without losing the quality of the histological details . all the xylene - substitutes have to be analyzed thoroughly , before concluding which alternative is better . treated specimen can be subjected to all stains and advanced histological procedures ( like immunohistochemistry ) , in order to consider this agent as a better and safer substitute for xylene .
phylogeny reconstruction and homology - based annotation are two of the most common modeling procedures in biology . both of them require the assembly of accurate multiple sequence alignments ( msa ) . in this work , we introduce a web server dedicated to 3d structure - based multiple rna sequence alignment using the sara - coffee package ( 1 ) . it is a suitable companion tool for any modeling technique that can benefit from a structurally accurate ribonucleic acid ( rna ) msa . this includes construction of profile stochastic context free grammar ( scfg ) using packages like infernal ( 2,3 ) . accurately aligning rna sequences is especially important in a context where recent developments in genomics have fueled the detection of thousands of expressed loci and challenged the long held view that non - coding rna ( ncrna ) functions were supported by a much smaller number of genes than their protein - coding counterparts . between 2008 and 2014 , the ensembl non - coding rna ( ncrna ) gene count has grown from 5732 ( october 2008 ) to 22 643 ( january 2014 ) , thus overtaking proteins . it remains unclear , however , which proportion of these genes simply results from spurious transcription . one should also bear in mind that the term ncrna encompasses a very heterogeneous gene population , including about 14 000 long ncrnas of mostly unknown function and a bit less than 9000 short ncrnas often better biologically characterized . this last group includes snornas , microrna precursors , transfer rna ( trna ) and in general most of the highly structured ncrnas catalogued in the rna families database ( rfam ) ( 4 ) . sara - coffee 's main characteristic is to combine sequence and structure pairwise alignment methods into a unified multiple sequence alignment , using sara ( 5 ) as a 3d structure pairwise alignment engine . when used in pure 3d structural aligner mode sara - coffee is limited by the small number of available rna pdb structures . these merely constitute 3% of all pdb entries ( 2'941 out of 99'775 in march 2014 ) and their pace of determination remains significantly slower than that of proteins with a doubling time of 7 years , as compared to 5 years for proteins . yet , in its sequence / structure hybrid mode , sara - coffee can be used to combine available 3d structural information with the very large number of novel uncharacterized ncrna sequence reported by genome sequencing projects . the problem of aligning rna sequences is rather complex . even though rna molecules often contain evolutionary conserved secondary structures , their primary structure signal is rarely as strong as the one resulting from the protein three - letters meta - alphabet . as a consequence , higher levels of sequence identity integrating additional information , e.g. secondary structure signal , into the alignment process is possible but can be computationally expensive . the first attempt to do so was described by sankoff and later improved using profile stochastic context free grammar ( scfg ) ( 2 ) . over the years , many methods have been reported for this purpose ; a non - exhaustive list includes foldalign ( 10 ) , stemloc ( 11 ) , consan ( 11 ) , locarna ( 12 ) , r - coffee ( 13,14 ) and mafft ( 15 ) . all of them constitute an attempt to capture the secondary structure signal contained in rna molecules in order to build more informative sequence alignment models . these models can then be passed to scfg profile building tools in order to build co - variation models like the ones used by rfam . these multiple alignment heuristics only address the issue of aligning sequences using experimental or predicted secondary structure information . in some recent work , we have shown that one can also combine sequence and experimental 3d structural information ( 1 ) into an msa . this process requires the ability to superpose rna 3d structures , a problem similar in nature to protein structure comparison and for which several pairwise comparison tools have been developed , including sara ( 5 ) , dial ( 16 ) , iparts ( 17 ) , arts ( 18 ) and r3d align ( 19 ) . our main motivation when developing sara - coffee has been to turn these pairwise 3d structure aligners into a method able to deliver 3d - based multiple sequence alignments . it must be stressed that this approach is generic enough to be applied to any of the above - mentioned rna 3d pairwise aligners . in a previous study , sara - coffee was extensively validated on a purpose built reference dataset named bralidart ( 1 ) made of 41 rna families containing between 4 and 71 members with known 3d structures . this dataset was assembled so as to only include high quality x - ray structures and exclude any discontinuous structure , on which the algorithm is expected to perform poorly . our benchmarks indicate that sara - coffee is significantly more accurate than alternative sequence based methods , even those using predicted secondary structures . overall , sara - coffee was reported to be over 10% points more accurate than primary structure based aligners , judging from its capacity to properly align pairs of interacting residues . on dense secondary structures in which 70% or more of the nucleotides form watson and crick base pairs sara - coffee outperforms all tested methods , including the ones using predicted secondary structure information . it is about 3% points more accurate than mxscarna ( 20 ) , the second best method in this benchmark . detailed structural analysis using a distance - root mean square deviation ( drmsd ) measurement also indicates that sara - coffee produces alignments significantly superior to all methods tested in the study . overall sara - coffee alignments have a drmsd 10 to 20% lower than alternative models ( i.e. 4.53 against 5.11 for locarna , the next best method in terms of 3d modeling ) . its algorithm can be described as a combination between sara ( 5 ) , a pairwise rna structural aligner and r - coffee ( 14 ) , a t - coffee ( 22 ) based multiple rna sequence aligner . the algorithm is described extensively in ( 1 ) and its general flow can be summarized as follows : by default , sara - coffee takes as input a set of rna sequences with known 3d structures . the first step of the algorithm involves aligning all possible pairs of the provided sequences using sara . the result is an alignment library populated with sara 3d - structure based pairwise sequence alignments . in practice , the alignment library is a list of all the residue pairs found aligned in any of the compiled pairwise alignments . the second step involves extracting contact information from each input structure using the -p mode of x3dna / find_pairs ( 21 ) that reports all base - pairs , including the non - canonical and higher - order ( 3 + ) . this contact information is then used to extend the alignment library so that its alignments become compatible with 2d and 3d contacts . this is achieved by adding pairs of aligned residues that are implied by the contacts but missing from the library . for instance , let us consider a contact between residues xy in sequence a and another contact between residues wz in sequence b. if x and w are found aligned in the library , then the contact based extension will involve adding the pair yz to the library , thus ensuring full compatibility between the library and the contact . if in the library , y ( or z ) are already aligned to other residues , this process will introduce incompatibilities that will be resolved through consistency analysis when incorporating the sequences into the final msa . once extended using contact information , the library can be fed to the default t - coffee algorithm . the top block indicates the reliability of each individual sequence alignment on a 0100 scale . dark red regions are the most reliable , the bottom line ( cons ) , indicates the most reliable columns . it starts by estimating the similarity between every pair of sequences counting words of size four and then uses neighbor joining to turn this distance matrix into a binary guide tree . the guide tree being binary , its resolution involves aligning at each node either two sequences , a sequence and a profile or two profiles , until reaching the root where the full msa is resolved . at each node the main characteristic of t - coffee is the ability to use the library described above instead of a standard substitution matrix . when using the library , the cost for aligning two symbols is set to be equal to the number of time these symbols are found aligned in the library , either directly or by combining any two pairs of aligned residues ( i.e. the pair xw of aligned residues may be supported by a combination of the two library pairs xk and kw , k being a residue from a third sequence ) . this consistency analysis helps deciding between conflicting library pairs that may result from the secondary structure based extension . when doing so , the library is built in a similar way , but in that case sara is only applied onto sequence pairs having both an experimental 3d structure . in all other cases , a pair - hmm ( proba_pair ) adapted from probcons is used to produce the pairwise comparisons and to populate the library with residue pairs having a high alignment posterior probability ( 22 ) . in the next step , the contact list for sequences with no experimental 3d structures is replaced with a secondary structure prediction , as provided by rnaplfold from the vienna package ( 23 ) . the sara - coffee web server is part of the t - coffee web platform ; its access is free and unrestricted , with no login procedure . the server is accessible from http://tcoffee.crg.cat/apps/tcoffee/do:saracoffee with any standard internet browser ( mozilla firefox 5 + , google chrome , internet explorer 8 + , safari 6 + and opera 11 + ) . anonymous jobs can nonetheless remain available from the submission terminal thanks to a cookies cache procedure . results are kept on the web server for a month after computation and are assigned a permanent url during this time . sequences with a known pdb structure must be named after their pdb identifier , including the chain index separated by an underscore character ( i.e. > pdbid_chain ) , other sequences can be given arbitrary names . white spaces are forbidden , all sequences are required to have a different name and the provided primary sequences must match the pdb seqres field . the input interface also gives access to an advanced mode that makes it possible to control several output options , including format , case , residue numbering , output order and interleaved format block length . once submitted , the server runs the default sara - coffee onto the provided dataset . results are returned via the same interface but can also be accessed via the history . the main limiting step of sara - coffee is the computation of sara pairwise 3d - structure based sequence alignment . for two trna structures , sara requires about 5 s. on a dataset of 71 trna with known 3d structures , the server takes about 4 h , it requires about 2 min on a dataset containing 5 trnas only like the one provided as a test . the result page displays the following items in order : msa : shows the resulting interleaved msa . this graphic is the html rendering of the file * .score_html , available for download from the next section . the msa is colored according to the t - coffee reliability scheme ( 24 ) where red and orange bits correspond to alignment portions for which there is a high consistency within the sara - coffee pairwise library , while lighter bits ( green , yellow and blue ) correspond to the less well supported portions , expected to be less accurately aligned.citation : link to the relevant publications when using this server.the result files produced by the submitted sara - coffee alignment . all files can be downloaded as a single zip file by clicking the download all link . they can also be automatically imported into the user dropbox account , if available.info : some information about the executed job.replay : this feature allows the users to re - run the job while modifying some input options or data.feedback : this feature encourages users to provide some feedback via social media . this graphic is the html rendering of the file * .score_html , available for download from the next section . the msa is colored according to the t - coffee reliability scheme ( 24 ) where red and orange bits correspond to alignment portions for which there is a high consistency within the sara - coffee pairwise library , while lighter bits ( green , yellow and blue ) correspond to the less well supported portions , expected to be less accurately aligned . all files can be downloaded as a single zip file by clicking the download all link . replay : this feature allows the users to re - run the job while modifying some input options or data . feedback : this feature encourages users to provide some feedback via social media . sequences with a known pdb structure must be named after their pdb identifier , including the chain index separated by an underscore character ( i.e. > pdbid_chain ) , other sequences can be given arbitrary names . white spaces are forbidden , all sequences are required to have a different name and the provided primary sequences must match the pdb seqres field . the input interface also gives access to an advanced mode that makes it possible to control several output options , including format , case , residue numbering , output order and interleaved format block length . once submitted , the server runs the default sara - coffee onto the provided dataset . results are returned via the same interface but can also be accessed via the history . the main limiting step of sara - coffee is the computation of sara pairwise 3d - structure based sequence alignment . for two trna structures , sara requires about 5 s. on a dataset of 71 trna with known 3d structures , the server takes about 4 h , it requires about 2 min on a dataset containing 5 trnas only like the one provided as a test . the result page displays the following items in order : msa : shows the resulting interleaved msa . this graphic is the html rendering of the file * .score_html , available for download from the next section . the msa is colored according to the t - coffee reliability scheme ( 24 ) where red and orange bits correspond to alignment portions for which there is a high consistency within the sara - coffee pairwise library , while lighter bits ( green , yellow and blue ) correspond to the less well supported portions , expected to be less accurately aligned.citation : link to the relevant publications when using this server.the result files produced by the submitted sara - coffee alignment . all files can be downloaded as a single zip file by clicking the download all link . they can also be automatically imported into the user dropbox account , if available.info : some information about the executed job.replay : this feature allows the users to re - run the job while modifying some input options or data.feedback : this feature encourages users to provide some feedback via social media . this graphic is the html rendering of the file * .score_html , available for download from the next section . the msa is colored according to the t - coffee reliability scheme ( 24 ) where red and orange bits correspond to alignment portions for which there is a high consistency within the sara - coffee pairwise library , while lighter bits ( green , yellow and blue ) correspond to the less well supported portions , expected to be less accurately aligned . all files can be downloaded as a single zip file by clicking the download all link . replay : this feature allows the users to re - run the job while modifying some input options or data . feedback : this feature encourages users to provide some feedback via social media . we describe the sara - coffee web server , a web based tool able to incorporate 3d structure information within rna multiple sequence alignments by combining sequence and structural information . sara - coffee has been shown to produce accurate alignment as judged from structural analysis . this server makes it possible to combine user 's data with publicly available rna 3d structures , so as to obtain the required models . future improvements will involve the possibility of uploading user 's defined 3d models as well as new output formats providing a local visualization of structural similarity . ] ; quantomics [ kbbe-2a 222664 ] ; center for genomics regulation ( crg ) ; la caixa fellowship program ( to m.c . ) ; spanish ministry of economy and competitiveness [ bes-2012051918 to p.b . ] . embl interdisciplinary postdoc ( eipod ) under marie curie actions ( cofund ) ( to g.b . ) ; department of pathology at the university of alabama at birmingham ( to e.c . ) . funding for open access charge : plan nacional [ bfu2011 - 28575 to c.n . and p.d . ] ; quantomics [ kbbe-2a 222664 ] ; center for genomics regulation ( crg ) ; la caixa fellowship program ( to m.c . ) ; spanish ministry of economy and competitiveness [ bes-2012051918 to p.b . ] . embl interdisciplinary postdoc ( eipod ) under marie curie actions ( cofund ) ( to g.b . ) ; department of pathology at the university of alabama at birmingham ( to e.c . ) ; computational resources are provided by the center for genomic regulation ( crg ) .
diagnosis of creutzfeldt - jakob disease ( cjd)1 is often challenging in elderly individuals because the various symptoms of this condition overlap with other conditions that are common in this population , such as alzheimer s disease or dementia with lewy bodies.2 however , we have had a patient who presented with atypical symptoms that were similar to those of anti - leucine - rich glioma inactivated 1 ( lgi1 ) encephalitis . therefore , a comprehensive understanding of cjd and anti - lgi1 encephalitis is critical , not only with regard to clinical features , but also the investigations required , which include cerebrospinal fluid ( csf ) , diffusion - weighted magnetic resonance imaging ( mri ) , and an electroencephalogram ( eeg ) . here we report a case of cjd with progressive cognitive impairment , psychiatric symptoms , and complex abnormal facio - brachio - crural movement , and then present a detailed analysis , which has not been done before . written informed consent was obtained from the patient s son and daughter for publication of this case report and the accompanying images , and ethical approval was given by the research ethics board at the first hospital of jilin university . a 65-year - old northern chinese man developed slowly progressive cognitive decline with dizziness , and was admitted to our hospital a week after symptom onset because of unstable gait and psychiatric symptoms . at admission , neurological evaluation revealed moderate cognitive disturbance of orientation and memory , along with a mini - mental state examination score of 19/30 . two days later , he developed drowsiness with facial grimacing and brief dystonic posturing of the left upper limb lasting 12 seconds . he was admitted to the intensive care unit , and found to have hyponatremia ( serum sodium 114 meq / l ) that was difficult to rectify . there was no history of tongue bite or urinary or bowel incontinence associated with these episodes . an eeg was performed to evaluate the frequently abnormal behavior of the left upper limb . his eeg video showed facial grimacing , head to the left , and external rotation of the left shoulder . the synchronized eeg was normal , with significant electromyographic artifacts , and the interictal eeg showed slow background with sharp - slow discharge in the right frontal lobe every 0.51.0 seconds ( figure 1 ) . diffusion - weighted mri showed a suspected lace sign in the occipital cortex.3 his csf study showed ten cells and normal sugar and protein levels . tests for virus antibodies , including anti - herpes simplex , anti - rubella , anti - cytomegalovirus , and anti - coxsackie virus , in the csf were negative . tests for paraneoplastic - associated antibodies in the csf , such as anti - ho , anti - yo , anti - ri , and anti - cv2 , were also negative . combining a detailed history and the eeg , mri , and csf findings , two diseases were considered , ie , anti - lgi1 encephalitis or cjd . the mri and eeg results supported a diagnosis of cjd , but the hyponatremia and clinical seizures were suggestive of anti - lgi1 encephalitis . to make the differential diagnosis , we tested further for autoimmune encephalopathy , obtaining a negative result for lgi1 antibody and a positive result for 14 - 3 - 3 brain protein . during this time therefore , we made a presumptive diagnosis of cjd according to the process together with elevated 14 - 3 - 3 protein in the csf,4,5 but without periodic paroxysmal sharp wave complexes ( pswcs ) as characteristic eeg findings.6 one week later , we performed a follow - up eeg that showed typical pswcs ( figure 2 ) . the patient was diagnosed as having cjd , but presented with progressive dementia , abnormal behavior including facial grimacing , external rotation of left shoulder , and hyponatremia , which is similar to the manifestations of anti - lgi1 encephalitis . in addition , in the early stages , his eeg did not show typical pswcs , which went against a diagnosis of cjd . our patient s main complaints were of cognitive impairment , psychiatric symptoms , and abnormal facio - brachio - crural movement with increased muscle tension in the left limb . however , soon after admission , he gradually lapsed into a drowsy state , with hyponatremia , a lace sign on mri , and focal periodic discharges on the eeg , indicating a broad differential diagnosis , such as lgi1 or cjd . the later findings of positive 14 - 3 - 3 protein in csf and typical pswcs on a second eeg were helpful in the clinical diagnosis of cjd . according to the world health organization diagnostic criteria for probable diagnosis of cjd , the presence of at least one typical feature on the eeg and 14 - 3 - 3 positivity in csf samples or at least two criteria of myoclonus , visual disturbances , cerebellar , pyramidal , or extrapyramidal findings , and akinetic mutism , together with progressive dementia , are required.7 zerr et al found that 14 - 3 - 3 protein is 94% sensitive and 84% specific for cjd.8 steinhoff et al found that periodic biphasic or triphasic synchronized sharp wave complexes had 64% sensitivity and 91% specificity for eeg examination during the intermediate or terminal phase.9 therefore , although our patient presented with atypical abnormal facio - brachio - crural movement and hyponatremia , which was suggestive of anti - lgi1 encephalitis , he met the diagnostic criteria for cjd . unfortunately , we were unable to perform an autopsy to confirm the diagnosis . however , in most cases , pswcs are absent in the initial phase , and the strict criteria for definition of pswcs in cjd suggest a duration of 100600 msec and an intercomplex interval of 5002,000 msec , which may assist differentiation.6 the eeg in cjd shows different abnormalities in different phases . during the initial phase of cjd , the eeg shows a mild slowing of background activity with diffuse 57 hz rhythms , and occasional bilateral or unilateral bursts of arrhythmic irregular wave complexes . during the intermediate phase , the eeg shows moderate slowing of background activity with diffuse 45 hz rhythms and frequent bursts of multifocal biphasic or triphasic wave complexes . finally , during the terminal phase , generalized pswcs are predominant on the eeg . in the early stages , our patient s eeg showed focal periodic wave complexes with an interval of 0.51.0 seconds , which was not helpful for a diagnosis of cjd . however , on progression of his illness , pswcs were found and led to the diagnosis of cjd , so prolonged observation may be required in such cases to detect the presence of typical pswcs on the eeg . in addition , a new clinical syndrome of faciobrachial dystonic seizures has been characterized recently , and linked with proteins associated with the voltage - gated potassium channel.10 this syndrome is specifically associated with antibodies to lgi1 protein . this anti - lgi1 encephalitis is characterized by peculiar movement disorder and hyponatremia , which may be confused with cjd . the patient described here presented with facial grimacing and brief dystonic posturing of the left upper limb lasting 12 seconds , but with a normal synchronized eeg , which excluded epileptic myoclonic seizures but was suggestive of nonepileptic faciobrachial dystonic seizures . in these circumstances , csf and eeg investigations are very important in the differential diagnosis , and include a negative lgi1 antibody , positive 14 - 3 - 3 brain protein , and pswcs on the eeg . in summary , focal pswcs on the eeg , faciobrachial dystonic seizures , and hyponatremia may occasionally be found in patients with cjd . the frequency of such findings is not known , with only few cases reported in the literature . hence clinicians should not be dissuaded from a diagnosis of cjd when faciobrachial dystonic seizures and hyponatremia are found . our case showed atypical manifestations and nonspecific eeg findings of cjd , which may be confused with anti - lgi1 encephalitis . thus , the presence of faciobrachial dystonic seizures and hyponatremia in a patient with progressive dementia and psychiatric symptoms may suggest a number of neurological diseases , but cjd needs to be considered .
in recent years , various uses of wax and fat microspheres in the pharmaceutical field have come into forefront , involving the microspheres technology . the goal of any drug delivery system is to provide a therapeutic amount of drug(s ) to the proper site in the body in order to promptly achieve and thereby to maintain the desired drug concentrations during treatment . this idealized objective can be achieved by targeting the drugs to a specific organ or tissue with the help of controlling the release rate of the drug during the transit time in gastrointestinal tract . poorly water - soluble drugs , which are lipophilic in nature mix , easily with fat and show good absorption rate . among the reported conventional methods different strategies have been developed in recent years to design different types of wax microspheres loaded with hydrophilic and lipophilic drugs using toxic solvents . the use of such solvents during formulation is of environmental concern and also faces challenge to human safety . to overcome these problems , in the present investigation , water is used to prepare wax microspheres by meltable dispersed emulsified cooling - induced solidification method . furthermore , the process was optimized to produce microspheres to give better yield with spherical geometry and predictable dissolution pattern . cetyl alcohol ( ca ) , used in the current study , has good pharmaceutical and biological properties . ca is hard , but oily to the touch , and is devoid of taste or smell , making it very useful as an ingredient in cosmetics , as a pharmaceutical excipient . it is an innocuous material generally regarded as essentially nontoxic and nonirritant , biodegradable , biocompatible , nonimmunogenic , gastroresistant , of high carrier capacity , and having controlled release of drug , low production costs , reproducible properties , and good shelf life . but the hypersensitivity reported in some cases may be due to impurities in commercial grades of ca . in pharmaceutical formulations , it is used in the preparation of suppositories , delayed release solid dosage forms , emulsions , lotions , creams , and ointment . regulatory status : it is included in the fda as inactive ingredient and in nonparenteral medicines licensed in the uk . i m is a nonsteroidal , anti - inflammatory agent with antipyretic , analgesic properties and is an indole derivative designated chemically as 1-(p - chlorobenzoyl)-5-methoxy-2-methyl-1h - indole-3-acetic acid . it is lipid - soluble , practically insoluble in water and sparingly soluble in alcohol . i m has a pka of 4.5 and is stable in neutral or slightly acidic media and decomposes in strong alkali . the suspension has a ph of 4.05.0 and it has a melting point between 155c and 161c and has molecular weight of 357.8 . i m is widely used in the treatment of active stages of moderate - to - severe stages of rheumatoid arthritis . due to its narrow therapeutic index , the frequency of adverse effects is dosing related . considering the long therapeutic regimen of osteoarthritis therapy , the administration of i m may induce adverse side effects on gastrointestinal tract ( git ) as well as central nervous system ( cns ) , renal and cardiac systems . the occurrence of these adverse effects can be reduced by the use of controlled release formulations . oral conventional dosage forms are administered 2 - 3 times a day to maintain adequate and effective therapeutic concentration in blood ; however , it fails to protect the patients against morning stiffness . development of controlled release formulation of i m has several advantages over the other conventional dosage forms , such as reduction in occurrence of high initial peak plasma concentrations , protection against morning stiffness , prolonged duration of action , improved bioavailability , patient compliance and reduction in adverse effects . the side effects could be lowered by controlling the drug release and by adjusting the absorption rate . previous experimental results have demonstrated that the waxes are biocompatible , nonimmunogenic material used for the entrapment of drug and its controlled drug release in the intestinal tract . delivering the drug in the intestinal environment from fatty ca microspheres could be manipulated by suitable coating techniques . the chief characteristics of enteric coating are their impermeability to gastric juice , but susceptibility to intestinal juice [ 13 , 14 ] . desired plasma levels can be achieved without the risk of side effects using once - a - day dose of controlled release preparation . these findings suggested that the kinetic control is an effective route for preventing the toxicity of i m . the aim of the present study was to formulate , characterize , and study the in vitro release of i m from microspheres and compare with commercially available oral formulation microcid sr ( 75 mg capsule ) furthermore , to investigate the pharmacokinetics and bioavailability of two different oral i m formulation ( optimized microsphere formulation and microcid sr 75 mg capsule ) following a single dosing in healthy albino sheeps in order to prove the bioequivalence between the preparations . indomethacin ( i m ) and mefanamic acid ( ma ) , the internal standard , were kindly donated by micro labs ( bangalore , india ) . cetyl alcohol ( ca - melting point 4951c ) , tween 80 , and all other chemicals and solvents used were of analytical grade and purchased from ranbaxy fine chemicals ( new delhi , india ) . commercially available oral capsule formulation ( microcid sr 75 mg , micro labs ltd . , india ) was used for the present study . 9 gm of ca was melted in a china dish kept on water bath . to the melted wax mixture , i m ( 3 gm ) previously passed through sieve no . 100 was dispersed in melted wax mass and stirred to obtain a homogeneous mixture . the resultant mixture was then poured into 150 ml of phthalate buffer solution ( ph 4.5 ) , previously heated to a temperature higher than melting point of ca ( > 50c ) . the surfactant , tween 80 ( 0.3% w / w ) , was added to the above mixture and stirred mechanically at 900 rpm using a stirrer ( rq-127a , remi , india ) . the mixture was stirred continuously above the melting point of ca at 900 rpm for 5 min . the temperature of the reaction mixture was cooled rapidly and brought down to 10c by the addition of cold water . the resultant solid spheres were collected by filtration and washed with water to remove surfactant residue . air - drying was carried out at room temperature for 48 h to give discrete , solid , and free flowing microspheres . a total of five formulations were prepared by varying the ca to drug ratios ( table 1 ) . tap density of the prepared ca microspheres was determined using tap density tester , and percentage carr 's index ( % i ) was calculated using the formula : ( 1)carr 's index(% i ) = [ ( tapped densitybulk density)tapped density]100 . angle of repose ( h ) was assessed to know the flow ability of ca microspheres , by a fixed funnel method : ( 2)tan()=heightradius . scanning electron microscope ( sem ) photomicrographs were recorded using joel - lv-5600 sem , usa . the obtained images were processed by image analysis software to characterize each individual microsphere by mean feret diameter ( fd ) ( average of 180 caliper measurements with an angle of rotation of 1 ) , aspect ratio ( ar ) ( ratio of longest feret diameter and its longest perpendicular diameter ) , and two - dimensional shape factor ( er ) by the following equation : ( 3)er=2rpm ( bl)2 , where r is the radius , pm is the perimeter , l is length ( longest feret diameter ) , and b is width ( longest perpendicular diameter to the longest feret diameter ) of the microspheres . all dynamic dsc studies were carried out on dupont thermal analyzer with 2010 dsc module . calorimetric measurements were made with the help of an empty cell ( high purity alpha alumina discs of dupont company ) as the reference . ftir spectra of pure drug , empty microspheres , and drug - loaded microspheres were obtained using kbr pellet method ( applying 6000 kg / cm ) . spectral measurements were obtained by powder diffuse reflectance on an ftir spectrophotometer ( shimadzu , ftir 8400s , japan ) in the wave number region of 4004000 cm to study drug excipient interactions . drug was extracted from ca microspheres using methanol , filtered and analyzed for drug content after suitable dilution . estimation of i m was accomplished by uv / visible spectroscopy ( shimadzu1601 , japan ) at 319 nm after sufficient dilution with ph 7.2 phosphate buffer . usp xxii dissolution apparatus type ii was employed to study percentage of drug release from various formulations prepared . accurately weighed quantities of drug - loaded microspheres ( im - equivalent to 75 mg ) of each batch were taken in 900 ml dissolution medium ( 2 h in ph 1.2 hydrochloric acid buffer and 6 h in ph 7.2 phosphate buffer ) and stirred at 100 rpm by maintaining at a temperature of 37 0.5c . the drug concentrations were determined by withdrawing the 10 ml of aliquots using guarded sample collectors periodically at an interval of 30 min for first 4 h and at 60 min interval for the next 4 h. release studies were carried out in triplicate . the optimized formulation was subjected for stability studies and stored in glass bottles at 25c/60% rh ( relative humidity ) , 30c/65% rh , and 40c/75% rh for a period of 90 days . 100 mg of microspheres from each batch of formulations was taken at the end of 30th , 60th , and 90th days and was subjected for drug content studies . in vivo release studies the sheep ages were in the range 57 years and their body weight ranged between 25 and 28 kg . a written approval was obtained from the institutional ethical committee of jss college of pharmacy , mysore , india . detailed verbal and written information on the study was provided to the veterinary surgeon , central animal facility , jss medical college hospital , and permission was obtained . the study was conducted as an open , randomized complete crossover design in which a single 75 mg dose of i m ( microcid sr 75 mg capsule and formulation f3 ) was administered to fasted , healthy adult males and females on two different occasions , separated by a wash - out period of 2 weeks between dosing interval . the content uniformity of marketed product and optimized formulation has been estimated as per usp specification . the contents of 5 units of microcid sr 75 mg capsule and formulation f3 were individually combined and weighed to the average weight of each unit . all the animals were shifted to the clinical trial laboratory from animal house at 6.00 am after overnight fast of 10 h. after shaving near the neck , an 18 gauge ( 1.3 45 mm , 96 ml / min ) cannula was inserted into a jugular vein and kept with heparinised saline lock for ensuing 24 h blood sampling . test medications ( marketed product and optimized formulation ) were administered to the sheeps , fed with banana and 200 ml water . light food was provided at 3rd h followed by two standard meals at 7th and 11th h following drug administration . blood samples ( 5 ml ) were collected at 0 h ( pre dose interval ) and at 0.5 , 1 , 1.5 , 2 , 2.5 , 3 , 3.5 , 4 , 5 , 6 , 7 , 8 , 12 , 16 , 20 , and 24 h postdose intervals . blood samples were centrifuged ( eltek tc 4100 d centrifuge , elektroshaft , india ) at 1500 rpm for 10 min . any other type of food was not permitted after 12 h administration of test medication . all subjects remained ambulatory and strenuous physical activity was prohibited during the first 12 h of blood sampling . plasma concentration of drug from the collected samples was quantified by modified hplc method . internal standard mefanamic acid ( ma ) ( 100 l ) and citrate buffer ( ph 3.0 , 500 l ) were added to 10 ml screw capped glass tubes containing 500 l of spiked plasma . the tubes were extracted gently with 7 ml of petroleum ether : dichloromethane ( 50 : 50 ) for 5 min on a rotary shaker and centrifuged at 900 rpm for 5 min . the organic phase was transferred to a watch glass and evaporated to dryness at 40c . the residue was resuspended in 100 l of mobile phase and 25 l was injected to the column . quantification was achieved by the measurement of the peak area ratio of the i m to the internal standard ( mefanamic acid ) . the limit of detection of i m in plasma was 100 ng / ml ( 500 l of plasma injected ) . the i m concentrations in plasma were assayed using a fully validated high performance liquid chromatography with ultraviolet detection ( hplc - uv ) method , with respect to adequate sensitivity , specificity , linearity , recovery , accuracy , and precision ( both within and between days ) . the hplc system consisted of hplc - shimadzu ( tokyo , japan ) lc-6a model , fitted with a -bondapack c18 ( 4.6 250 mm ) column of particle size 5 m ( supelco , bellefonte , pa ) . the flow rate was maintained at 1 ml / min , and the drug concentration was detected using a uv / visible detector ( spd-6av ) . the mobile phase consisted of 80% methanol and 0.02 m sodium acetate buffer ( 60 : 40 v / v ) . the stability of the samples under frozen conditions , at room temperature , and during freeze - thaw cycle was also determined . the peak plasma concentration ( cmax ) and time needed to reach peak plasma concentration ( tmax ) were computed directly from plasma level profiles as a measure of the rate of absorption of the drug from each product . the elimination rate constant ( kel ) was calculated from the terminal elimination phase of logarithm of drug concentrations against time curve by the method of least square regression analysis . the biological halflife ( t1/2 ) was determined by the relation : ( 4)t1/2 = 0.693k . the extent of absorption for the drug ( microcid sr 75 mg capsule and formulation f3 ) in different subjects from the area under the plasma concentration time curve from zero to 24 h ( auc024 ) were calculated by the trapezoidal rule method . area under the plasma concentration time curve from zero to infinity ( auc0 ) was calculated using the formula ; ( 5)auc0=auc0t+c24k , where c24 is drug concentrations in plasma at 24 h. the drug plasma concentration and pharmacokinetic parameters were analyzed by paired t - test and analysis of variance ( anova ) at 95% confidence limit . difference between two related means was considered statistically significant when their p values were equal to or less than 0.05 . evidence has [ 1012 ] shown in the recent years that wax / fatty materials have the physical properties and behavior suitable to prepare gastroresistant , biocompatible , biodegradable microspheres to release the entrapped drug in the intestinal lumen [ 14 , 15 , 18 ] . in the present study , a modified novel meltable dispersion emulsified cooling - induced solidification method was employed using inert fat ( ca ) and nontoxic solvents to entrap the drug . in the present study , various parameters were studied such as drug and ca ratio , stirring speed and time , amount of surfactant added , volume of the aqueous phase used , effect of ph on drug entrapment , temperature of the aqueous phase , and rapid cooling studies . therefore the influence of the above parameters was highlighted . when the ph value of the external aqueous phase was acidic , the solubility of the drug was reduced and the encapsulated amount of the drug increased . the maximum drug load was obtained at ph 4.2 ( phthalate buffer ) . as the ph increased from 4.2 to 7.0 , the percent of i m loading was reduced from 21.52 to 3.92% . in the present study , it was found that 150 ml of aqueous phase is suitable for producing the spherical microspheres . as the volume of external phase increased , the yield was reduced and the resultant microspheres were irregularly shaped . when the volume of the aqueous phase was less than 150 ml , the resultant microspheres were highly aggregated in nature and highly impossible to distinguish as an individual microsphere . in order to avoid the formation of irregularly shaped larger particles , in the present method , incorporation of drug into ca microspheres required the addition of tween 80 as a surfactant , at an optimum concentration to reduce the interfacial tension between the hydrophobic material and external aqueous phase . an attempt was made to incorporate drug in the ca microspheres without the addition of a surfactant . but the process has a failed , as it resulted in an aggregate cake - like mass during the solidification of ca . this may be due to repulsion resulting from high interfacial tension between the hydrophobic ca material and external aqueous phase . it was found that tween 80 having an hlb value of 15 was suitable to increase substantially dispersion of ca in external aqueous phase and promote drug incorporation in the ca microspheres . to obtain an optimal surfactant concentration , various concentrations ranging from 0.1 to 0.6% ( w / w ) of the total formulation discrete microspheres with good flow properties using an optimum concentration of surfactant 0.3% w / w ( tween 80 ) were used . concentrations of tween 80 ranging from 0.1 to 0.2% w / w failed to produce reproducible microspheres . the resultant ca microspheres were composed of irregular masses , which were not possible to distinguish as individual microspheres [ 1012 ] . temperature of the aqueous phase was maintained at 5c higher than the melting point of the ca in the corresponding formulations . from sem studies it was observed that the resultant microspheres were free from surface irregularities , except some wrinkles . it was also observed that when the temperature of the aqueous phase was less than 5c than the melting point of the ca , big flakes were produced . in the present study , to produce the spherical discrete microspheres , an optimum drug to ca ratio of 1 : 3 w / w was used ( table 1 ) . it was found that higher amount of drug to ca ratio ( 2 : 3 ) produces aggregate masses during the cooling process . sem photographs also indicated the presence of the crystals on the surface of the microspheres . hence an optimum 1 : 3 ratio was used to prepare microspheres [ 10 , 12 , 14 ] . it was observed that the average size of the microspheres ranged between 12 to 32 m as presented in table 2 . the important factor that influences a stirring speed of 900 rpm and stirring time of 3 min were used to obtain reproducible microspheres . it was observed that with the increase in the stirring speed from 900 to 1100 rpm there was a decrease in the average size of the spheres and recovery yield of the microspheres . it is due to small - sized wax microspheres , which were lost during successive washings . when the stirring speed was lower than 900 rpm , larger pellets were formed . it was also found that an increase in stirring time , from 2 to 4 min ( at a stirring speed of 900 rpm ) , there was a decrease in the recovery yield of microspheres . when the stirring time was lower than 2 min , it was observed that some amount of melted material adhered to the sides of the beaker during the cooling process resulting in lower recovery of yield . microparticulate drug delivery systems are formulated as single unit dosage forms in the form of capsule or tablet . the values of angle of repose were well within the range , indicating reasonable good flow potential for the microspheres . the tapped density values ranged between 0.42 g / cm and 0.51 g / cm . the results of % compressibility index ranged from 10.19% to 13.98% , suggests good flow characteristics of the microspheres ( table 2 ) . sem photographs showed that the ca microspheres were spherical in nature and had a smooth surface with inward dents and shrinkage , which is due to the collapse of the wall of the microspheres ( figure 1 ) . microphotography reveals the absence of crystals of the drug on the surface of microsphere , indicating uniform distribution of the drug within the microspheres . the rate of solvent removal from the microspheres exerts an influence on the morphology of the final product . interestingly , for microspheres dried at room temperature for 24 h , the sphericity values were nearer to the value 1 , whereas for microspheres cured for 24 h at 4c , the obtained sphericity values ranged between 1.131.19 . the removal of residual moisture content from microspheres during curing exerts an influence on the morphology of the final product . dsc studies were performed on pure drug , empty , and drug - loaded microspheres , have shown sharp endothermic peaks . it was observed that presence of endothermic peak of the drug at 161.4c in the drug - loaded ca microspheres indicates that the drug is uniformly distributed in the wall of the microspheres . the characteristic ir absorption peaks of i m comparing the ir spectra at 3413 ( aromatic c h stretching ) , 2618 ( carboxylic acid stretching ) , 1695 ( c = o stretching ) , 1600 ( c = c stretching ) , 1452 ( o ch3 deformation ) , and 1236 cm ( o h deformation ) were not altered after successful encapsulation of drug , indicating no chemical interactions between the drug and ca used . a comparison and an interpretation of this region in our spectra agree with their conclusions [ 14 , 18 ] . the percent of drug loading in the formulations was found to be in the range of 18.6721.52% . it was low in the formulation f4 ( 19.46 ) and more in f3 ( 23.52 ) . the encapsulation efficiency ( % ) was found to be more in formulation f3 ( 94.34% ) as compared to f1 ( 89.54% ) , f2 ( 89.63% ) , f5 ( 88.98% ) , and f4 ( 87.67% ) . from this result , it can be concluded that the formulation f3 had more encapsulation efficiency . from the release studies depicted in figure 4 , it was observed that there is no significant release of drug at gastric ph from fat microspheres . at the end of 8th h , in vitro drug release from f3 ( 96.32% ) was slower than microcid sr ( 98.98% ) in the intestinal environment . drug was released in a biphasic manner consisting of initial fast release followed by a slow release in intestinal ph from the ca microspheres [ 2 , 10 , 12 , 18 ] . the decreased in vitro drug release from ca microspheres might be due to more hydrophobicity and influence of molecular weight of ca . in vitro drug release was considerably retarded from the ca microspheres when compared to microcid sr . the rate of drug release followed first order release kinetics and numerical data fitted into peppas model showed that the mechanism of drug release from ca microspheres was fickian diffusion ( f1 - 0,412 , f2 - 0.415 , f3 - 0.398 , f4 - 0.421 , f5 - 0.431 , and microcid sr-0.476 ) . after an initial burst effect , the subsequent release of drug from microspheres was slow . microcid sr 75 mg capsule and formulation f3 were subjected for stability studies for 90 days . it was observed that in vitro drug release from microcid sr 75 mg capsule and formulation f3 at the end of 90 days ( 8th h ) were 99.43 and 95.09% , respectively . however , no significant change in in vitro drug release from both the products was noticed after the study period , indicating good stability for the prepared formulation . drug content uniformity for microcid sr 75 mg capsule and formulation f3 was found to be 74.83 mg and 74.80 mg , respectively . the percents of drug content uniformity of microcid sr 75 mg capsule and formulation f3 are 99.76 and 99.49% , respectively . hence , the percent of drug content uniformity in both products were well within the limits as per united state pharmacopoeia and national formulary specification . recovery of the i m from the plasma was calculated by comparison of peak height ratio after direct injection of i m or ma to the peak height of the same concentrations of the analytes extracted from plasma . in both cases the extraction solvent selected in this investigation gave higher recoveries and clean extracts than other solvents tested . plasma spiked with 500 ng / ml of i m and 1000 ng / ml of ma the retention times for i m and ma were 5.52 and 8.23 min , respectively . sensitivity of hplc assay qualitative confirmation of the purity of i m and ma peaks were obtained ( table 3 ) . the limit of quantification was 50 ng / ml of i m in plasma when 0.5 ml plasma was placed . the obtained mean correlation coefficients for the standard curves ( n = 6 ) was 0.998 . assay was shown to be sensitive , capable of reliably detecting i m concentrations in plasma as low as 50 ng / ml . interferences from endogenous compounds were overcome by using an acidic buffer ( citrate buffer ph 3.0 ) to alter the ph of the aqueous phase before extraction . to prevent the substantial interferences from endogenous compounds , strong acid - like hcl was employed . the mean plasma concentration as a function of time is shown in figure 5 and the calculated pharmacokinetic parameters of microcid sr and f3 formulations are given in table 4 . after oral administration of both products , more mean cmax value was observed for microcid sr 75 mg capsule ( 2134 29.6 ng / ml ) than formulation f3 ( 1989 20.30 ng / ml ) . however , the difference in the cmax values obtained for microcid sr 75 mg capsule and formulation f3 was statistically insignificant . mean plasma concentrations of i m for both products in all experimental conditions were within the therapeutic concentration range ( 3003000 ng / ml ) . the cmax values for both products do not exceed the above limit in all animals . it was observed the plasma concentration of i m falls below detection limit ( 50 ng / ml ) after 24 h in all animals following administration of either product . on the basis of the therapeutic concentration range of i m , it could be concluded that the therapeutic effects of both formulations would be probably be maintained for about 12 h following a single dose administration . thus it could be predicted that the two controlled release formulations included in this study are associated with a similar onset of therapeutic response following a single dose administration under fasting conditions . furthermore , it could be predicted that both controlled release formulations in this study are associated with a similar onset of therapeutic response , following a single dose administration under fasting conditions . the time taken to reach peak plasma concentration tmax of i m was little higher in case of microcid sr compared to formulation f3 , but no statistical significance differences between two products are observed ( table 4 ) . the calculated mean t1/2 values for microcid sr and formulation f3 were 2.62 0.02 h and 2.67 0.02 h , respectively . there was not much difference in the t1/2 for i m between both formulations , and no statistical significance differences were observed between both products . mean rate of absorption ka for microcid sr was 0.3934 0.002 h and for formulation f30.3856 0.002 h and mean elimination rate constants kel for microcid sr and for formulation f3 were 0.2843 0.004 h and 0.2678 0.004 h , respectively . however a small difference between both products related to cmax , tmax , t1/2 and reduced fluctuations ( peak to trough ratios ) of the plasma concentrations . all these effects probably may be due to the dissolution rate limited drug release and hence absorption . from the study it was observed that reduced fluctuations combined with the elevated mean plasma concentration from both products offer advantage in protecting patients against morning stiffness . the systematic availability of i m can be determined by comparison of the area under the plasma concentration ( auc ) versus time curves . the mean auc024 values for microcid sr and formulation f3 were 12478 104.21 ng / ml h and 12145 87.32 ng / ml h , respectively . slow in vitro release of i m from the products microcid sr and f3 formulations may be responsible for the decreased auc values when compared to the reported conventional dosage forms . the average value of the individual and mean auc024 ratio at 95% confidence limit ( 0.81.25 ) was within acceptable limits for bioequivalent products . in order to obtain in vitro - in vivo correlation , drug absorption profiles were compared for microcid sr and formulation f3 using the cumulative fraction of the drug absorbed in vivo against cumulative fraction of the drug dissolved in vitro up to 8 h. from the study it was noticed that both products showed an adequate correlation . currently accepted criteria in the us for bioequivalence for most dosage forms require that the mean pharmacokinetic parameters of the test dosage forms should be within 80120% of the reference dosage form using 90% confidence interval . pharmacokinetic parameters clearly indicate that the parameters of f3 are in good agreement with microcid sr . the observed mean auc0values for microcid sr and formulation f3 were 12632 132.12 ng / ml.h and 12452 96.32 ng / ml.h which does not show any significant statistical difference between the products . on the basis of fda recommendation , the two products , microcid sr and formulation f3 , no untoward effects were observed by any of the subjects after the administration of either product . thus , the two formulations can be considered similar , because all the subjects are very well tolerated . these observations clearly indicate the absence of high peak plasma concentrations ( > 5000 ng / ml ) , which are very often associated with adverse effects due to drug accumulation , because of the accumulation effect . the products microcid sr and formulation f3 investigated in the present study were found to be bioequivalent . the objective of the study was to prepare and evaluate wax microspheres loaded with i m by optimized meltable dispersion emulsified cooling - induced solidification method for controlled release . the method employed was simple , rapid , and economical and does not imply the use of toxic organic solvents . the results of the drug entrapment and micromeritic properties exhibited fairly good spherical nature as evidenced by sem photograph . the compatible state of the drug - loaded wax microspheres was evaluated by ftir and dsc . both formulations were found to be bioequivalent and both formulations showed an adequate correlation between cumulative fractions dissolved in vitro and cumulative fractions absorbed in vivo . optimized formulation f3 and marketed product microcid sr showed similarity in drug release profiles and in vivo bioequivalent behavior . from the present work , it can be concluded that the prepared wax microspheres demonstrate the potential use of cetyl alcohol for the development of controlled drug delivery systems for water insoluble or lipophilic drug .
the most common location is the mesentery , followed by the omentum , mesocolon and retroperitoneum1 - 3 ) . although approximately 60% of these patients are younger than 5 years , a significant number of abdominal lymphangiomas do not manifest until adulthood1 ) . regardless of the diagnostic modalities used , these facts contribute to the difficulty of diagnosing lymphangiomas at the preoperative stage in adult patients . lymphangioma of the gallbladder is quite rare with only a few cases being reported in the literature . we recently encountered an interesting case of a cystic lymphangioma of the gallbladder , which was discovered during the work - up for the patient 's biliary pain and because of her abnormal liver test , and this is an unusual presentation for this type of lesion . a 34-year - old woman was referred to our hospital with complains of abdominal pain that suddenly developed in the right upper quadrant and also a febrile sensation she felt for the previous 2 days . the patient 's body temperature , blood pressure , pulse rate and respiratory rate were 37.2 , 120/80 mmhg , 80/minute and 20/minute , respectively . a physical examination demonstrated tenderness and a soft palpable mass in the right upper quadrant of the abdomen . the laboratory data was as follows : white blood cell count : 13,600/mm ( 82.1% neutrophils , 10.6% lymphocytes and 6.4% monocytes ) , hemoglobin : 11.6 g / dl , total bilirubin : 1.83 mg / dl ( normal range : 0.1 to 1.0 mg / dl ) , direct bilirubin : 1.15 mg / dl ( normal range : 0.1 to 0.2 mg / dl ) , aspartate aminotransferase : 336 iu / l ( normal range : < 40 iu / l ) , alanine aminotransferase : 548 iu / l ( normal range : < 40 iu / l ) , gamma - glutamyl transpeptidase : 112 iu / l ( normal range : < 50 iu / l ) , alkaline phosphatase : 145 iu / l ( normal range : 39 to 117 iu / l ) , and amylase : 38 u / l ( normal range : 25 to 125 iu / l ) . the viral markers ( hbsag , anti - hbs antibody and anti - hcv antibody ) were negative . the tumor markers ( cea , ca19 - 9 and ca125 ) were also within their normal ranges . abdominal ultrasonography showed a multi - septated cystic mass in the gallbladder fossa and an adjacent compressed gallbladder ( figure 1 ) . computed tomography ( ct ) of the abdomen also revealed a cystic mass measuring 64.5 cm in size in the gallbladder fossa ; further , this was also seen on abdominal ultrasonography ( figure 2 ) . endoscopic retrograde cholangiography was performed to determine if there was any communication between the lesion and the biliary tract , as well as to evaluate for the presence of biliary pathology that could lead to her biliary pain and the abnormal liver test , e.i . , on cholangiography , there was no communication between the bile tract and the lesion and no abnormal findings of the gallbladder , with the exception of a laterally compressed gallbladder ( figure 3 ) . a small amount of sludge was released from the bile duct after the endoscopic sphincterotomy . according to the above imaging studies , the patient underwent laparoscopic cholecystectomy . a laparoscopic examination revealed the presence of a cystic mass , measuring 532 cm , attached to the gallbladder wall . the histological examination showed that the cystic lesion originated in the subserosa of the gallbladder and it consisted of variable sized clear spaces that were lined by flat endothelium with some lymphoid tissue in the wall ( figure 4 ) . immunohistochemical staining showed that the cells lining the cystic space tested positive for factor viii - related antigen and there was a negative reaction for cytokeratin . following the surgical resection , the patient recovered well postoperatively and she showed no evidence of recurrence at 6 months follow - up . an intraabdominal lymphangioma is a rare benign neoplasm that accounts for less than 5% of all lymphangiomas2 ) . lymphangiomas can arise from the mesentery , retroperitoneum , colon , small intestine , pancreas , omentum , mesocolon and gallbladder1 - 8 ) . the clinical presentations of intraabdominal lymphangioma are either asymptomatic or this tumor is accompanied by nausea , vomiting and abdominal fullness . the symptoms are due to either pressure on the adjacent structures by the enlarging mass , or to such complications as hemorrhage , infection , perforation , torsion and rupture . however , there are no clinical features that can be used to differentiate intraabdominal lymphangioma from the other intraabdominal masses2 - 4 ) . the treatment of choice is a complete excision because incomplete removal can lead to a recurrence2 ) . to the best of our knowledge , there have been only six reported cases of lymphangiomas arising from the gallbladder4 - 8 ) . only a physical examination demonstrated the presence of a mass in the right upper quadrant of the abdomen . in one case , the patient presented with a six month history of chronic right upper quadrant pain radiating to the back . there was no mention of symptoms in the remaining two cases . in the previous cases unlike the previously reported cases , our patient had acute abdominal pain and a tender mobile mass in the right upper abdominal quadrant , but she also showed an abnormal liver test that indicated an obstructive pattern . accordingly , it is believed that the abnormal liver test was due to this small amount of sludge , which was induced by the gallbladder dysfunction resulting from the compression of the gallbladder by the mass . unfortunately , the gallbladder sludge was not demonstrated on the imaging studies because all the diagnostic procedures used in this study focused only on the cystic mass and not the gallbladder . abdominal ultrasonography , ct and mri imaging have all been used to diagnose abdominal lymphangioma6 - 10 ) . it is helpful to image the internal structure of the mass , particularly the internal septa . if the lesion is accompanied by bleeding or infections , then the ultrasonography and ct images may show a solid component that is difficult to distinguish from other lesions such as a cystadenoma or a cystadenocarcinoma . mri shows multiloculated cystic lesions with different signal intensities on the t1-and t2-weighted images and these different intensities are due to the different fat and fluid ratios within each cyst . in addition , the septa are enhanced on the delayed phase images , whereas the cystic portions are not . based on the histological findings , lymphangiomas are classified as simple , cavernous or cystic2 ) . the histological characteristics of a cystic lymphangioma include the following : 1 ) flat endothelial lining of the cyst rather than the cuboidal or columnar epithelium ; 2 ) the presence of lymphoid tissue in the cyst wall ; and 3 ) the presence of smooth muscle in the cyst wall1 , 2 ) . the histological findings of our case were consistent with these features . in addition , the cystic mass tested positive to factor viii - related antigen and it was negative for cytokeratin . a lymphangioma is characterized by an endothelial lining that stains positively for factor viii - related antigen or cd31 . the mesothelioma stains positively for cytokeratin and epithelial membrane antigen , and this is in contrast to lymphagioma11 ) . we report here on the first case of a cystic lymphangioma of the gallbladder that was associated with biliary sludge . the cystic lymphangioma was diagnosed during the evaluation of the patient 's sudden abdominal pain and tender movable mass in her right upper quadrant , and her abnormal liver test .
creating an extraocular aqueous humour filtration is still the most popular surgical strategy in glaucoma worldwide . however , because of the well known bleb - related problems , a quest for new bleb - free procedures has been pursued for several years . for example , canaloplasty ( cp ) and the trabeculocanalicular devices implantable through a minimally invasive approach ( the so called migs ) are two surgical strategies not ending in external filtration . cp consists of an ab - externo dilation of schlemm 's canal ( sc ) ( via a transconjunctival and transscleral approach ) obtained by intracanalicular injection of high molecular weight ( hmw ) viscoelastic and placement of a permanent intracanalicular tension suture . migs devices are placed in the anterior chamber ( ac ) angle under gonioscopic view and via a clear cornea incision , sparing then the conjunctiva for later interventions . irvine , ca ) is a migs device made of nitinol , ( a nickel and titanium alloy widely used in ophthalmic and other medical applications ) , currently approved for intraocular use in europe . this device is an 8 mm long crescent - shaped open structure , curved to match the shape of sc . once implanted , the microstent bypasses the trabecular meshwork and dilates sc over 3 clock hours to provide direct aqueous access from the ac to multiple collector channels [ 4 , 5 ] . to date the large majority of these reports showed that , in the short - to - mid term , both procedures are effective in achieving significant reduction of intraocular pressure ( iop ) , with a better efficacy profile for filtration surgery and a better safety profile for cp [ 69 ] . the hereby presented study was aimed to compare the 2-year clinical outcome of cp versus a bleb - free migs ( i.e. , the hydrus microstent ( hm ) ) , within the frame of a retrospective comparative study . this was a retrospective , nonrandomized comparative case series ( with the approval of the local ethics committee requiring no trial registration number ) . medical records of consecutive patients , where either cp or hm implantation was uneventfully performed in one eye from january 2011 to january 2012 at the university hospital of parma ( italy ) , were reviewed . all surgeries were performed by two surgeons ( stefano a. gandolfi and nicola ungaro ) , and all study subjects signed a dedicated informed consent prior to surgery . the series included subjects under local treatment for primary or secondary ( e.g. , pseudoexfoliative and pigmentary ) open - angle glaucoma , referred to as the glaucoma service of our institution parma for uncontrolled iop . the eyes were addressed to either one of the two procedures since , according to the egs guidelines , the estimated postsurgery target iop was arbitrarily set in the mid - to - high teens range by the two surgeons ( stefano a. gandolfi and nicola ungaro ) . the following data were collected from a total of 45 patients ( 45 eyes , 24 cp , and 21 hm ) with a minimum follow - up of two years : ( a ) demographics , ( b ) iop , ( c ) best corrected visual acuity ( bcva , tested by logarithmic chart at 4 metres ) , ( d ) visual field mean defect ( md , humphrey 24 - 2 sita - standard program , carl zeiss meditec inc . , dublin , ca ) , ( e ) the number and type of hypotensive medications , ( f ) and the need for further glaucoma surgery . iop was measured using goldmann applanation tonometer and following the standard operating procedures ( sop ) of the european vision clinical research network ( evicr.net ) which certified our institution ( certificate number : ecr37/2014 ) , namely , ( a ) patients in sitting position at the slit lamp ; ( b ) fluorescein staining with a standard fluorescein paper strip ; ( c ) two rapidly consecutive readings , averaged with 2 mmhg iop difference , with a third reading being performed when the difference was > 2 mmhg . failure if , two years after surgery , they needed no hypotensive medications , some hypotensive medications , or further glaucoma surgery to attain the target iop , respectively . cp was performed according to the standard fashion described in previous reports [ 11 , 12 ] . briefly , after conjunctival dissection at the 12 o'clock limbus , a 5 5 mm partial thickness ( 50% ) scleral flap was dissected followed by a 4 4 mm inner scleral flap at 95% depth . the dissection was then carried into the clear cornea to create a 0.3 mm descemet 's window ; the inner scleral flap was then removed and the inner wall of the canal was peeled off together with trabecular meshwork , until percolation of aqueous humour was observed . a microcatheter ( itrack-250a , iscience interventional , inc . , menlo park , ca ) was then inserted 360 in the canal ; a 10 - 0 prolene suture was tied to the tip of the catheter , and the catheter was retracted backward and , in order to achieve viscodilation of the canal , sodium hyaluronate 1.4% ( healon gv , advanced medical optics , inc . , santa ana , ca ) was simultaneously injected . the 10 - 0 prolene suture was finally tightly tied in a loop with a slip knot , to obtain an inward traction . the scleral flap was secured back to the sclera with 10 - 0 nylon sutures to create a water - tight closure . hm implantation was performed as follows : after a peribulbar injection of 5 ml of lidocaine , the patients were placed under the microscope and the head tilted to allow a clear view of the angle structures with a gonioprism . a 1.21.5 mm clear cornea incision was properly made to access the targeted site for microstent placement . the bevelled tip of the cannula was used to perforate the trabecular meshwork , and the microstent was implanted into schlemm 's canal by advancing the tracking wheel with the index finger , leaving 1 - 2 mm ( the inlet segment ) remaining in the ac . in one of the selected cases , the microstent had to be retracted and reinserted in a different location . upon confirmation of position in the canal , the delivery system was withdrawn and viscoelastic was removed ; the ac was inflated with balanced salt solution to achieve normal iop . statistical data were processed with the spss package ( spss inc . released 2007 ; spss for windows , version 16.0 . student 's t - test was for mean confrontation between groups , chi - squared test was for double and triple enter contingency tables . for 2 2 contingency tables , the normal distribution of the analyzed data was stated by verifying for each parameter that means were almost equal to medians and asymmetric within 2 . twenty - four eyes of 24 patients ( 16 men , age range : 3466 years ) who underwent cp and twenty - one eyes of 21 patients ( 15 men , age range : 3769 years ) who underwent hm implantation were included in the study . we considered all the patients who successfully completed surgery and the 2-year follow - up ( therefore no loss to follow - up ) . no significant difference was found between the two groups with respect to demographics ( age : p = 0.31 ; gender : p = 0.59 ; race : all caucasians ; right versus left eye p = 0.67 ) . the diagnosis was as follows : n = 28 primary open - angle glaucoma ( poag ; 16 cp and 12 hm ) ; n = 15 pseudoexfoliation syndrome ( pex : 8 cp and 7 hm ) ; and n = 2 pigmentary glaucoma ( pg : 2 hm ) . at preoperative evaluation ( baseline ) , no significant differences in the number of ongoing hypotensive active substances ( p = 0.23 ) and in the number of eyes previously treated with argon laser trabeculoplasty / selective laser trabeculoplasty ( ast / slt ) were detected between groups ( p = 0.34 ) . eye parameters at baseline and 2 years after surgery are detailed in table 1 ( mean sd ) . refers to the iop ( on current hypotensive therapy ) measured before surgery at the time of completion of the inpatient 's record . iop final refers to the iop values measured 2 years after surgery ( with a 30-day time window ) , with or without medications . no intergroup difference was detected in either efficacy ( iop ) or safety ( i.e. , bcva and md ) outcomes . conversely , an intragroup analysis showed that iop significantly diminished in both groups upon surgery ( p < 0.001 ) . the iop changes of individual eyes in each treatment group are shown in a scatterplot ( figure 1 ) . table 2 shows the rate of each clinical outcome at the end of the follow - up . the distribution of the clinical success ( complete or qualified ) was not significantly different between the two groups ( pearson chi - square , p = 0.52 ; likely ratio , p = 0.51 ) . also the event failure ( i.e. , the need for further glaucoma surgery ) was similar in the two groups either in terms of the number of procedures or the number of temporal latencies . namely , two cases in the hm group were performed after 12 and 18 months of follow - up ; two cases in the cp groups 12 and 13 months postoperatively . concerning the number of hypotensive active substances administered at the end of the follow - up , the mean values were in the cp group 0.7 differences referred to the intensity of the regimen ( i.e. , none , 1 or more active substances ) are shown in figure 2 ; no statistical difference was observed ( pearson chi - square = likely ratio ; p = 0.74 ) . the final distribution among the subgroups of clinical outcome of the eyes previously treated by ast / slt is displayed in table 3 . a laser treatment was paralleled by a lower rate of complete success in the cp group versus the hm group with borderline significance ( fisher exact test , p = 0.04 ) . as far as complications are concerned , a transient hyphema proved to be the most commonly described adverse event ( 7/24 eyes in the cp group and 4/21 eyes in the hm group ) . an early postoperative iop peak ( 30 mmhg within the first 48 hours ) was recorded in 3 eyes in the cp group and in 1 eye after hm implantation . a yag laser procedure was performed in 6 eyes in the cp group ( goniopuncture ) and in 4 eyes in the hm group ( lysis of peripheral anterior synechiae ) during follow - up . in particular , procedures aiming to restore the physiological outflow through the trabecular meshwork / sc complex are being developed . in the hereby presented study , we retrospectively compared the midterm clinical outcomes of cp , that is , an ab - externo approach to redilate the sc , versus an ab - interno procedure such as the implantation of a scaffold ( hm ) . the percentage of complete success in the cp group ( 50% ) proved to be comparable with the 55% and 46% reported after a similar follow - up by lewis and coworkers ( 2009 ) and brusini ( 2014 ) , respectively [ 13 , 14 ] . matlach and coworkers ( 2015 ) found a slightly lower rate of complete success ( 39% ) , but the two series are less directly comparable because of the lack of a cut - off iop to define the postoperative success in our study . since the shortage of the so far published data , the results collected in our hm group can not be valuably compared with prior series . a recent report showed the high rate of success ( 80% ) of hm combined with cataract surgery . if we had performed combined surgery , we would have probably achieved better results , too . however , since scaffolds aim to restore the outflow within the physiological range , the midteens iop values observed postoperatively in our hm group are consistent with the expected mechanism of action of the procedure . because of the retrospective nature of the study , no preset cut - off iop values were planned . in fact , the target iop was individually set by the surgeons according to what was suggested by the egs guidelines for mild - to - moderate glaucoma damage with high starting iop ( i.e. , a target iop in the mid - to - high teens ) . comparing the two treatment groups , in our study the efficacy profile of cp was statistically comparable to that of hm . both procedures are theoretically endowed within similar mechanisms of action . therefore , our results are not at all surprising . a slightly ( albeit not significant ) better trend for clinical success in the cp group could be inferred by a qualitative evaluation of data in table 2 and figure 2 : two years after surgery , 50% of eyes treated by cp maintained the target iop without medications ; 57% of hm implanted eyes required any medical treatment to attain similar iop . a greater sample size , together with a longer follow - up , could potentially offer statistical significance to this observation . also the event failure ( i.e. , the need for further glaucoma surgery ) occurred similarly in the two groups , either in terms of number of cases ( two in each treatment group ) or number of temporal latencies . however , since cp is not a conjunctival - sparing procedure , a further limbal filtration surgery , if needed , is likely to be less successful and technically compelling . conversely , the clear cornea approach gives the hm procedure a less invasive profile in terms of surgical impact on the eye . in fact , when further surgery was needed in our study eyes , a tube was implanted after cp , meanwhile a plain nonpenetrating deep sclerectomy was successfully performed after hm . both procedures offered a comparable low complication rate in our study . in the literature , cp was widely associated with a low rate of side effects , mostly when compared to filtering surgery . albeit more recent , also migs have been associated with a good safety profile in the short term to midterm . in conclusion , our retrospective comparison showed that both cp and hm were safe and effective blebless procedures in early - to - mid stage open - angle glaucoma . however , along with the limited sample size and follow - up , its retrospective nature is a major point of weakness for the present study . this entailed the lack of preset : ( a ) randomization procedure , ( b ) cut - off values for target iop , and ( c ) intermediate follow - up time points . the administration of a survey to evaluate quality of life , as proposed by klink and coauthors in a quite similar setting , could represent a further improvement to the present findings . glaucoma surgeries now include procedures aiming to restore the physiological outflow through the trabecular meshwork / sc complex . this latter in particular can be successfully redilated via either an ab - externo ( the canaloplasty ) or an ab - interno ( the scaffolds ) approach.outcomes of canaloplasty are quite well described and compared to other filtering techniques ; much less is reported concerning scaffolds . glaucoma surgeries now include procedures aiming to restore the physiological outflow through the trabecular meshwork / sc complex . this latter in particular can be successfully redilated via either an ab - externo ( the canaloplasty ) or an ab - interno ( the scaffolds ) approach . outcomes of canaloplasty are quite well described and compared to other filtering techniques ; much less is reported concerning scaffolds . within the frame of this retrospective comparative study , one finds the following : two years after surgery , both cp and the hm were effective in decreasing iop;both procedures offered a low complication rate;a slightly ( albeit not significant ) better trend for a two years after surgery , both cp and the hm were effective in decreasing iop ; both procedures offered a low complication rate ; a slightly ( albeit not significant ) better trend for a
breast cancer is one of the most common malignancies and has become a serious threat to women s health around the world . in 2012,1.67 million women were diagnosed with breast cancer worldwide , accounted for 25.2% of all cancers among women . meanwhile , 522,000 women died of breast cancer which accounted for 14.7% of all female cancer deaths in 2012 . interestingly , the risk of this disease varies significantly across the world . comparing with western countries , the incidence and mortality of breast cancer in china are at relatively lower levels . according to the statistics of international agency for research on cancer ( iarc ) , the age - standardized incidencerate of female breast cancer in china was 22.1/100,000 in 2012 , while the age - standardized mortality was 5.4/100,000 . however , because of the large population size , the number of cases in china is higher than that in most countries . chen reported that there were almost 269,000 women joined the group of breast cancer in china , and the deaths were 69,500 at the same time . in the past decades , incidences of female breast cancer in most countries and regions have increased by 30%-40% , but this increasing trend was more obvious in china . from 1980 to 1990 , the crude incidence has increased by 72% with an annual growth of 3%-4% , which was far above the world average . on the other hand , the crude mortality of breast cancer in china also increased by approximately 100% since 1970s ( 5.90/100,000 in 2004 - 2005 vs. 2.95/100,000 in 1970s ) . the fast increase of breast cancer risk in china is partly associated with its economy booms and rapid changing lifestyles . comparing with cancers of high fatality such as lung cancer and liver cancer , the prognosis of breast cancer is much better . population - based cancer survival could reflect the prognosis of cancer and evaluate the progress of disease control , and is also helpful in making policies to ensure improved and equitable cancer care . however , only a few cancer registries have carried out population - based survival statistics for breast cancer among the chinese people in recent years , results showed that the survival rates have been significantly improved , but still much lower than many developed countries such as the united states , australia and some european countries . being lack of historical cancer registry data , the epidemiologic characteristics of female breast cancer in china , such as long - term change of incidence , mortality and survival rates , still need further investigation . jiangsu province is an eastern coastal province in china with a population of 79 million . although being one of the developed areas in china , jiangsu province is an area with high incidence and mortality of malignant tumors . according to the second national death retrospective survey in early 1990s , the mortality of cancer was 160/100,000 , which is 48.15% higher than the national average ( 108/100,000 ) . as a big province in china , the long - term change of female breast cancer still needs further illustration , furthermore , populationbased survival analysis of this disease has not been conducted previously yet . the purposes of this study are to describe the incidence , mortality and the temporal trend of female breast cancer in jiangsu province , and to analyze the survival status of breast cancer patients in the province . data were collected from jiangsu province cancer registry , which is affiliated with jiangsu provincial center for disease control and prevention . cancer registry in jiangsu province was first initiated in 1970s in qidong county and has experienced quick development since 2000 . now the whole province has been covered by cancer registry system , however , only a few counties could provide population - based registry data dating back to early 2000 . there were 7 registries providing integrate data in 2003 , then with the improvement of cancer registry work , the registered points were gradually increased in the following years and until 2011 there were 29 registries providing integrate data . for survival analysis , patients who were diagnosed as breast cancer between january 1,2003 and december 31,2005 were recruited and followed up for at least 5 years . a mix of active and passive follow - up methods was used to identify the vital status of patients from the date of diagnosis . in total , 7 counties provided complete survival information and passed quality evaluation . among the 7 counties , 1 is in the south of jiangsu province , 3 in the middle and 3 in the north . information of breast cancer patients including personal information ( name , age , birth date , address , etc . ) and diagnostic information ( onset date , diagnostic basis , date of death , icd- 10 code , etc . ) were collected using standardized cancer registry card . according to the data quality standards from international agency for research on cancer / international association of cancer registration ( iarc / iacr ) , software including msfox pro , ms - excel and iarccrg tools ( iarc / iacr ) was used to inspect and assess the quality of data . the reliability , validity and integrity were evaluated by major indices including proportion of morphological verification ( mv% ) , percentage of cases diagnosed with death certification only ( dco% ) and mortality to incidence ratio ( m / i ) . in this study , the mv% of female breast cancer was 84.72% , dco% was 0.52% and m / i was 0.28 , which indicated that data quality met the criteria of data quality of breast cancer . crude incidence and mortality were calculated by using the number of new cases or death cases divided by the population inregistry areas during the same period . rates were standardized by using 2000 chinese standard population ( asrc ) and segi s world population ( asrw ) . time trends during 2003- 2011 were estimated by annual percent change ( apc ) , which was calculated with the following formula : apc=100(e-1 ) , coordinated with the model y=+x+ , wherein is constant term , is the slope calculated from a linear regression of log rates in a calendar year and means random error . life - table method was utilized to compute the observed survival ( os ) and ederer method was used to estimate expected survival via the stata software 12.0 ( stata corp lp , college station , texas , usa ) . in addition to os , relative survival ( rs ) , a comprehensive index which can represent the excess mortality of cancer when other mortality risk was removed , was also calculated by dividing the os of patients by the expected survival of a comparable group in the general population . data were collected from jiangsu province cancer registry , which is affiliated with jiangsu provincial center for disease control and prevention . cancer registry in jiangsu province was first initiated in 1970s in qidong county and has experienced quick development since 2000 . now the whole province has been covered by cancer registry system , however , only a few counties could provide population - based registry data dating back to early 2000 . there were 7 registries providing integrate data in 2003 , then with the improvement of cancer registry work , the registered points were gradually increased in the following years and until 2011 there were 29 registries providing integrate data . for survival analysis , patients who were diagnosed as breast cancer between january 1,2003 and december 31,2005 were recruited and followed up for at least 5 years . a mix of active and passive follow - up methods was used to identify the vital status of patients from the date of diagnosis . in total , 7 counties provided complete survival information and passed quality evaluation . among the 7 counties , 1 is in the south of jiangsu province , 3 in the middle and 3 in the north . information of breast cancer patients including personal information ( name , age , birth date , address , etc . ) and diagnostic information ( onset date , diagnostic basis , date of death , icd- 10 code , etc . ) were collected using standardized cancer registry card . according to the data quality standards from international agency for research on cancer / international association of cancer registration ( iarc / iacr ) , software including msfox pro , ms - excel and iarccrg tools ( iarc / iacr ) was used to inspect and assess the quality of data . the reliability , validity and integrity were evaluated by major indices including proportion of morphological verification ( mv% ) , percentage of cases diagnosed with death certification only ( dco% ) and mortality to incidence ratio ( m / i ) . in this study , the mv% of female breast cancer was 84.72% , dco% was 0.52% and m / i was 0.28 , which indicated that data quality met the criteria of data quality of breast cancer . crude incidence and mortality were calculated by using the number of new cases or death cases divided by the population inregistry areas during the same period . rates were standardized by using 2000 chinese standard population ( asrc ) and segi s world population ( asrw ) . time trends during 2003- 2011 were estimated by annual percent change ( apc ) , which was calculated with the following formula : apc=100(e-1 ) , coordinated with the model y=+x+ , wherein is constant term , is the slope calculated from a linear regression of log rates in a calendar year and means random error . life - table method was utilized to compute the observed survival ( os ) and ederer method was used to estimate expected survival via the stata software 12.0 ( stata corp lp , college station , texas , usa ) . in addition to os , relative survival ( rs ) , a comprehensive index which can represent the excess mortality of cancer when other mortality risk was removed , was also calculated by dividing the os of patients by the expected survival of a comparable group in the general population . in selected cancer registry areas of jiangsu province , from 2003 to 2011 , the observed total population size was 140,465,998 and 69,911,221 were females . meanwhile , 17,605 women were diagnosed with breast cancer , accounted for 11.72% and ranked the 4th of all female cancer cases . the annual average crude incidence was 25.18/100,000 with an asrc and asrw of 19.03/100,000 and 17.92/100,000 , respectively . there were 4,883 women died from breast cancer during the same period , accounted for 5.09% and ranked the 6th of total female cancer deaths . the annual average crude mortality was 6.98/100,000 with the asrc and asrw of 4.93/100,000 and 4.80/100,000 , respectively ( table 1 ) . significant upward trends were observed for both incidence and mortality from 2003 to 2011 , although there are some fluctuations between years . as listed in table 1 , the crude incidence increased from 13.17/100,000 to 31.62/100,000 with an apc of 11.37% ( p < 0.01 ) , and the crude mortality increased from 4.86/100,000 to 7.74/100,000 with an apc of 5.78% ( p after age standardization , the increasing trends became smooth but still obvious ( figure 1 , figure 2 ) . mortality trend of female breast cancer in jiangsu province , 2003 - 2011 . incidence and mortality for female breast cancer in jiangsu province , 2003 - 2011 ( 1/10 ) table 2 displays the age - specific incidence and mortality of female breast cancer in registry areas of jiangsu province . it was obvious that both incidence and motility increased with age . among women after 30 years old , the incidence increasedrapidly with age growth and reached the peak at age of 55 - 59 years ( 63.01/100,000 ) , then stayed at a high level and decreased slightly until 85 + years again ( 51.24/100,000 ) . similarly , the mortality also reached its first peak at age of 55 - 59 years ( 20.08/100,000 ) , and the highest rate occurred at age of 85 + years ( 32.16/100,000 ) ( figure 3 ) . incidence and mortality of female breast cancer in jiangsu province , 2003 - 2011 age - specific incidence and mortality of female breast cancer in jiangsu province , 2003 - 2011 . from january 1,2003 to december 31,2005 , there being 1,392 breast cancer cases recruited for survival analysis in selected registry areas . all subjects were followed for at least 5 years andthe rate of loss to follow - up was 2.5% . the os and rs from 2003 to 2005 are displayed in table 3 . the age - standardized 1-,3- and 5-year os was 79.4% [ 95% confidence interval ( 95% ci ) : 76.0 - 82.9],58.4% ( 95% ci : 54.6 - 62.5 ) and 45.9% ( 95% ci : 42.2 - 49.9 ) , respectively , while rs was 81.4% ( 95% ci : 77.8 - 85.1),62.7% ( 95% ci : 58.3 - 67.5 ) and 52.0% ( 95% ci : 47.2 - 57.3 ) , respectively . both os and rs were found to be dropped with survival years . os and rs of female breast cancer in 7 registries of jiangsu province ( patients diagnosed during 2003 - 2005 ) the os and rs of different ages are also shown in table 3 . for instance , the 5-year os of patients at age of 15 - 44 years was 60.3% , however , the os dropped to 26.7% for those at age of 75 + years . the rs showed the same pattern , decreased from 60.7% to 40.8% . the os and rs in different areas in jiangsu province areshown in table 4 . the lowest survival rates were found in northern jiangsu province , where the 5-year os was 41.0% and rs was 46.8% . on the contrary , the highest survival rates were observed in southern jiangsu province where the 5-year os was 53.7% and rs was 62.4% . age - standardized os and rs of female breast cancer by areas in jiangsu province ( 7 registries , patients diagnosed 2003 - 2005 ) in selected cancer registry areas of jiangsu province , from 2003 to 2011 , the observed total population size was 140,465,998 and 69,911,221 were females . meanwhile , 17,605 women were diagnosed with breast cancer , accounted for 11.72% and ranked the 4th of all female cancer cases . the annual average crude incidence was 25.18/100,000 with an asrc and asrw of 19.03/100,000 and 17.92/100,000 , respectively . there were 4,883 women died from breast cancer during the same period , accounted for 5.09% and ranked the 6th of total female cancer deaths . the annual average crude mortality was 6.98/100,000 with the asrc and asrw of 4.93/100,000 and 4.80/100,000 , respectively ( table 1 ) . significant upward trends were observed for both incidence and mortality from 2003 to 2011 , although there are some fluctuations between years . as listed in table 1 , the crude incidence increased from 13.17/100,000 to 31.62/100,000 with an apc of 11.37% ( p < 0.01 ) , and the crude mortality increased from 4.86/100,000 to 7.74/100,000 with an apc of 5.78% ( p after age standardization , the increasing trends became smooth but still obvious ( figure 1 , figure 2 ) . mortality trend of female breast cancer in jiangsu province , 2003 - 2011 . incidence and mortality for female breast cancer in jiangsu province , 2003 - 2011 ( 1/10 ) table 2 displays the age - specific incidence and mortality of female breast cancer in registry areas of jiangsu province . it was obvious that both incidence and motility increased with age . among women after 30 years old , the incidence increasedrapidly with age growth and reached the peak at age of 55 - 59 years ( 63.01/100,000 ) , then stayed at a high level and decreased slightly until 85 + years again ( 51.24/100,000 ) . similarly , the mortality also reached its first peak at age of 55 - 59 years ( 20.08/100,000 ) , and the highest rate occurred at age of 85 + years ( 32.16/100,000 ) ( figure 3 ) . incidence and mortality of female breast cancer in jiangsu province , 2003 - 2011 age - specific incidence and mortality of female breast cancer in jiangsu province , 2003 - 2011 . from january 1,2003 to december 31,2005 , there being 1,392 breast cancer cases recruited for survival analysis in selected registry areas . all subjects were followed for at least 5 years andthe rate of loss to follow - up was 2.5% . the age - standardized 1-,3- and 5-year os was 79.4% [ 95% confidence interval ( 95% ci ) : 76.0 - 82.9],58.4% ( 95% ci : 54.6 - 62.5 ) and 45.9% ( 95% ci : 42.2 - 49.9 ) , respectively , while rs was 81.4% ( 95% ci : 77.8 - 85.1),62.7% ( 95% ci : 58.3 - 67.5 ) and 52.0% ( 95% ci : 47.2 - 57.3 ) , respectively . os and rs of female breast cancer in 7 registries of jiangsu province ( patients diagnosed during 2003 - 2005 ) the os and rs of different ages are also shown in table 3 . for instance , the 5-year os of patients at age of 15 - 44 years was 60.3% , however , the os dropped to 26.7% for those at age of 75 + years . the rs showed the same pattern , decreased from 60.7% to 40.8% . the os and rs in different areas in jiangsu province areshown in table 4 . the lowest survival rates were found in northern jiangsu province , where the 5-year os was 41.0% and rs was 46.8% . on the contrary , the highest survival rates were observed in southern jiangsu province where the 5-year os was 53.7% and rs was 62.4% . age - standardized os and rs of female breast cancer by areas in jiangsu province ( 7 registries , patients diagnosed 2003 - 2005 ) the incidence and mortality of female breast cancer vary significantly around the world , for instance , in northern america , the incidence is as high as 76.7/100,000 , however , it is only 19.3/100,000 in eastern africa . the causes to the disparities mentioned above are complex , which have been indicated by previous researches including society , lifestyle , ethnicity , heredity , culture , environment and so on . the incidence of breast cancer in jiangsu province from 2003 to 2011 ( asrc=19.03/100,000 ) was found slightly lower than the chinese national average ( asrc=23.27/100,000 ) , and mortality ( asrc=4.93/100,000 ) was similar with the national level ( asrc=4.93/100,000 ) . compared to other countries , the incidence of breast cancer in jiangsu province was slightly above less developed regions , such as tanzania , mongolia and gambia , whereas it was below the world average and developed regions , such as america , france , japan and singapore . similarly , the mortality in jiangsu province was also lower than the international average and some developed regions . although the incidence and mortality of breast cancer were relatively low in jiangsu province compared with some regions in the world , they have increased rapidly in recent years . from 2003 to 2011 , the age - standardized incidence increased from 10.55/100,000 to 23.39/100,000 and the mortality increased from 3.75/100,000 to 5.27/100,000 , which shared the same tendency of china where the age - standardized incidence increased from 22.18/100,000 to 25.89/100,000 and the mortality increased from 4.80/100,000 to 6.56/100,000 during 2004 to 2010 ( 13,30 - 32 ) . china has been experiencing the transition in both economy and lifestyle , the development of economy , the acceleration of life pace , the westernization of lifestyle , the decline of fertility and breast feeding among women , the changing of traditional diet structure and significantly increased prevalence in obesity , all of these have jointly contributed to the sharp increase of breast cancer risk in jiangsu province as well as the whole china ( 33 - 35,22 ) . according to limited published results , the total 5-year rs in china in 2003 - 2005 was estimated to be 73.0%,77.8% in urban and 55.9% in rural areas . the 5-year rs in jiangsu province was 52.0% , which was similar to the average level in rural areas of china , which could be explained by the fact that most areas selected for survival analysis were rural in the present research . the survival rate of breast cancer varies around the world . in comparison with other countries or regions , the 5-year rs of female breast cancer in jiangsu province was lower than that of developed regions , such as germany , france , korea , united states , japan , canada , but was higher than less developed regions such as philippines , mumbai , india , gambia , kampala and uganda . even in china , xiang reported that the 1-,3- and 5-year rs in shanghai from 1988 to 1991 was 89.7%,77.2% and 71.7% , respectively . the 5-year rs in beijing was reported as 66.3% and 74.2% during 1982 - 1983 and 1987- 1988 , respectively . in qidong , 1-,3- and the factors contributed to such variances between regions and countries are multiple , and some studies indicated that socio - economic inequalities , type of health insurance and race may affect cancer survival rate . the survival rates of patients in prosperous regions were higher than those in poverty - stricken regions . moreover , patients who have private insurance had higher survival than other patients . besides , variations in the prevalence of associated risk factors , public awareness of health knowledge , environmental pollution , and differences in initial treatment , supervision and support networks may be other reasons of the geographic changes . in this study , the difference among survival in south , middle and north jiangsu province also supported that economic , social determinants and lifestyle may be significant influencing factors of survival rates . in this study , the highest 5-year rs was observed among the group of age 15 - 44 years and the lowest 5-year rs was observed at age of 75 + years . elderly patients are usually in poor physical condition and may also suffer from other diseases , and thus are prone to receive more conservative treatments . besides , people in this age may also lack of exercise , and previous research had shown that more physical activity did contribute to the decline of breast cancer - specific mortality as well as all - cause mortality . these reasons might explain why the survival rate of female breast cancer declines with age . early detection of breast cancer is of utmost importance to increase survival rate and reduce mortality rate . found that 5-year rs detected at early stage ( stage i ) was much higher than those detected at late stage ( stage iv ) . but it is unfortunate that china has no nationwide screening program for breast cancer and earlier - stage detection . therefore , more resources should be allocated to primary prevention and earlier diagnosis , such as screening practices , particularly mammography screening which is more widely used in western countries . moreover , public awareness of the benefits of mammography and clinical breast examination in earlier - stage detection should be improved . in addition , more accessible health services should be provided for chinese women in order to increase the enthusiasm of screening . firstly , most cancer registries in jiangsu province were rural areas before 2006 , therefore the differences in incidence , mortality and cancer survival between rural and urban areas could not be compared . secondly , because lacking of historical data , the long - term variations of survival rates in jiangsu province could not be analyzed yet . lastly , detailed information of cancer cases , such as cancer stage and histological type , were incomplete in most registries , and thus we were not able to further analyze the association between survival rates and disease . breast cancer has increased rapidly among chinese women but the survival is still relatively low especially among rural areas in china . in order to reduce the burden of this disease , more resources should be allocated to primary prevention and earlier diagnosis , and more accessible health services should be provided in order to increase the positivity of screening among chinese women .
thoracic endovascular aortic repair ( tevar ) could reduce perioperative mortality and pulmonary , cardiac and renal complications when compared to open repair . furthermore , most of the past series data included devices that will soon not be marketed anymore . in an ironic way , these data are loosing their value exactly in the moment they have the most to say . in this paper we present our experience in the management of patients with descending thoracic aortic aneurysm undergoing tevar , including preoperative imaging modalities , endovascular procedures techniques , follow up , results and complications . preoperative planning and sizing accurate planning is mandatory when a tevar is considered , because precise quantitative measurements are needed besides the usual qualitative diagnosis ( figure 1 ) . three - dimensional rendering of the entire aorta in a patient with a descending thoracic aortic aneurysm obtained with the osirix software . diameters , lengths and angles need to be measured very accurately together with the presence of thrombus , calcifications , anomalies , etc . an example of sizing required to plan an endovascular procedure for a thoracic aortic aneurysm . a timely diagnosis and an accurate planning and sizing allow to offer an effective treatment to many patients , however , in spite of a flawlessly executed operation the most severe complication after these procedures is certainly spinal cord ischemia which can cause symptoms as dramatic as paraplegia . endovascular procedure all the procedures were performed in the operating room , using a portable digital c - arm image intensifier with road - mapping capabilities ( series 9600 , oec medical system or moonray , simad medical technology ) . cerebrospinal fluid drainage has a protective role in open surgical repair of descending thoracic and thoracoabdominal aortic aneurysms , while its role in tevar is less defined . our current indications for cerebrospinal fluid drainage in tevar includes : long coverage ( predicted use of more than one endograft ) . coverage of high risk area including t10-t12 . intraoperative trans - esophageal echocardiography monitoring is routinely used during all thoracic endovascular procedures . in our experience trans - esophageal echocardiography is useful under many aspects as it allows to double check for the most appropriate landing zone of the endograft ( in particular when the left subclavian artery origin has to be spared ) . as a second instance trans - esophageal echocardiography is capable of documenting thrombosis or the presence of slow flow inside the aneurysm sack after deployment of the endograft , thus avoiding repeated angiography and the use of high doses of contrast media . in our specific experience in the treatment of type b dissection , it allows to detect whether the guide wire is correctly positioned inside the true lumen or it has moved inside a patent false lumen . as a second , and most important instance , trans - esophageal echocardiography easily documents the presence and position of proximal entry tear , the presence of additional distal tears ( eventually not detected by angiography ) and thrombosis or reperfusion of the false lumen . our preferred choice is to perform the endovascular procedure under general anesthesia ; in selective cases , depending of the patient general condition and comorbidity , the strategy of access site for endograft insertion and the institution of cerebrospinal fluid drainage , we also perform local or spinal anesthesia . patients are placed in the dorsal decubitus position , and the operative field is prepared and draped . it is mandatory that the operating field is prepared in such a way to allow a laparotomy for access and abdominal aorta and/or iliac arteries control . in most cases the common femoral artery the contra - lateral femoral artery is percutaneously punctured for diagnostic guidance . in case of extremely diseased ( calcific ) external iliac arteries we routinely use the common iliac through a paramedian extraperitoneal approach as the access site ; we use to directly puncture the artery through a purse string . in rare cases the abdominal aorta has been used as the access site , with direct puncture and then closure with a purse string suture . a 260-cm long , 0.035-inch precurved super stiff guidewire ( lunderquist , cook medical inc . ) is advanced up to the aortic arch under fluoroscopic guidance . the endograft is then passed over the guide wire into the appropriate position within the descending thoracic aorta or aortic arch under fluoroscopic guidance . during deployment , a mild systemic hypotension is induced pharmacologically by the anaesthesiologist with a bolus of fast - acting venous or arterial vasodilators such nitrates , urapidil . after deployment of the endograft on the selected location a completion angiogram is performed . special care is mandatory when removing the introducer sheath , as rupture of the external iliac artery at its origin is more likely to happen with thoracic endografts . it is particularly advisable to leave the guide wire in place until the sheath is completely removed in order to perform an emergent endoclamp in case of iliac rupture . the objective of a device intended for treatment of an aortic aneurysm is clear cut : it must prevent aneurysm rupture . this is achieved by effectively excluding the aneurismal sac from circulation , in the absence of blood flow and pressure within the sac , thrombus will form and with time the sac itself may regress or disappear altogether . for aortic dissection the degree of uncertainty is greater , in uncomplicated acute cases without dilatation of the aortic wall , closure of the proximal intimal tear may achieve healing of the aorta . however the device itself may produce additional tears at both the proximal and distal level of the fragile diseased aorta . most currently available endografts are not primarily intended for the treatment of this condition especially if they carry proximal or distal uncovered stents , hooks or barbs . for chronic cases , it must be remembered that effective obliteration of the false lumen may compromises the circulation to arteries that arise from the false lumen itself ( i.e. left renal artery ) , flow to all important vessels must therefore be verified and additional procedures ( i.e. stenting ) may be necessary to restore flow in these vessels . in most other cases the use of aortic endografts is off label and experimental ; for instance experience with endovascular treatment of traumatic lesions , penetrating ulcers , intra - mural hematoma is increasing and once an endograft has been successfully implanted , the patients need to undergo follow up imaging to confirm that everything is working properly , and if something is wrong , to allow for a timely and safe procedure to fix the problem . the presence of metal parts ( that are very radio - opaque ) in the endovascular prostheses makes the postoperative scans not only important for the patient s safety but also fascinating to look at ( figure 3 ) . once again appropriate post - processing of the images is mission- critical for an accurate diagnosis . three - dimensional computed tomography angiography ( 3d cta ) of thoracic aortic aneurysm exclusion . in the box is visible the endoluminal reconstruction of 3d cta : note that proximal stent lays at the origin of left carotid artery without covering it . since then we have performed a total of 322 procedures . of these 116 involved the aortic arch with partial / total rerouting of the supra - aortic vessels , 172 involved the descending thoracic aorta , and 34 were hybrid procedures for pathology of the thoracoabdominal district . a total of 126 procedures were conducted for degenerative aneurysms of the descending thoracic aorta . considering specifically the descending thoracic aorta our technical success rate , i.e. deployment of endograft with complete exclusion of the lesion / minimal endoleak , was 99.4% ( with one case requiring emergent open conversion ) . the rate of pulmonary complication , i.e. the need for more than 24 hours of mechanical ventilation , was 1.2% . the rate of cardiac complication , i.e. new onset myocardial necrosis demonstrated by positive cardiac biomarkers tests , has been 0.6% . the rate of renal complication , i.e. the need for temporary or permanent renal replacement therapy , was 1.9% . cerebrovascular accident , defined as a new neurologic deficit lasting more than 24 hours confirmed by imaging , occurred in 1.2% of patients . the rate of spinal cord ischemia , manifesting either as paraplegia or paraparesis , was 5.2% . delayed onset spinal cord ischemia had a tendency towards amelioration with adequate pressure support and cerebrospinal fluid drainage . in spite of the great enthusiasm that endovascular techniques provoked and that are confirmed by the excellent results of our case series , it must be remembered that particularly at the level of the thoracic aorta these techniques are by no means completely safe . a french report from ricco jb and coworkers for example , analyzed a countrywide experience over two years and showed an high rate of complications . damage to access vessels several literature reports pointed out that serious and even fatal problems may arise from introduction of the device from the femoral artery . in particular rupture or avulsion of the external iliac artery have been reported , this usually becomes dramatically evident only at the time the large introducer sheath used for endograft delivery is withdrawn . we therefore liberally switch to extra - peritoneal surgical exposure of the common iliac artery or even the distal aorta if it is difficult to advance the device through the femoral arteries . cerebrovascular accidents are among the most common and dreaded complications of endovascular therapy of thoracic aortic disease . they are mainly linked to atheromatous embolization into the cerebral arteries caused by guide wires , catheters , introducer sheaths manipulation or the endograft itself . it should be remembered that angiography for diagnostic purposes alone carries a 1 - 2% risk of complications . second generation commercial grafts are surely less prone to this complication than the home made devices used initially . this is due to the better flexibility of the grafts and sheaths and to the smaller profiles , that results in improved navigability through the access vessels . greater experience of the operators may also play a role both in the selection of candidates with adequate anatomical characteristics and during the procedure itself . anyhow , minimal manipulation of the wires and catheters and a meticulous technique do play a significant role in the prevention of perioperative stroke . endoluminal repair allows the avoidance of aortic cross clamping and its sequelae ; however , the intercostal arteries covered by the endograft can not be reimplanted . the reported incidence of both immediate and delayed paraplegia in patients undergoing endovascular procedures of thoracoabdominal aortic aneurysms can be as high as 12% of cases . the coverage of a long thoracic aortic segment has been reported to be a significant risk factor for spinal cord injury . patients who have open abdominal aortic aneurysms repair also appear to be prone to such a risk because of the marginal spinal cord collateral blood supply secondary to the ligation of lumbar arteries performed during the surgical procedure . also , proximal collateral circulation of the spinal cord may be put at risk by the occlusion of the left subclavian artery ( landing zone 2 ) that abolishes the contribution to the blood supply provided by the anterior spinal artery , a branch of the ipsilateral vertebral artery . finally , spinal cord injury could be precipitated by the late sealing of a type ii endoleak or late thrombosis of collateral pathways . in a recent study we analyzed our most recent 5-year experience with repairs of thoracic aortic pathology to evaluate the incidence and investigate the determinants of spinal cord ischemia in endovascular procedures , identify patients at risk , and assess the role and efficacy of prophylactic adjuncts and therapeutic measures . our data showed that the lack of collateral spinal cord blood supply through the lumbar arteries in patients with previous aortic repair appears to be a relevant risk factor even though it did not reach statistical significance in our series . the intentional occlusion of the left subclavian was not a predictor of paraplegia and neither was the exclusion of an extensive thoracic aortic segment . among the perioperative variables , only a mean arterial pressure < 70 mmhg was a statistically significant determinant of delayed neurologic deficit ( p<.0001 ) . a delayed neurologic deficit developed in four patients , completely resolved after the institution of csf drainage , steroids administration , and arterial pressure pharmacologic adjustment . our experience addresses the importance of hemodynamic control to prevent postoperative neurologic deficits and encourages aggressive , postoperative care of these patients . in our practice , we now try to maintain a perioperative mean arterial pressure of > 90 mmhg and use cerebrospinal fluid drainage in patients deemed at high risk , including those who received abdominal aortic aneurysms repair . in this respect , patients with synchronous thoracic and abdominal aortic aneurysms , which we earlier treated simultaneously for both aneurysms , currently undergo staged procedures to better allow the development of collaterals for spinal cord blood supply . in the case of delayed paraplegia , prompt cerebrospinal fluid drainage , if not previously instituted , is also used to keep the cerebrospinal fluid pressure < 10 mm hg and possibly reverse the deficit . aorto - esophageal fistula as follow - up is becoming longer and the reported series larger , new serious complications are emerging . one of the most ominous one is endograft infection ( figure 4 ) , especially if sustained by an aortoesophageal fistula ( figure 5 ) . on the left : axial computed tomography scan of an infected thoracic endograft . note the presence of air bubbles ( arrows ) clearly indicating the presence of bacterial flora . it may be caused by erosion of the endograft through the aortic wall into the esophagus , and we may speculate that excessive over - sizing may contribute . sometimes it presents as a fatal hemorrhage however sometimes the presentation is less dramatic at it may be characterized by pain , dysphagia , hematemesis , and septic symptoms . successful long term aneurysm exclusion requires the durability of proximal and distal fixation sites against the bloodstream forces , the fatigue of the materials and the morphological behavior of the aneurysm . the society for vascular surgery / american association for vascular surgery ( svs / aavs ) standards defined endograft migration any movement relative to anatomical therapy . to address this problem , a variety of fixation methods such as hooks , barbs , free - flows and longitudinal support devices have been developed and each presented peculiar advantages and drawbacks . due to the extreme friability of the aortic wall of a dissected aorta , the use of trans- rupture of the longitudinal support wire is a well known complication that affected the first generation thoracic gore tag device stressed by the bending forces arising in the arch or in kinked aneurysms . the descending thoracic aorta is the ideal morphological site for application of endograft technology being the more large straight tubular arterial segment of human body . it has been the first site of aortic aneurysm endograft exclusion as published by volodos et al . yet in 1988 , 3 years before parodi et al . reported the first series of abdominal aortic aneurysm stent - graft repair . since the pioneering experience of volodos and the first renowned short series of thoracic aortic aneurysm endovascular repair published by dake et al in 94 , many series , registries and trials have confirmed feasibility and reduced invasivity of thoracic aortic aneurysms endovascular repair leading to a real worldwide revolution in the therapeutic approach to the descending aortic diseases . the endovascular era opened new attractive scenarios with many hopes so that , currently , the open repair of an aneurysm limited to the descending aorta , especially to its middle part has became a really unusual occurrence in the most of european vascular centers . even in our center , in the last years we observed a progressive shift in the selection of patients for tevar of the descending thoracic aorta . at the beginning of our experience ( the first 4 - 5 years ) tevar was limited to those patients presenting with one or more serious comorbitidies , while open surgery remained the choice for those in fair clinical conditions . in particular tevar was the treatment of choice in the elderly ( > 77 years ) , in patients with a depressed ejection fraction ( < 40% ) , in patients with severe pulmonary disease ( home oxygen therapy , sever obstruction ) and with a life expectancy of less than 5 years . starting 3 years ago , mainly within investigational protocols and trials , we have gradually increased the rate of patients undergoing tevar . derived from this new attitude , we experienced really appealing results of tevar also in fit surgical patients , especially with critical intercostal arteries left uncovered . however , studies clearly addressing the respective roles of open and endovascular repair are still not available , and the worldwide spreading of expensive endovascular materials and facilities , with the extensive follow - up for endografting add to the cumulative escalation of overall health care costs . furthermore the stents have been designed to have a durability of 10 years based on iso ( international standardization organization ) stress testing and the series with the greatest number of patients reach maximum 5 years of follow - up , and are by now associated to a high rate of reinterventions . in conclusion , in order to treat both patients unfit for open surgery and patients with long life - expectancy especially with challenging anatomies for endograft deployment , both tevar and open repair should be part of therapeutic armamentarium of every modern vascular surgeon efficiently approaching thoracic vascular diseases .
affect data were collected from 554 residents ( 71 % female ) of a large , urban geriatric center as part of a longitudinal study . mean age for the sample was 83.3 years ( sd = 6.0 ) . sixty - nine percent of the participants resided in apartments and 31 % resided in the nursing home . the philadelphia geriatric center positive affect and negative affect scale was used to assess affective states . this 10-item measure consists of positive and negative subscales rated on likert scales ranging from 1 to 5 . other assessments included a modified schedule for affective disorders and schizophrenia ( msads ) , the geriatric depression scale ( gds ) , fuld 's adaptation of the blessed memory - information - conccntration task ( bmic ) , physical self - maintenance scale ( psms ) , and cumulative illness rating scale ( cirs ) . the msads is a semistructured clinical assessment used in this case to determine patients ' level of depression : major , dysphoric , or nondepressed . the gds docs not contain somatic , vegetative symptoms of depression , which are often symptoms of age rather than depression in this population . scores may range from no depression ( 0 ) to severely depressed ( 30 ) and a score of > 10 suggests clinically significant depression . the bmic is a 33-item measure of cognitive impairment , which is scored by the number of incorrect responses . a score of > 10 suggests cognitive impairment . the psms is a measure of activities of daily living , which assesses the degree of functional disability . it includes 8 items regarding the amount of assistance needed to complete everyday activities such as eating , bathing , and toileting . items are scored need no help ( 1 ) , need some help ( 2 ) , and need help or ca n't do alone ( 3 ) . a score of 12 or greater suggests clinically significant functional impairment . the cirs is a summary of illnesses categorized into 13 independent body systems . each system is rated by the patient 's physician on a 5-point scale of severity ranging from none ( 0 ) to extremely severe ( 4 ) . means of these assessments are shown in table i. in general , this sample is nondepressed , cognitively unimpaired , and functionally able despite moderate to moderately severe physical illness . sample descriptive data and correlations with markers of frailty are shown in table i as expected , gds , cirs , psms , and pain were negatively correlated with pa and positively correlated with na ( all p<0.001 ) . however , among nondepressed , or euthymic persons , the correlation between pa and na was r=-0.17 , which suggests that much of the strength of the negative relationships between the two in the population as a whole can be attributed to their associations with clinical depression . one - way analyses of variance ( anovas ) were conducted to compare the means of pa and na among cuthymics , dysphorics , and persons with major depression . na was highest among persons with major depression and lowest among cuthymics ( table ii ) . pa was relatively stable from baseline to 1-year follow - up ( r=0.53 ) and 2-year follow - up ( r=0.51 ) among euthymics . among persons with any depression , major or dysphoria , the correlation was r=0.48 at the 1-ycar follow - up and r=0.28 at the 2-year follow - up , indicating that pa was lower at follow - up in depressed persons . in contrast , na was not stable over time among euthymics , whereas it appeared to be relatively stable in depressed persons . receiver operating characteristics ( roc ) curves were calculated for pa and na to determine their sensitivity and specificity in identifying any depression ( determined by gds scores > 10 and/or msads diagnosis of any depression ) and for identifying msads diagnosis of major depression , and were used to calculate cutoff scores for pa and na ( table iv ) . using these cutoff scores , it was determined that 73% of persons with any depression had some form of affective disturbance ( ie , high na and low pa ; high na and high pa ) . among persons with any depression with some form of affective disturbance , eighty - two percent of persons with major depression reported some affective disorder and 52% reported experiencing low pa and high na ( table v ) . in addition to being a moderately good identifier of depression , both pa and na contributed to the prediction of depression . logistic regression analyses indicated that high na and low pa ( anhedonia ) are associated with risk of depression after 1 year ( model x=12.91 , p=0.002 ) . low pa increases the odds of depression by 15% ( exp b=0.87 , p=0.02 ) and high na increases odds of depression by 38% ( exp b=1.38 , f=0.01 ) . when pa , na , and the hamilton rating scale for depression1 were used to track changes among persons receiving pharmacological treatment for depression . changes in hamilton scores were correlated with both changes in pa ( r=-0.43 , p=0.002 ) and changes in na(r=0.40 , p=0.004 ) . subjects can complete them quickly and understand immediately the reasons for wishing to use them repeatedly . in particular , our findings indicate that the absence of pa is of equal importance to the presence of na in identifying patients with clinically significant depression . we suggest that these brief parallel measures might be particularly useful when the course of illness is being followed over time and when increasing the prevalence of positive experiences is one of the explicit treatment goals . in conclusion , pa as well as na are salient indicators of depression , and may tap into depression in older populations who may underreport depressive symptoms . pa and na scales are a simple and conclusive way to measure affective states over time that may provide salient information regarding the ongoing course and prognosis of depression .
fifty years of development and progress have led to people with hemophilia having access to potentially unlimited supplies of therapeutic products , in the form of coagulation factor concentrates ( cfcs ) safe from historical hazards . despite this , emerging evidence of subclinical bleeding with current prophylactic regimens indicates , arguably , that all patients are undertreated.1 the conventional classification of hemophilia is also under question , with evidence that levels of 15%20% are needed to protect against all bleeds.2 acquiring this level of protection requires access to more cfcs until the distant promise of a cure attained through gene therapy.3 the first era of treatment products limited access to factor viii for hemophilia a in particular , due to the difficulty in extracting factor viii from limited plasma supplies . the current era in the established economies is dominated by the provision of recombinant products . these promise to eliminate the access problem , as their supply is , hypothetically , unlimited as enhancements in the technology are applied.4 the issues pertaining to hemophilic therapeutic access currently include : treatment costsapproval processes for authorization and reimbursementtreatment hazards . approval processes for authorization and reimbursement these intertwined issues , and the continuing pressures on health care costs , are influencing the current paradigms for these areas . the traditional delineation between market authorization and reimbursement agencies is under pressure in the european union ( eu ) through processes seeking to establish common paths for the approval and funding of products.5 this has succeeded an ongoing body of literature assessing the pharmacoeconomics of treatment choices , including the treatment of hazards such as inhibitors to cfcs.6 concurrently , the market approval processes for cfcs have been more closely aligned to the traditional assessment methods for medicines . we will review these developments and attempt to synthesize proposals for the continued progression of access to care for hemophilia . over the past 20 years , a paradigm of evidence - based medicine ( ebm ) has become established in the approval and oversight of medical interventions , primarily through a recognition , through the work of archie cochrane , that the adoption of therapeutic interventions was historically detached from evidence of their efficacy.7 a key feature of ebm has been the acceptance of a hierarchy of measures contributing to evidence , viewed as a pyramid and headed by the randomized clinical trial ( rct ) . rcts had been developed , primarily through the work of hill8 in previous decades , but it was only following the inception of ebm that they became the yardstick / standard for the assessment of efficacy and safety . product authorization agencies initially charged with ensuring the safety of therapies rapidly absorbed the ebm paradigm as their remit was widened to include the assessment of efficacy and therapeutic claims . in some landscapes , particularly the eu , the regulatory oversight of blood and plasma products was excluded from the mainstream framework until relatively recently . as governments in the social - market economies have detached from the direct ownership of plasma fractionation activity , the relevant marketing authorization ( ma ) agencies have assumed higher levels of oversight over their activities , including the assessment of efficacy . in this regard , mas such as the us food and drug administration ( fda ) and the eu s medicines agency ( ema ) have attempted to implement the tenets of ebm . recognizing the limitations imposed by a small population of patients in the rare blood disorder congenital fibrinogen deficiency , the fda granted approval for a treatment product based on a study on 15 patients in which clinical efficacy was assessed through a surrogate endpoint.9 similar flexibility was shown by the ema in approving activated factor vii for the treatment of glanzmann s thrombasthenia through various overlapping inputs of a patient population of less than 10.10 this has been tempered by overzealous assessments from the cochrane collaboration , which did not endorse prophylaxis as a preferred modality to treatment on demand for hemophilia11 until demonstrated through a rct.12 this also demonstrated progressive joint disease in the control population , a regrettable outcome given the 30 years of experience of the benefits of prophylaxis and one confirmed by follow - up of the patient population.13 the continued use of these randomized studies is leading to undertreatment of patients and permanent injury14 as patients are randomized into on - demand treatment with all its consequences ebm may be viewed as the predominant kuhnian paradigm15 in therapeutics . as in all such paradigms , it is dubious if the originators of the ebm hierarchy envisaged the requirement for rcts as the yardstick by which efficacy would be established . this traditional paradigm provides a comfort zone for regulators , while evident tensions arise , such as the issues around the removal and subsequent restoration of the ma of aprotinin for minimizing blood loss during cardiac surgery.16,17 the brisk controversy around this issue shows that the interpretation of rct data is still as subjective as any other human endeavor . the recent boldt scandal18 in which clinical trials used in ma and reimbursement process were retracted from the literature because of fraudulent practices demonstrates the vulnerability of the paradigm . depending on which version of the ebm pyramid is examined , systematic reviews and their quantification through meta - analyses are often pivotal in guiding practice , including therapeutic choices in hemophilia , and in ma procedures.19 they also influence hypotheses around which rcts are constructed to answer a specific question , eg , does prophylaxis produce better long - term outcomes in hemophilia patients than on - demand therapy ? as mentioned earlier , the particular cochrane review on this issue continued to assert that this was an open question until the late 2000s , against a background of 40 years of clinical experience . this type of cochrane question also seems counter to traditional health principles that prevention ( prophylaxis ) is better than cure ( on - demand therapy ) . the discrepancies between hypothesis - generating meta - analyses and subsequent rcts20 also contribute to reservations about the conventional ebm hierarchy . in many rcts / meta - analyses addressing important questions in hemophilia treatment , a key assumption has been equivalence in the treatment effects of different cfcs and homogeneity in the treated population . the first assumption is viewed with reservation on the basis that biological medicines such as cfcs can not be considered as generic or biosimilar.21 some investigators have addressed the issue by stratifying patients according to the type of cfc administered,22 demonstrating heterogeneity in treatment effects analyzed a posteriori which may be a reflection of differences between products . in addition , differences in the pharmacokinetic profiles of infused cfcs suggest that homogeneity in the patient population can not be assumed.23,24 overall , the limitation of rcts to providing information only about averages has fuelled the thrust toward incorporating the assessment of efficacy into a new paradigm of personalized medicine for hemophilia.25 a striking example of the application of personalized prophylaxis with the achievement of improved outcomes at lower costs shows the potential of this approach.26 a convergence between the ebm paradigm of clinical trials and personalized medicine may be possible in the product authorization area through the use of n of 1 trials . n of 1 trials are considered to provide the strongest level of evidence about the existence of a causal relationship between a treatment and an outcome.27 the n of 1 trial is an rct carried out in one patient , who undergoes pairs of treatment periods where one period includes the experimental treatment under investigation and the other period includes a placebo / comparator treatment . the treatment periods are randomly assigned for each cycle , eg , pair 1 , placebo , treatment ; pair 2 , treatment , placebo ; pair 3 , treatment , placebo . kravitz et al28 have estimated a hypothetical 33% enhancement in therapeutic precision when using n of 1 trials versus the conventional clinical trial structure . in an australian study , significant cost - savings relative to conventional designs were reported.29 a similar outcome has been estimated by kravitz et al30 in the united states . given the variability in pharmacokinetic23,31 and bleeding32 profiles in hemophilia , a similar analysis would be relevant for the possible use of n of 1 trials in this and other rare bleeding disorders . it would contribute to the growing literature on the pharmacoeconomics of hemophilia and widen the perspective to include personalized treatment , as we shall discuss further . the criteria for eligibility for n of 1 trials make them especially suited for rare , chronic conditions33 such as hemophilia . in practical terms , trials for cfcs include basic pharmacokinetic measurements and comparison to approved products . trials also include assessment of efficacy , generally through a clinical endpoint such as joint bleeds . we suggest that both these parameters may be studied through the basic type of n of 1 design described earlier . it should be noted that crossover trial designs , of which n of 1 trials are an example , have been applied to hemophilia investigations in a number of settings.3436 the ability to combine n of 1 trials to estimate population treatment effects37 demonstrates that they can also constitute a tool for evidence - based clinical guidelines as with conventional rcts and meta - analyses , while influencing such guidelines through a patient - centric and individualized approach . in the current era of patient - centeredness and comparative effectiveness research , n of 1 trials are proposed as important parts of the methodological armamentarium , facilitating individualized care and improving patient outcomes.38 the new era of personal - ized medicine stands to supplant the old ebm paradigm , and although some consider a synthesis of ebm and pm to be possible and natural,39 we concur with de leon s view40 that the differences between these approaches make them incommensurable.15 the fda41 and the ema42 have committed to considering n of 1 trials and other approaches for assessing health technologies for rare chronic disorders43 but at least one peer health technology assessment ( hta ) body in europe has expressed reservations regarding their applicability.44 hence , tensions are evident between the ma and hta bodies , which we will now review . the fathers of ebm have stated that practicing evidence based medicine will identify and apply the most efficacious interventions to maximise the quality and quantity of life for individual patients ; this may raise rather than lower the cost of their care.45 despite this , it is difficult not to conclude that , over the past decades of its dominance , the ebm paradigm has been absorbed as a major tool for cost - containment in health reimbursement systems . a major shaper of this development has been the cochrane collaboration , which has included in its goals making cochrane the home of evidence to enable informed decision making.46 the authors conclusions in cochrane reviews of interventions for rare bleeding disorders4749 are peppered with references to cost issues and the need to demonstrate cost - effectiveness . concurrently , the establishment of rcts as the yardstick for ma for specific indications has increased the cost of drug development , and the unfortunate withdrawal of most governments from the area of clinical trials has shifted the costs entirely to industry . one necessary feature for carrying out such trials is the free provision of the drugs under investigation , as well as any added comparator drugs , to the trial subjects . some funding agencies have noted how this leads to considerable savings to the health systems involved.50,51 we ponder on the necessary conflict of interest , which must arise as mas recognize the implications of their measures on the provision of free medicines in the health systems they serve , noting that as of february 2015 , 260 studies were registered for investigations on hemophilia.52 this gradual convergence between the historically distinct regulatory and reimbursement components of pharmaceutical provision is particularly discernible in the ongoing process of developing common evaluation pathways for the approval and hta processes in the eu.53 this convergence is being pursued solely in the area of clinical efficacy . the various hta bodies established to advise government payers in europe have different approaches to assessing effectiveness . this is a broader and more pragmatic concept than efficacy as measured by an rct,54 the methodology favored by mas . agencies such as the uk national institute for health and clinical excellence and the australian pharmaceutical benefits advisory committee use the quality - adjusted life year as a metric to allow all interventions to be directly related through a preestablished threshold for cost - effectiveness and reimbursement.55 the german institute for quality and efficiency in health care ( institut fr qualitt und wirtschaftlichkeit i m gesundheitswesen iqwig ) , on the other hand , uses an efficiency frontier approach that compares all possible interventions for one specific indication , divorced from any prioritization with other issues . the iqwig s choice of methodology is based on the agency s position that prioritization is a political , not a scientific choice.56 the eu hta bodies attempt at convergence has resulted in a hta core model,57 still unavailable publicly , which is informed through a detailed questionnaire supplied to product sponsors.58 it is difficult to discern the basis of a harmonized approach to hta evaluation through this document , particularly in the crucial areas around assessing therapeutic benefits and effectiveness measures . it has been noted that hta bodies employ a wider range of inputs in their evaluation than a narrow efficacy assessment as used by most mas,59 but a wide body of convergence is evident , with bodies like iqwig giving primacy to the rct as the first level of evidence.44 concurrently , the use of health - related quality of life measures utilized by hta bodies is also the subject of regulatory guidance.60 this poses the question as to why mas do not widen their assessments to include such measures , as recent policy documents suggest they are committed to do.41,42 it would seem more logical for hta bodies to assess the extent to which products should be reimbursed , through a synthesis of cost and effectiveness , using the same effectiveness measures that resulted in the approval , by mas , of a ma application . while some merit exists for a parallel track in order to abbreviate the overall market access process , the most time - consuming phase is the drug development process . the subsequent approval process for efficacy and effectiveness should not be hindered through bureaucratic inefficiency , as is clearly the case with some agencies experiencing considerably longer approval times than others with comparable resources.61 two recent case studies in the eu demonstrate the potential effect of hta processes on the position of cfcs in national markets . in the first case , a systematic review of available evidence of most aspects of hemophilia care was performed to inform the swedish council on hta.62 an expert group of eminent swedish treaters concluded that , for all 12 clinical areas reviewed , the scientific evidence to determine practice was lacking and relevant studies were mostly absent . the group employed a mainstream ebm paradigm in selecting studies for the review , according a primacy to rcts and a consideration to prospective controlled studies . the swedish hta was carried out but the results of the analysis were not made public , while sweden s generous provision of treatment for hemophilia continues.63 the price of recombinant factor viii , the dominant modality for hemophilia care in sweden , has been stable over the past 3 years , while government reimbursement agencies in similar social market health systems have developed mechanisms for significant reductions in price.64 in the second case , iqwig in germany has been commissioned by the federal ministry of health to produce a rapid report on the treatment of hemophilia patients.65 the process for the generation of such a report is described on page 17 of general methods 4.1.44 and may include input from patients . it must be presumed that the ebm hierarchy favored by iqwig will influence this product . the report will inform the process whereby reimbursement of cfcs through insurance occurs in germany . in this process , a maximum reimbursable price requested for new novel / innovative products is granted only if iqwig s assessment agrees that the product provides an added benefit relative to currently reimbursed cfcs as described on page 142 of general methods 4.1.44 as of february 2015 , this process has been tested with two cfcs for hemophilia . the sponsor of the recombinant factor viii turoctocog alfa claimed added benefits in the form of additional safety from infectious agents and enhanced provision and convenience to patients through validated storage at room temperature . these claims were not accepted by the iqwig and the request for a maximum price on these bases was rejected by the gemeinsamer bundesausschuss ( g - b ) ( german federal committee),66 which is the actual decision maker in the german government . scrutiny of the g - b s reasons reveals that , acting on the iwig s advice , this body considers all factor viii cfcs to be equivalent in safety and efficacy , and therefore similarly appropriate to act as comparators to turoctocog alfa.67 the g - b also did not consider the sponsor s representation that the inclusion of an additional product , reimbursable at the maximum price , would improve the security of supply , on the basis that this did not constitute a therapeutic advantage as required by law . in the recently announced decision regarding simoctocog alfa , a recombinant factor viii differentiated through generation in human cell lines , iqwig also turned in a negative assessment,68 asserting that the sponsor had only performed single - dose studies , over a relatively short time frame in a crossover design . this decision does not augur well for iqwig s consideration of possible n of 1 trials as these would be similarly configured . this decision by iqwig appears to consider that the ma for the agent did not cover effectiveness adequately , another example of where hta and ma bodies diverge on critical issues . analysis of this decision is important for the future of access to hemophilia care in germany , europe s largest market for cfcs . the price of products in germany is still relatively high compared to that obtained in centralized , competitive tender processes , as may be seen on page 7 of the study by g - b,66 while traditionally , prescribers are free to treat patients with their product of choice . strong lobbying by industry directly to prescribers ensures high visibility of products , including the new generation of innovative , value - adding cfcs . furthermore , the high reference cfc reimbursement price , even at the maximum limit allowed by the insurance process , has a strong influence in those countries that reference germany for their drug prices.69,70 hence , events in germany are influential on the hemophilia community within and outside its borders . the iqwig s criteria for added benefit leading to extra reimbursement will be scrutinized strongly as other cases , offering more discernible benefits than were apparent with the first cfc submissions , are subjected to the process . in particular , the outcomes for innovative products such as cfcs modified to result in longer half - lives will be assessed with great interest to see whether outcomes recorded primarily through pharmacokinetics will be accepted as additionally beneficial by iqwig . an appropriate balance between cost - containment and the reward of clinically beneficial innovation is desirable . we note that , while iqwig s dismissal of the arguments around turoctocog alfa s benefits regarding safety and convenience may be valid , their apparent requirement for direct comparison with a comparator ie , trials is troubling , given iqwig s preference for rcts . we consider the use of historical controls in lieu of subjects in control arms to be acceptable , as we shall discuss further . the attention of the approval process for cfcs is currently focused on new , innovative products manufactured by biotechnology . over the past few years , several of these products have been developed and studies of their use in patients , subsequently used to acquire market authorization and access , have been published ( table 1 ) . the requirements of mainstream ebm and other aspects required by hta bodies such as health related quality of life are visible in several of these studies . in particular , the establishment of a therapeutic claim for prophylaxis has been justified , in some trials , by randomizing patients to either prophylaxis or on - demand treatment with the cfc under investigation . this approach merits scrutiny . it appears that the sponsoring manufacturers in these trials are seeking a label therapeutic claim , specifically , for prophylaxis . we would propose that , in the context of an rct for prophylaxis versus on - demand therapy , on - demand therapy has the status of a placebo . clearly , the question of the effectiveness of prophylaxis , or any aspect of aspect of cfc replacement therapy , can not be assessed with a true placebo , as this would constitute an example of the parachute scenario.84 the assignment of on - demand treatment as a control is not as drastic , but we would pose the following : a scrutiny of table 1 indicates that not all applications approved by the fda included randomized trials . we note that this was novoeight ( turoctocog alfa ) , which was subsequently approved by the ema with the same data but rejected by the g - b on the basis , among other reasons , that there were no comparative data with an acceptable comparator.the efficacy and superiority of prophylaxis versus on - demand therapy had been established for several decades before it was demonstrated , to the satisfaction of the cochrane collaboration , with a rct in children the joint outcome study.12 this study showed increased and progressive13 joint deterioration in children who bleed more frequently due to being randomized to the on - demand arm . the same investigators are now conducting a similar study the spinart trial in adults.85 the first year s results of a 3-year randomization shows that on - demand treatment is highly inferior to prophylaxis , begging the question as to why this trial is not discontinued on ethical grounds . all trials including the spinart trial continue to confirm the benefit of prophylaxis under all clinical circumstances.the basis for any rct is the existence of equipoise , defined as a state of genuine uncertainty about the value of a treatment course within the expert medical community.86 in this instance , against the background of evidence from the published rcts,12,74,87 the question arises is there genuine uncertainty in the minds of the clinical investigators performing rcts on new cfcs that these will be inferior in preventing bleeds when they are shown to be effective in treating bleeds ? we would contend that this is not the case . although it is a regulatory / industry mantra that bioequivalence can not be presumed for cfcs , there is no evidence , and no biological pathological hypothesis , that can give rise to such uncertainty.in an instance where a trial for an antiplatelet antithrombotic was placed on hold by the fda because of concerns about the choice of placebo as comparator,88 the agency did not accept concerns about the relatively high cost of a therapeutically similar comparator as justification for a placebo - controlled trial . subsequently , the trial design was pursued by the investigators who persuaded the fda to allow a placebo - controlled design , only to discontinue enrollment after the committee overseeing the trial found a 40% reduction in treatment versus placebo effects.89 this indicates that it may be investigators , and companies sponsoring the trials , who are influencing trial design.we note that the father of equipoise , benjamin freedman proposes that placebo controls may be justified in a number of circumstances , including when validated optimal treatments are not available to the treatment population because of cost - constraints.90 freedman hastens to add that this principle may only be applied when background conditions of justice exist within the health system where the trial is occurring and that when the system does not establish entitlement to even a minimum care level , the principle must not be used to justify the use of placebos on the poor.this brings us back to the case of new cfcs . scrutiny of the studies in table 1 , and others , which have employed a rct design to support a prophylaxis claim have included investigators and patient populations drawn from underprivileged countries where the level of hemophilia care is minimal . while it is not possible to assess which patients were used for the specific prophylaxis versus on - demand comparison , it is valid to assume that in most of the resourced environments this would be refuted on ethical grounds . we note the requirements for biomedical research in human subjects which are encapsulated in the declaration of helsinki.91 this declaration is unfortunately no longer supported by the fda for trials carried outside the united states,92 on the basis that adherence to the international conference guideline of good clinical practice.93 we would suggest that the international conference on harmonisation ( ich ) good clinical practice guideline is not an ethical code but a procedural regulatory manual based on a synthesis of the regulatory frameworks of the united states , japan , and europe , the major pharmaceutical markets . it does not include any of the declaration of helsinki s adjurations regarding the protection of subjects from unnecessary allocation to placebos . we have scrutinized the requirements of both mas and hta bodies , and we see no indication that , even in the event of a prophylaxis versus on - demand therapy rct being demanded , such a trial can not be done with two prophylaxis arms , one for the investigative treatment and one for an approved comparator . it is clear , for example , that the iqwig s current posture would allow a comparator to be used from all the hemophilia cfcs approved in germany . as required by the declaration of helsinki , any subjects on these clinical trials , including those in resource - poor countries , are to be kept on the treatment after the trial has ended , an issue of clear significance when comparator products are used . a scrutiny of table 1 indicates that not all applications approved by the fda included randomized trials . we note that this was novoeight ( turoctocog alfa ) , which was subsequently approved by the ema with the same data but rejected by the g - b on the basis , among other reasons , that there were no comparative data with an acceptable comparator . the efficacy and superiority of prophylaxis versus on - demand therapy had been established for several decades before it was demonstrated , to the satisfaction of the cochrane collaboration , with a rct in children the joint outcome study.12 this study showed increased and progressive13 joint deterioration in children who bleed more frequently due to being randomized to the on - demand arm . the same investigators are now conducting a similar study the spinart trial in adults.85 the first year s results of a 3-year randomization shows that on - demand treatment is highly inferior to prophylaxis , begging the question as to why this trial is not discontinued on ethical grounds . all trials including the basis for any rct is the existence of equipoise , defined as a state of genuine uncertainty about the value of a treatment course within the expert medical community.86 in this instance , against the background of evidence from the published rcts,12,74,87 the question arises is there genuine uncertainty in the minds of the clinical investigators performing rcts on new cfcs that these will be inferior in preventing bleeds when they are shown to be effective in treating bleeds ? although it is a regulatory / industry mantra that bioequivalence can not be presumed for cfcs , there is no evidence , and no biological pathological hypothesis , that can give rise to such uncertainty . in an instance where a trial for an antiplatelet antithrombotic was placed on hold by the fda because of concerns about the choice of placebo as comparator,88 the agency did not accept concerns about the relatively high cost of a therapeutically similar comparator as justification for a placebo - controlled trial . subsequently , the trial design was pursued by the investigators who persuaded the fda to allow a placebo - controlled design , only to discontinue enrollment after the committee overseeing the trial found a 40% reduction in treatment versus placebo effects.89 this indicates that it may be investigators , and companies sponsoring the trials , who are influencing trial design . we note that the father of equipoise , benjamin freedman proposes that placebo controls may be justified in a number of circumstances , including when validated optimal treatments are not available to the treatment population because of cost - constraints.90 freedman hastens to add that this principle may only be applied when background conditions of justice exist within the health system where the trial is occurring and that when the system does not establish entitlement to even a minimum care level , the principle must not be used to justify the use of placebos on the poor . scrutiny of the studies in table 1 , and others , which have employed a rct design to support a prophylaxis claim have included investigators and patient populations drawn from underprivileged countries where the level of hemophilia care is minimal . while it is not possible to assess which patients were used for the specific prophylaxis versus on - demand comparison , it is valid to assume that in most of the resourced environments this would be refuted on ethical grounds . we note the requirements for biomedical research in human subjects which are encapsulated in the declaration of helsinki.91 this declaration is unfortunately no longer supported by the fda for trials carried outside the united states,92 on the basis that adherence to the international conference guideline of good clinical practice.93 we would suggest that the international conference on harmonisation ( ich ) good clinical practice guideline is not an ethical code but a procedural regulatory manual based on a synthesis of the regulatory frameworks of the united states , japan , and europe , the major pharmaceutical markets . it does not include any of the declaration of helsinki s adjurations regarding the protection of subjects from unnecessary allocation to placebos . the declaration s intent is evident . we have scrutinized the requirements of both mas and hta bodies , and we see no indication that , even in the event of a prophylaxis versus on - demand therapy rct being demanded , such a trial can not be done with two prophylaxis arms , one for the investigative treatment and one for an approved comparator . it is clear , for example , that the iqwig s current posture would allow a comparator to be used from all the hemophilia cfcs approved in germany . as required by the declaration of helsinki , any subjects on these clinical trials , including those in resource - poor countries , are to be kept on the treatment after the trial has ended , an issue of clear significance when comparator products are used . the processes for the approval to market and provide access for medicines , including cfcs for hemophilia , have been traditionally distinct and separate . over the past decade , the continuing pressures on health care budgets , irrespective of their source , have contributed toward an ongoing convergence for some of these processes . concurrently , the implantation of the ebm paradigm for both these areas has revealed inadequacies which impede access to care and increased treatment costs . the increasingly diverse and competitive landscape of cfc products is leading to clinical trials that exhibit some doubts regarding their ethical basis . the criteria for approval of both authorization and reimbursement should be wider than the conventional rct framework . they should include the assessment of effectiveness as well as efficacy , in individual patients through mechanisms such as n of 1 trials . such mechanisms , when properly conducted , promise to actually enhance the evidence base of cfc therapies , relative to current requirements for patient numbers , which are arbitrarily specified and have caused mounting treater concern.94 we support the use of patient - centered outcomes by hta agencies when they are synthesizing recommendations regarding the added benefit coming from the plethora of new products . such outcomes should include measures of health - related quality of life , which may need to be more nuanced than the instruments used currently by these agencies . all bodies involved in the delivery of cfc treatments should embed ethical principles in their assessments , which negate harm to patients , and these should be transparent to manufacturers seeking a therapeutic claim and to investigators responsible for clinical trials . these concepts , synthesized in figure 1 , should optimize patient and societal outcomes in a compassionate and efficient health care system .
metaplastic breast carcinoma ( mbc ) is rare with an incidence less than 1% of all breast malignancies . it denotes tumors with mixed primary epithelial and sarcomatous components as well as mixed adenocarcinoma with squamous cell carcinoma and tends to be larger in size when diagnosed , with higher tumor grade and triple - negative for hormone receptors . expression of estrogen , progesterone , and human epidermal growth factor receptor 2 ( her-2/neu ) receptors has been reported in 12% , 9% , and 15% , respectively , of patients with mbc . the rare incidence of muscular metastasis in all malignancy , as in breast carcinoma , has been reported to range from 0.2% to 17.5% in large autopsy series . surgical excision was recommended in selected patients such as those with painful , isolated , easily approachable mass . herein , we report a rare case of mbc with multiple muscle metastasis ( mmm ) . a 65-year - old taiwanese woman had a history of debulking surgery , bilateral oophorectomy with omentectomy , total anterior hysterectomy with radical pelvic lymph nodes dissection due to ovarian carcinoma ( mucinous - type carcinoma , stage ic ) 1 year ago . patient 's medical compliance was poor and failed to complete her chemotherapy ( cyclophosphamide 750 mg / m , carboplatin 300 mg / m ) . recently , she noted a palpable right breast mass , which enlarged rapidly to about 15 cm in size and nearly occupied the whole right breast in 2 months . neoadjuvant chemotherapy with the regimens of taxotere ( 75 mg / m ) , epirubicin ( 75 mg / m ) , and cyclophosphamide ( 500 mg / m ) was given for 6 cycles with poor response , followed by a modified radical mastectomy ( mrm ) with dissection of axillary lymph nodes and skin grafting . the histopathologic examination revealed a metaplastic carcinoma with myoepithelial and squamous differentiation associated with adenomyoepithelioma ( figure 1a c ) . immunohistochemistry study showed that the tumor cells are positive for epithelial markers cytokeratin ( ae1/ae3 ) stain , and myoepithelial markers , including cytokeratin 5/6 ( ck 5/6 ) , p63 , and s100 stains ( figure 2a e ) . expressions of hormone receptors , including er , pr , and her-2/neu , were all negative ( figure 3a d ) . the dissected axillary lymph nodes showed metastastic carcinoma with hormone triple - negative in 3 out of 26 nodes . ( a ) right lower field shows adenomyoepithelioma with an inner layer of eosinophilic ductal cells and an outer layer of clear myoepithelial cells arranged in a glandular pattern . at the periphery ( left upper field ) , there is myoepithelial carcinoma arranged in cords and nests ( h&e stain , 200 ) . ( b ) tumor cells have abundant eosinophilic cytoplasm and hyperchromatic nuclei with various shapes , including round , ovoid , plasmacytoid , polygonal , and spindle shapes ( h&e stain , 200 ) . ( c ) a part of the breast tumor reveals squamous cell carcinoma ( h&e stain , 200 ) . ( d ) the metastatic tumor over the left forearm reveals round - shaped to spindle - shaped cells , similar to primary tumor shown in ( b ) ( h&e stain , 200 ) . primary mbc ( a , h&e stain , 100 ) reveals diffuse expression of cytokeratin ( b , 100 ) , ck5/6 ( c , 100 ) , p63 ( d , 100 ) , and is focally positive for s100 protein ( e , 100 ) immunohistochemically . the tumor cells of metastastic forearm ( f , h&e stain , 100 ) are also extensively reactive to cytokeratin ( g , 100 ) and show nuclear staining of p63 ( h , 100 ) . both the primary mbc ( a , 100 ) and metastastic forearm ( e , 100 ) were negative for er ( b and f , 100 ) , pr ( c and g , 100 ) , and her-2/neu ( d and h , 100 ) immunostains . seven months later , the patient complained about pain and numbness over left forearm , right lower back , and bilateral lower extremities . sonography of left forearm revealed a tumor with hypervascularity and cystic component , measuring about 6 5 cm in size . positron emission tomography / computed tomography ( pet / c ) scan showed hypermetabolic masses in the left proximal forearm , right psoas , and quadratus lumborum muscles ( figure 4 ) . surgical excision of left forearm mass ( figure 5 ) was performed for symptomatic relief of intractable pain . however , excision for the right psoas and quadratus lumborum muscles lesion was not feasible due to high risks of morbidities . histopathologic examination of left forearm tumor revealed metastatic carcinoma , similar to previous breast cancer with negative er , pr , and her-2/neu immunostaining ( figure 3e h ) , positive for epithelial markers cytokeratin ( ae1/ae3 ) stain , and myoepithelial markers as p63 ( figure 2f h ) . further boosted radiotherapy was added , but the metastatic tumors in the right psoas and quadratus lumborum muscles progressed in size . eventually , lung metastasis occurred 10 months and she died 12 months later after mrm . pet / ct scan showed hypermetabolic masses in the left proximal forearm and right psoas to quadratus lumborum muscles . gross picture of the left forearm tumor : whitish in color , well encapsulated , 5 3 3 cm in size . ck = cytokeratin , er = estrogen receptor , h&e = hematoxylin and eosin , her-2/neu = human epidermal growth factor receptor 2 , mbc = metaplastic breast carcinoma , pet / ct = positron emission tomography / computed tomography , pr = progesterone receptor . the breast cancer can metastasize anywhere in the body , primarily to the bone , lung , regional lymph nodes , liver , and brain , with the most common site being the bone . muscle metastasis is rare in breast malignancies compared with bone metastasis and often manifested as a disseminated disease with multiple organ metastasis . pet / ct is considered the most sensitive tool for the diagnosis of muscular metastasis , and has been reported to detect more unsuspected muscular metastasis than computed tomography and magnetic resonance imaging . intramuscular hot spots on pet / ct scans should be considered a sign of metastatic lesions even in the absence of any abnormality on computed tomography scans . muscular pain is a sentinel sign of bone or skeleton muscle metastasis and should be considered a premonition for mbc , as in our case , who died 3 months later after the diagnosis of mmm . skeletal muscle is relatively resistant to metastatic disease because of its hostile microenvironment , such as muscle motion resulting in mechanical tumor destruction , power of hydrogen from inhospitable muscle , muscle 's ability to remove tumor - produced lactic acid associated with angiogenesis , and activation of lymphocytes and natural killer cells in skeletal muscle . mbc invasion directly into pectoral muscles is uncommon , but distal skeleton muscle metastasis is even more rarely . sites of metastasis that has been reported include extraocular , paraspinal , gluteal , and abdominal wall muscles , which induced symptoms including vision diplopia or intractable pain according to tumor locations . breast malignancy is treated as a systemic disease because the pathophysiological mechanism of metastasis is thru lymphatic drainage to systemic lymph nodes and hematogenous spread to the distal areas . however ; the treatment in breast carcinoma , including mbc , with mmm is according to pathologic immunohistochemistry and hormone receptors to choose 3 fundamentally different kinds of drugs , including cytotoxic chemotherapies , hormone therapies , and targeted therapies . in our review of english literature , this is the first case of mbc with distal mmm resulting in intractable pain and numbness , treated with surgical excision for symptomatic relief . biopsy is adequate for diagnosis ; however , total surgical excision , although not curable , should be adopted for symptomatic relief of intractable pain and numbness . the prognosis of mbc with mmm is very poor in our patient who died 1 year after mrm , and the treatment goals are currently for symptomatic relief . chemotherapy and radiotherapy ( skin burn , muscle contracture complications ) are commonly used and might also be effective . in conclusion , mbc with isolated and easily approachable metastatic mass , total surgical excision is an alternative option for pain relief with improvement of life quality .
stress - induced cardiomyopathy ( scmp ) is a nonischemic reversible cardiomyopathy characterized uniquely by a left ventricular ( lv ) wall motion abnormality termed " apical ballooning " and the prognosis is generally excellent.1)2 ) however , several critical complications are possible . the formation of lv thrombus is one of these rare complications which can result in systemic embolism.3 ) here , we report a case of scmp complicated by lv mural thrombus which gradually increased in mobility as lv contractility recovered and which eventually required surgical removal . a 66-year - old woman who presented with severe dysphagia visited our emergency room . on admission her mental status was alert , and she had a blood pressure of 115/76 mm hg , a respiratory rate of 20 per minute , a pulse rate of 96 per minute , and a body temperature of 36.4. she did not have any history of heart disease , hypertension or diabetes mellitus , but had undergone subtotal gastrectomy due to stomach cancer . urgent gastrofibroscopy was performed and a piece of chicken trapped at the lower esophagus was removed . additionally , a small polyp was found at the previous surgical anastomosis site which subsequently confirmed pathologically as early gastric cancer . the patient also complained of mild dyspnea and diffuse chest discomfort on admission , and an electrocardiogram ( ecg ) showed st elevation with qt prolongation ( qtc = 480 msec ) , and t wave inversion on leads v2 - 6 ( fig . troponin t and creatinine kinase - mb ( ck - mb ) levels were elevated to 0.29 ng / ml and 20.46 ng / ml , respectively ( reference range ; < 0.01 ng / dl for troponin t , < 3.61 ng / ml for ck - mb ) . transthoracic echocardiography ( tte ) revealed mid and apical lv segmental wall motion abnormalities with apical ballooning that did not correspond to a coronary artery territory , as well as a left ventricular ejection fraction of 41% ( lv end - diastolic volume / lv end - systolic volume = 84/49 ml ) ( fig . in addition , a 19 18-mm - sized non - mobile echogenic mass suspicious for a mural thrombus was found at the apex of the left ventricle ( fig . as the features were consistent with scmp complicated by lv mural thrombus , anticoagulation as well as conventional heart failure therapy was initiated . 1b ) and follow - up tte demonstrated resolution of the mid and apical lv segmental wall motion abnormalities and fully recovered lv systolic function . however , the lv mural thrombus had partially detached from the lv wall with recovery of lv contractility and was adherent to the ventricular wall by a narrow stalk . the remarkably increased mobility of the lv thrombus was thought to carry a very high thromboembolic risk ( fig . the mass , which had its base attached to the trabecular endocardium of the apico - inferior wall of the lv cavity , was completely removed by suction tip through the aortic valve . the mass appeared to be an amorphous bizarre shape , measuring 1.7 1.2 0.7 cm in size ( fig . the patient recovered without any other complications and was discharged 18 days after the operation . scmp was first described in japan in 1991.1 ) it is characterized by a reversible lv systolic dysfunction with distinct ' apical ballooning ' accompanied by slightly increased cardiac enzyme levels and electrocardiographic changes including qt prolongation , st elevation , or t wave inversion . despite many investigations , the mechanism of scmp is not yet fully understood and many investigators have suggested a catecholamine - mediated mechanism as the cause of scmp.2 ) although the long term prognosis of patients with scmp is usually excellent,3 ) well - known complications of scmp are cardiogenic shock , congestive heart failure and sudden cardiac death . besides these morbidity , ventricular tachycardia , lv rupture , apical thrombus formation and embolism have been reported as rare complications of scmp.3 ) according to previous studies , the prevalence of lv thrombus in cases of scmp is approximately 2.5% , and the occurrence rate of embolic complications in this condition is reported to be as high as 33% despite proper anticoagulation therapy.4)5 ) the pathogenesis of lv thrombus formation in scmp is still not fully known and some theories have been suggested hemostasis,6 ) catecholamine induced platelet activation,7)8 ) and catecholamine induced cardiomyocyte damage9 ) as underlying mechanism . the incidence of lv thrombus formation in anterior ami was reported to be 30 - 40% before the era of reperfusion therapy,10)11 ) and the embolic rate of the condition was 10 - 15%.12 ) the relatively low incidence of lv thrombus formation ( 2.5% vs. 30 - 40% ) and high embolic risk ( 33% vs. 10 - 15% ) in scmp compared to ami might be related to faster and better recovery of lv function . as lv wall motion recovers , the mobility of mural thrombus may increase rapidly by partial detachment from the ventricular wall , which can lead to increased embolic risk . in scmp , the mural thrombus that was initially immobile can become highly mobile within a short time . and this phenomenon can explain why initially mobile lv thrombus in scmp is not associated with a higher embolic event rate than initially immobile lv thrombus in previous studies.5 ) of 11 previously reported cases of scmp that were complicated by a mobile lv thrombus , lv thrombus was immobile or even absent in the initial examination of 6 cases.13)14)15)16)17)18)19)20 ) thus , the embolic risk of lv thrombus in scmp may not be determined based on the initial mobility of the thrombus . in all reported cases with scmp , anticoagulation therapy was promptly initiated after the detection of the thrombus . despite appropriate anticoagulation therapy , however , cardiac embolic events occurred in one third of patients with scmp complicated by lv thrombus . in contrast , when the thrombus was removed by surgery using a trans - apical approach there was no embolic event in two reported cases.19)20 ) in the present case , a trans - aortic approach following oblique aortotomy was performed . although the apical approach can provide good and easy access to the thrombus , myocardial injury is inevitable . using a trans - aortic approach following aortotomy , visualization or extraction of the thrombus through the aortic valve may be somewhat difficult , but myocardial damage can be avoided , and this approach may be more beneficial for the heart , especially in a clinical setting . to the best of our knowledge , this is the first reported case of scmp complicated by lv thrombus which was surgically removed by a trans - aortic approach after oblique aortotomy . although lv thrombus is a rare complication of scmp , the embolic risk is relatively high because of rapid change of thrombus mobility . therefore , short - term tte follow - up after anticoagulation therapy might be recommended in this clinical setting , and if the thrombus becomes highly mobile as lv systolic function improves , surgical removal could be a reasonable therapeutic option .
the current study is a secondary analysis of baseline data from a randomized clinical trial of a school - based intervention for youth at risk for type 2 diabetes ( 12 ) . seventh grade youth from six schools in a new england city were invited to participate if they had bmi 85th percentile and a family member with diabetes . seventh graders were targeted because of their increased risk for type 2 diabetes at puberty . youth were excluded if they had an existing chronic disease ( other than asthma ) or were involved in another clinical trial . for interested students , parents were contacted to describe the study and obtain informed consent in line with university institutional review board requirements . between april 2004 and december 2006 , 380 students were screened for eligibility , with 234 students meeting the inclusion criteria and 28 families refusing to enroll ( the most common reason for refusal was lack of interest ) . of the 206 students who consented / assented to participate , 4 students were later ineligible because of low bmi , 1 was promoted to eighth grade , 1 was expelled , 7 refused , and 5 moved after consenting ; these youth were not statistically different in sex and age from participants . data for the remaining 188 participants were collected by trained research staff in school - based clinics . for purposes of these analyses , data from 15 students who did not self - categorize as either hispanic or african american were excluded before analyses . their representative groups were white ( n = 5 ) , more than one race ( n = 5 ) , other ( n = 1 ) , and unspecified ( n = 4 ) . data analyses were performed for the remaining 173 participants . weight in kilograms and percent body fat were measured with a scale and body composition analyzer ( model bf-350 ; tanita corporation of america , arlington heights , il ) . the leg - to - leg bioimpedance method was used to determine percent body fat . waist circumference was determined at the umbilicus at the end of a normal expiration , and hip measures were taken at the widest portion of the hip using a gulick tape measure . oral glucose tolerance tests ( ogtts ) were performed with a standard glucose load ( 1.75 g glucose / kg body wt up to a maximum of 75 g ) ( trutol 100 ; nerl diagnostics , east providence , ri ) . insulin resistance was estimated by homeostasis model assessment ( homa ) of insulin resistance ( homa - ir ) using the equation , homa - ir = fasting insulin ( microunits per milliliter ) fasting glucose ( millimoles per liter)/22.5 . a value > 2.2 is indicative of insulin resistance . fasting a1c levels were determined using the dca 2000 analyzer ( bayer , tarrytown , ny ) . lipids , including total cholesterol , hdl cholesterol , and triglycerides were measured ( cholestech ldx system ; cholestech , hayward , ca ) . ldl cholesterol was calculated using the formula , ldl cholesterol = ( total cholesterol hdl cholesterol ) ( triglycerides/5 ) ( 14 ) . those who had laboratory values within the prediabetes range were referred to their primary care providers for further evaluation and follow - up . the health behavior questionnaire ( 15 ) was used to measure dietary intention ( 13 items , intentions to choose foods considered heart healthful ) , usual food choices ( 14 items , usual food selections ) , perceived support for physical activity ( 18 items , social support for physical activity among family members , teachers , and friends ) , and social reinforcement for healthy food choices ( 7 items , social support for heart - healthy food from family members , teachers , and friends ) . these scales use dichotomous forced - choice formats among two foods or yes / no . positive values indicate healthier choices or greater support for healthy choices , and negative values indicate poorer choices or support . internal consistency values for the current study are as follows : dietary intent , = 0.69 ; usual food choices , = 0.68 ; support for physical activity , = 0.60 ; and social reinforcement for healthy food choices , = 0.87 . in addition , the dietary self - efficacy scale ( 5 items , e.g. , how sure are you that you can eat a baked potato instead of french fries ? ) and physical self - efficacy scale ( 5 items , e.g. , how sure are you that you can choose to jog during recess ? these scales use a 3-point likert - type scale , with 1 = not sure , 2 = a little sure , and 3 = very sure . internal consistency for the current study was = 0.85 for dietary self - efficacy and = 0.64 for physical activity self - efficacy . the revised godin - shephard activity survey ( 16 ) measures self - reported activity . subjects report the number of times in an average week that they spent > 15 min in activities classified as mild ( 3 mets ) , moderate ( 5 mets ) , or strenuous ( 9 mets ) . the met is the standard unit of work measure used in exercise physiology that involves the ratio of oxygen consumption , body weight , and unit of time . the number of times students engaged in each activity is multiplied by the met level and summed to provide a weekly total . dietary intake was estimated by averaging two 24-h recalls ( one weekend and one weekday ) . interviews were conducted at school by a registered dietitian or diet technician , and food models were used to improve estimation of portion sizes . nutrient intake was analyzed using nutritionist pro software ( version 2.4.1 ; first data bank , san bruno , ca ) . values were compared with national health and nutrition examination survey 20012002 average intakes and dietary reference intakes for age and sex but not race / ethnicity ( 17 ) . the effects of sex and ethnicity were examined on all available variables , using tests for noncontinuous variables and standard least - squares anova models for continuous variables . to correct for skewness , homa values were transformed using the logarithm function . weight in kilograms and percent body fat were measured with a scale and body composition analyzer ( model bf-350 ; tanita corporation of america , arlington heights , il ) . the leg - to - leg bioimpedance method was used to determine percent body fat . waist circumference was determined at the umbilicus at the end of a normal expiration , and hip measures were taken at the widest portion of the hip using a gulick tape measure . oral glucose tolerance tests ( ogtts ) were performed with a standard glucose load ( 1.75 g glucose / kg body wt up to a maximum of 75 g ) ( trutol 100 ; nerl diagnostics , east providence , ri ) . insulin resistance was estimated by homeostasis model assessment ( homa ) of insulin resistance ( homa - ir ) using the equation , homa - ir = fasting insulin ( microunits per milliliter ) fasting glucose ( millimoles per liter)/22.5 . a value > 2.2 is indicative of insulin resistance . fasting a1c levels were determined using the dca 2000 analyzer ( bayer , tarrytown , ny ) . lipids , including total cholesterol , hdl cholesterol , and triglycerides were measured ( cholestech ldx system ; cholestech , hayward , ca ) . ldl cholesterol was calculated using the formula , ldl cholesterol = ( total cholesterol hdl cholesterol ) ( triglycerides/5 ) ( 14 ) . those who had laboratory values within the prediabetes range were referred to their primary care providers for further evaluation and follow - up . the health behavior questionnaire ( 15 ) was used to measure dietary intention ( 13 items , intentions to choose foods considered heart healthful ) , usual food choices ( 14 items , usual food selections ) , perceived support for physical activity ( 18 items , social support for physical activity among family members , teachers , and friends ) , and social reinforcement for healthy food choices ( 7 items , social support for heart - healthy food from family members , teachers , and friends ) . these scales use dichotomous forced - choice formats among two foods or yes / no . positive values indicate healthier choices or greater support for healthy choices , and negative values indicate poorer choices or support . internal consistency values for the current study are as follows : dietary intent , = 0.69 ; usual food choices , = 0.68 ; support for physical activity , = 0.60 ; and social reinforcement for healthy food choices , = 0.87 . in addition , the dietary self - efficacy scale ( 5 items , e.g. , how sure are you that you can eat a baked potato instead of french fries ? ) and physical self - efficacy scale ( 5 items , e.g. , how sure are you that you can choose to jog during recess ? these scales use a 3-point likert - type scale , with 1 = not sure , 2 = a little sure , and 3 = very sure . internal consistency for the current study was = 0.85 for dietary self - efficacy and = 0.64 for physical activity self - efficacy . the revised godin - shephard activity survey ( 16 ) measures self - reported activity . subjects report the number of times in an average week that they spent > 15 min in activities classified as mild ( 3 mets ) , moderate ( 5 mets ) , or strenuous ( 9 mets ) . the met is the standard unit of work measure used in exercise physiology that involves the ratio of oxygen consumption , body weight , and unit of time . the number of times students engaged in each activity is multiplied by the met level and summed to provide a weekly total . dietary intake was estimated by averaging two 24-h recalls ( one weekend and one weekday ) . interviews were conducted at school by a registered dietitian or diet technician , and food models were used to improve estimation of portion sizes . nutrient intake was analyzed using nutritionist pro software ( version 2.4.1 ; first data bank , san bruno , ca ) . values were compared with national health and nutrition examination survey 20012002 average intakes and dietary reference intakes for age and sex but not race / ethnicity ( 17 ) . the effects of sex and ethnicity were examined on all available variables , using tests for noncontinuous variables and standard least - squares anova models for continuous variables . to correct for skewness , homa values were transformed using the logarithm function . the 173 adolescents ( 80 male and 93 females ) were 1115 years old ( 12.9 0.7 years ) . of the total , 84 ( 49% ) were hispanic , and 89 ( 51% ) were african american . we found significant differences between racial / ethnic groups related to guardians ' marital status ( p = 0.002 ) , education ( p < 0.001 ) , and self - rated health score ( p = 0.002 ) , with hispanic families more likely to report marriage , a lower level of education ( i.e. , less than high school ) , and poorer self - rated health ( scores ranged from 1 = poor to 4 = excellent ) . as seen in table 1 , average bmi ( 31.6 6.4 kg / m ) and percent body fat ( 39.5 10.6% ) were high . as shown in table 2 , fasting insulin levels ( 37.9 21.3 u / ml ) were high , and 100% of the participants had high homa - ir ( 8.5 5.2 ) . glucose ( 0-h ) , a1c , triglycerides , total cholesterol , hdl cholesterol , and ldl cholesterol were within normal ranges . prediabetes ( glucose 100125 mg / dl [ 4.66.9 mmol / l ] ) was present in 15% ( 26 participants ) ( 18 ) . anthropometric measures : total and by ethnicity and sex metabolic measures : total and by ethnicity and sex as seen in table 3 , adolescents reported fairly high support for physical activity ( 9.6 5.4 ) . however , they reported fairly low self - efficacy for physical activity ( 2.4 2.3 ) and perceived benefits for activity ( 3.9 0.70 ) . adolescents reported fairly high self - efficacy for diet ( 6.4 6.0 ) and dietary knowledge ( 7.0 5.8 ) , but they reported poor usual food choices ( 1.7 5.6 ) and dietary intent ( 1.5 5.7 ) . health behaviors : total and by ethnicity and sex self - reported energy intake was lower than expected ( table 4 ) ; however , the percentage of kilocalories from fat was > 30% for the entire sample . participants had lower intake than the recommended average requirements for vitamin e , vitamin k , calcium , potassium , fiber , and magnesium ( 17 ) . dietary reference intakes were met for iron , sodium , protein , and carbohydrate . compared with the average u.s . child aged 913 years , our students reported lower intake of all nutrients except vitamins a and c ( 17 ) . nutrient intake : total and by ethnicity and sex as seen in tables 1 and 2 , age , height , fasting glucose , and 2-h ogtt were significantly higher in male than in female participants ( all p < 0.01 ) . as expected , female participants had higher percent body fat than male participants , but there were no sex differences in the presence of prediabetes . table 3 indicates that male participants reported greater physical activity and healthier usual food choices ( both p = 0.005 ) . although kilocalorie intake was not significantly different between sexes ( table 4 ) , male participants had higher intake of potassium , calcium , phosphorus , and magnesium than did female participants ( all p = 0.01 ) . none of the female participants met the dietary reference intake values for these nutrients and male participants met only the suggested levels for phosphorus ( 17 ) . as seen in tables 1 and 2 , african american youth were significantly taller and heavier than hispanic youth , whereas hispanic youth had higher triglycerides and lower hdl cholesterol ( all p = 0.05 ) . hispanic youth reported significantly less physical activity and less diet self - efficacy than african american youth ( table 3 ) . hispanic youth reported significantly higher intake of total fat , monounsaturated fat , and polyunsaturated fat than african american youth ( table 4 ) . with loghoma as the dependent variable , we fitted an ancova model to test for main effects and interactions , using a backward elimination algorithm to arrive at the best model . the final model ( f1,144 = 3.69 , p = 0.01 , r = 0.071 ) included sex , bmi , and the sex bmi interaction , with a steeper slope for girls than for boys . ethnicity was not retained in the best model as a significant predictor for insulin resistance . the 173 adolescents ( 80 male and 93 females ) were 1115 years old ( 12.9 0.7 years ) . of the total , 84 ( 49% ) were hispanic , and 89 ( 51% ) were african american . we found significant differences between racial / ethnic groups related to guardians ' marital status ( p = 0.002 ) , education ( p < 0.001 ) , and self - rated health score ( p = 0.002 ) , with hispanic families more likely to report marriage , a lower level of education ( i.e. , less than high school ) , and poorer self - rated health ( scores ranged from 1 = poor to 4 = excellent ) . as seen in table 1 , average bmi ( 31.6 6.4 kg / m ) and percent body fat ( 39.5 10.6% ) were high . as shown in table 2 , fasting insulin levels ( 37.9 21.3 u / ml ) were high , and 100% of the participants had high homa - ir ( 8.5 5.2 ) . glucose ( 0-h ) , a1c , triglycerides , total cholesterol , hdl cholesterol , and ldl cholesterol were within normal ranges . prediabetes ( glucose 100125 mg / dl [ 4.66.9 mmol / l ] ) was present in 15% ( 26 participants ) ( 18 ) . anthropometric measures : total and by ethnicity and sex metabolic measures : total and by ethnicity and sex as seen in table 3 , adolescents reported fairly high support for physical activity ( 9.6 5.4 ) . however , they reported fairly low self - efficacy for physical activity ( 2.4 2.3 ) and perceived benefits for activity ( 3.9 0.70 ) . adolescents reported fairly high self - efficacy for diet ( 6.4 6.0 ) and dietary knowledge ( 7.0 5.8 ) , but they reported poor usual food choices ( 1.7 5.6 ) and dietary intent ( 1.5 5.7 ) . health behaviors : total and by ethnicity and sex self - reported energy intake was lower than expected ( table 4 ) ; however , the percentage of kilocalories from fat was > 30% for the entire sample . participants had lower intake than the recommended average requirements for vitamin e , vitamin k , calcium , potassium , fiber , and magnesium ( 17 ) . dietary reference intakes were met for iron , sodium , protein , and carbohydrate . compared with the average u.s . child aged 913 years , our students reported lower intake of all nutrients except vitamins a and c ( 17 ) . nutrient intake : total and by ethnicity and sex as seen in tables 1 and 2 , age , height , fasting glucose , and 2-h ogtt were significantly higher in male than in female participants ( all p < 0.01 ) . as expected , female participants had higher percent body fat than male participants , but there were no sex differences in the presence of prediabetes . table 3 indicates that male participants reported greater physical activity and healthier usual food choices ( both p = 0.005 ) . although kilocalorie intake was not significantly different between sexes ( table 4 ) , male participants had higher intake of potassium , calcium , phosphorus , and magnesium than did female participants ( all p = 0.01 ) . none of the female participants met the dietary reference intake values for these nutrients and male participants met only the suggested levels for phosphorus ( 17 ) . as seen in tables 1 and 2 , african american youth were significantly taller and heavier than hispanic youth , whereas hispanic youth had higher triglycerides and lower hdl cholesterol ( all p = 0.05 ) . hispanic youth reported significantly less physical activity and less diet self - efficacy than african american youth ( table 3 ) . hispanic youth reported significantly higher intake of total fat , monounsaturated fat , and polyunsaturated fat than african american youth ( table 4 ) . with loghoma as the dependent variable , we fitted an ancova model to test for main effects and interactions , using a backward elimination algorithm to arrive at the best model . the final model ( f1,144 = 3.69 , p = 0.01 , r = 0.071 ) included sex , bmi , and the sex bmi interaction , with a steeper slope for girls than for boys . ethnicity was not retained in the best model as a significant predictor for insulin resistance . in this study , we described metabolic indicators and health behaviors in a sample of african american and hispanic youth at high risk for type 2 diabetes by virtue of bmi and family history . the average adolescent in our study manifested insulin resistance , while maintaining normal glucose levels , but without the elevated lipids associated with metabolic syndrome ( 19 ) . in addition , the relationship between bmi and insulin resistance was stronger for girls than for boys . although our participants were selected for being at high risk for type 2 diabetes , the prevalence of insulin resistance at 100% is surprising and differs from a previous study of overweight youth , in which the prevalence of insulin resistance was only 25% ( 20 ) . however , our sample may have been at higher risk because of a family history of diabetes , which was not a requirement in other studies . the high percentage of hispanic youth in our population may have also influenced the increased prevalence of insulin resistance , because a genetic predisposition to diabetes has been reported in some hispanic populations ( 3 ) . our data suggest that , despite lower percent body fat , male youth had higher blood glucose levels at fasting and after a glucose load than did female youth . female youth reported poorer food choices and lower intake of calcium , similar to other studies ( 21 ) , suggesting that they engage in less healthy eating behaviors than male youth . it is important to note , however , that underreporting of total kilocalories consumed is common , particularly among overweight adolescent girls ( 22 ) . in addition , female youth reported engaging in less physical activity than male youth , in line with studies showing a sex difference in physical activity within ethnic groups and across ages ( 10 ) . specifically , a precipitous decline in activity levels has been reported in girls between ages 9 and 19 ( 23 ) . because inactivity is related to overweight , programs to improve girls ' knowledge and attitudes about the benefits of activity and finding sex - acceptable ways to increase high - intensity activity among girls should be priorities . in our sample , hispanic youth had poorer lipid profiles than african american youth , including higher triglyceride and lower hdl cholesterol levels . this finding is similar to a recent study , in which hispanic youth had twice the prevalence of metabolic syndrome than non - hispanic white youth , with significantly higher levels of triglycerides and lower hdl cholesterol ( 24 ) . it is possible that the difference in lipid profile is related to diet ; hispanic youth in our study reported a higher fat intake than african american youth , and it has been shown that hispanic youth with greater anglo acculturation have diets higher in fat ( 11 ) . in addition , hispanic youth reported lower diet self - efficacy and less physical activity than african american youth . few studies have examined activity levels in overweight minority groups , but there is evidence that hispanic and african american girls have the lowest levels of moderate to vigorous activity compared with those of other racial / ethnic groups ( 10 ) . higher dietary fat intake and lower levels of physical activity reported by the hispanic youth in our sample suggest that they have poorer health behaviors related to weight and cardiovascular health than african american youth . further research is needed to determine the effects of acculturation on nutrition , activity , and behavioral and metabolic parameters in high - risk youth . nutrition , activity , and health behavior measures were based on self - report and may be subject to socially desirable responses . the sample was self - selected to participate in an intervention trial and was obtained from a population with a high prevalence of overweight ( 3 ) . therefore , relationships among metabolic parameters and health behaviors may not generalize to other populations . in addition , despite reminders , it is possible that some participants were not fasting for blood draws , resulting in higher rates of prediabetes and homa - ir . last , we did not measure acculturation , which may help to explain some results for the hispanic youth . despite these limitations , our findings suggest the need to develop strategies to identify insulin resistance , such as periodic screening with an ogtt , in high - risk youth , especially hispanic and female youth . few affordable and accessible child - focused programs are available or have proven to be very successful . researchers and practitioners , therefore , have the responsibility to develop interventions to prevent and treat overweight , insulin resistance , prediabetes , type 2 diabetes , and their consequences in this population . it is not yet clear whether family involvement is necessary for reducing adolescent obesity , but previous studies have shown that in younger children , promoting family reinforcement of healthy behaviors and increasing physical activities that are attractive to specific ethnic and sex groups is important ( 25 ) .
a recent article from the group in berlin reports on a retrospective review of observational data comparing their experience using tranexamic acid as an enforced alternative to aprotinin . their data suggest an increase in morbidity and mortality in the tranexamic acid treated patients . the first was to cause all of the safety and efficacy data for aprotinin to be independently examined by regulatory authorities in both north america and europe . this process is coming to its conclusion and it is anticipated that , based on a positive benefit - risk ratio , the canadian authority will renew the marketing license for aprotinin before the end of this year . the european agency is also starting a review but it is not anticipated this process will be completed until 2011 . the second effect of the withdrawal of aprotinin was that clinicians had to find an alternative blood - sparing agent for use during major cardiac surgery . epsilon aminocaproic acid has no approval in europe or canada for human administration , leading to the exclusive use of tranexamic acid in these countries . this article showed a plateau effect on drains losses with a total dose of 3 grams tranexamic acid but with no observed effect on transfusions . the second problem is that there is no evidence for a benefit of tranexamic acid to reduce transfusion burden in patients at higher risk for transfusions , such as those taking aspirin prior to surgery and those having prolonged bypass periods associated with more complex , typically combined valve and revascularisation surgery . the current article mirrors a meta - analysis showing re - exploration for bleeding is reduced by aprotinin but not tranexamic acid in such patients . finally , and of crucial importance , there have never been any specifically powered studies to investigate the safety of tranexamic acid . over the past months a number of articles have suggested the use of tranexamic acid is not without risk . in an extension of a previous analysis from toronto , the authors concluded that mortality after cardiac surgery other than primary revascularisation was greater in those patients given tranexamic acid compared to those given high dose aprotinin . an increase in mortality when tranexamic acid was given instead of aprotinin is also a conclusion from the current article . neurological outcomes is a long standing safety concern as we know administration of tranexamic acid is associated with clinically significant cerebral vasospasm with acute cerebral haemorrhage . the current article shows a three - fold increase in patients having seizures who were allocated to receive high dose tranexamic acid as part of their management during surgery where a cardiac chamber was opened . the statistical analysis used in the current study was similar to that used to show a deleterious effect of aprotinin , which has subsequently been shown to be flawed . the lysine analogues have marked structural homology with gamma amino butyric acid ( gaba ) and act as competitive inhibitors in the central nervous system . second , several other groups have independently made the observation of increased seizure activity , mainly in patients having open cardiac chamber procedures . the european regulatory authority is currently deliberating on not only the licensing for aprotinin but also tranexamic acid . with the increasing body of evidence , it is becoming clearer that aprotinin therapy is of greatest benefit in patients at highest risk ( the originally intended patient population ) . the data also suggest that tranexamic acid in a dose of about 3 to 5 grams may be useful to reduce transfusion burden in patients not taking platelet active medication and having primary myocardial revascularisation . this patient population appears not to have observed safety issues when tranexamic acid was administered . the current study adds to the data questioning if tranexamic acid administration has a place in higher risk cardiac surgery and especially in surgery where a cardiac chamber is opened . in the past 5 years dr has acted as a paid consultant to bayer schering , cubist and curacyte , the pharmaceutical companies who have the potential blood - sparing agents aprotinin under review and eccallentide and cu 2010 respectively under development .

spinal cord injury ( sci ) is a damage to the spinal cord that results in the loss of mobility and sensation below the level of injury . the disorder is characterized according to the amount of functional loss , sensational loss , and inability to stand and walk . for example there are 12.7 and 59 new cases per million in france and the united states of america , respectively . it may be the result of trauma , especially motor vehicle accident , penetrating injuries , or diseases . as a result of this type of disability , they can transport themselves from one place to another using a manual wheelchair with a speed and energy expenditure similar to normal subjects . although , the use wheelchair provides mobility to such patients , it is not without problems . the main problems are the restriction to mobility from architectural features in the landscape , and a number of health issues due to prolonged sitting . decubitus ulcers , osteoporosis , joint deformities , especially hip joint adduction contracture , can result from prolonged wheelchair use . it has been suggested that by decreasing urinary tract infections , improving cardiovascular and digestive systems functions and psychological health walking is a good exercise for paraplegics in order to maintain good health . in contrast , most patients prefer not to use an orthosis , or use it occasionally . the main problem with orthosis use is the high energy demands it places on the users during ambulation . in contrast to mobility speed with a wheelchair , the mobility speed of a sci patient with an orthosis is significantly less than that of normal walking . donning and doffing of the orthosis is another important problem associated with the use of an orthosis . the high amount of the force applied on the upper limb musculature is another issue , which affects the use of an orthosis . depending on the style of walking , between 30% and 55% of body weight is applied on the crutch during walking . the high extent of the force , which is transmitted to the upper limb joints , increases the incidence of some diseases as well as shoulder pain . fear to fall , especially during hand function performances , is another problem of using an orthosis . although standing with an orthosis may have some benefits for the patients , it has a number of problems . therefore , the main question that remains is wether or not walking and standing with an orthosis can fulfil the afore - mentioned benefits . unfortunately , the information mentioned in some textbooks regarding the benefits of using an orthosis for sci individuals are based on the survey studies . so , the aim of the present review was to find some evidence regarding the effect of using orthoses on physiological improvement of subjects with sci . moreover , it was aimed to find the performance of paraplegic subjects during walking with orthoses and the problems associated with the use of orthoses . an electronic search was done via the pubmed , embase and isi web of knowledge data bases from 1960 to 2010 . the selection of papers for review was accomplished in two steps . in the first step , relevant articles were selected based on whether the title / abstract addressed the research questions of interest based on some key words such as , spinal cord injury , physiological benefits , walking , standing and orthosis . in the second step papers whose language of publication was english , addressing the adults and children with paraplegia and/or quadriplegia , and those in which subjects used orthoses or frame to improve some parameters such as , bone mineral density ( bmd ) , respiratory system function , cardiovascular system function , and joints range of motion were selected . from an initial list of 100 articles , 40 articles were fully retrieved and reviewed , based on key words and parameters included . the results of the research articles were fully reviewed and categorized based on the mentioned benefits . the results of the various research studies regarding the performance of the orthoses were categorized based on energy consumption , and gait and stability analysis . the results of reviewing the articles are shown in the following tables 1 - 13 . the findings of various studies regarding the effects of standing and walking on bone mineral density the findings of various studies regarding the effects of standing and walking on skin integrity the findings of various studies regarding the effects of standing and walking on improving bowel and bladder function and urinary tract infection the findings of various studies regarding the effects of standing and walking on improving joint range of motion and decreasing muscle spasticity the findings of various studies regarding the stability of paraplegic subjects in a quiet standing position with various orthoses argo = advance reciprocal gait orthosis , nrgo = advance reciprocal gait orthosis without cable , kafo = knee ankle foot orthosis , mlo = medial linkage orthosis , ml = mediolateral , ap = anteroposterior , cop = centre of pressure , n = newton , m = meter , mm = millimeter the findings of various studies regarding the stability of paraplegic subjects while undertaking various hand tasks kafo = knee ankle foot orthosis , argo = advance reciprocal gait orthosis , nrgo = advance reciprocal gait orthosis without cable , ml = mediolateral , ap = anteroposterior , cop = centre of pressure , n = newton , s = second , m = meter , mm = millimetre the findings of various studies regarding the force applied on the foot and crutch during walking with various orthoses rec = reciprocal gait mechanism , swi = swing through gait mechanism , hgo : hip guidance orthosis , rgo = reciprocal gait orthosis , hkafo = hip knee ankle foot orthosis , argo = advanced reciprocal gait orthosis , argo ( 1)=argo orthosis aligned in 6 degrees of abduction , argo ( 2)=argo orthosis aligned in 0 degrees of abduction , argo ( 3)=argo orthosis aligned in 3 degrees of abduction , argo ( 4)=argo orthosis aligned in 6 degrees of adduction , n / bw = newtone / body weigh the findings of various studies regarding the gait parameters of the subjects in walking with various orthoses wbc = weight bearing control orthosis , abd = abduction , add = adduction , flex = flexion , ext = extension the findings of various studies regarding some gait parameters during walking with various orthoses wbc = weight bearing control orthosis , hgo = hip guidance orthosis , kafo = knee ankle foot orthosis , argo = advance reciprocal gait orthosis the findings of various studies regarding the results of some gait parameters in walking with various orthoses kafo = knee ankle foot orthosis , hkafo = hip knee ankle foot orthosis , rgo = reciprocal gait orthosis , hgo = hip guidance orthosis , vrso = vannini rizzoli stabilizing orthosis , fes = functional electrical stimulation , argo = advance reciprocal gait orthosis , nrgo = advance reciprocal gait orthosis without cable , wbc = weight bearing control orthosis the findings of various studies regarding some results of energy consumption tests kafo = knee ankle foot orthosis , hkafo = hip knee ankle foot orthosis , hgo = hip guidance orthosis , rgo = reciprocal gait orthosis , sci = spinal cord injury the findings of various studies regarding the energy consumption of paraplegic subjects during walking with various orthoses kafo = knee ankle foot orthosis , wbc = weight bearing control orthosis , argo = advance reciprocal gait orthosis , nrgo = advance reciprocal gait orthosis without cable the findings of various studies regarding the physiological cost index ( pci ) of paraplegic subjects during walking with various orthoses hgo = hip guidance orthosis , argo = advance reciprocal gait orthosis , nrgo = advance reciprocal gait orthosis without cable , mmlo = moorong medial linkage orthosis , wbc = weight bearing control orthosis it may be a result of decreasing the compression loads applied on the long bones , or may be related to the lack of muscles stress applied on the bones . most of the research done regarding the effects of using orthosis on bmd have shown that walking and standing with orthoses do not influence the magnitude of osteoporosis as much as expected . . it may be due to the maintain of the loads on the spine while sitting in a wheelchair . there is only one study , which specifically mentioned that walking with orthosis brought a lot of physiological benefits for the subjects without presenting any evidence . unfortunately many textbooks in this field refer to that paper without considering the results of other research studies . the other important parameters regarding the influence of standing and walking on bmd is the duration of using an orthosis . it was shown that walking and standing with an orthosis must be life - long , and must be repeated several times a week to have any effects on bone osteoporosis ( at least five session a week and one hour every cession ) . the use of mechanical orthoses and the neurological status ( muscles stress ) , to remain ambulatory are two important parameters which influence bmd . however the findings of various research studies have shown that the effect of the latter is more important ( table 1 and 2 ) . it seems that the type of injury , wether or not complete , influences the bmd . therefore , every effort should be made to prevent turning an incomplete sci into a complete one ( table 3 ) . last but not least important point regarding the effects of using orthosis on the bmd of sci patients is that some of the research studies , which their outcome differs from sci , have been carried out on patients with spina bifida and myelomeningocele patients . the results of various studies regarding the effects of using orthosis on spasticity are shown in table 4 . unfortunately , most of the subjects did not return the questionnaires . according to the findings of different investigations undertaken on sci subjects there are a number of ways , which can be used to measure spasticity clinically and biomechanically such as using ashworth scale , counting beats of clonus , tardieu scale , muscle stretch reflexes , and functional tests . it seems that standing and walking with an orthosis extends the hip and knee joints , and stretches the surrounding muscles . so , applying body weight through leg reduces muscle spasm more efficiency than stretching the muscles only in a supine position . however , there is no evidence to support this view . it has been stated that in standing position , the pelvic tends to tilts more anteriorly than in sitting position . this increase lumber lordosis , and finally stabilizes the spine in an extended posture . in this posture , the force applied on the internal organs decreases , and as a result the performance of respiratory organs increases . abdominal organs fall downward and forward during standing , because there is no an abdominal muscle to increase the stability of the abdominal walls anteriorly . at the end , the force applied on diaphragm decreases , and respiratory function improves . however , it was shown by ogilive that the use of orthosis and ambulation did not affect the respiratory function of participants 24 months after continued use of orthoses ( table 5 ) . improvement of the function of the cardiovascular system is a further benefits mentioned in the literature for ambulation with orthosis . douglas et al mentioned that walking with orthosis influenced the performance of the cardiovascular system in 133 patients with sci . there was no clear description of the method , which was used to monitor the function of cardiovascular system in the subjects participating in the study . the decrease of urinary tract infections and improvement in the function of bowel and bladder are the other benefits mentioned to be achieved from orthosis ambulation . there are only two research studies based on national survey of samples of individuals with sci possessing symptoms of paraplegia or quadriplegia ( table 3 ) . the subject participated in these studies mentioned that walking with orthosis decreased the number of urinary tract infections , and regulated the functions of bowel and bladder . unfortunately , there is no clinical research , which has evaluated the effect of using orthosis on improving the performance of bowel and bladder function . another benefit , which was mentioned by douglas et al regarding the benefits of using orthosis , is the prevention of joint deformity and improvement of joint range of motion . they claimed that during standing the body weight is applied vertically downward and symmetrically upon both feet . in standing position the gravitational positioning of flexed joints decreased , and as a result the risk of deformity of lower limb joint decreased as well . moreover , middleton et al mention that maintaining range of motion and preventing of joint deformity were the two most important outcomes presented by the participants . however , they did not show any evidence to support their findings ( table 4 ) . according to the results of various research studies , the main problems associated with the use of orthosis is the high energy demand it places on the users during ambulation ( tables 11 , 12 , 13 ) . moreover , the walking speed in a sci individuals with an orthoses is significantly less than that of healthy individuals and also in contrast to mobility with a wheelchair ( tables 11 , 12 , 13 ) . although , the type of orthosis and style of walking influence the magnitude of energy consumption , there is a huge difference between the energy consumptions between walking with and without orthosis . as is shown in tables 11 , 12 , 13 there is a big difference between the performances of the subjects in walking with various types of orthoses . some parameters such as the type of orthosis , the position of lesion in vertebral column , age of subjects , and the style of walking influence the performance of the subjects . the high magnitude of the force applied on upper limb musculature is another issue that affects the use of orthosis . depend on the style of walking , between 30% and 55% of body weight is applied on the crutch during walking ( table 7 ) . the high magnitude of the force , which is transmitted to upper limb joints , increases the incidence of some diseases and the pain of shoulder . donning and doffing of orthoses is another important problem associated with the use of an orthosis . herman and biering found that only three out of 45 patients continued using their orthosis after 10 years . the reason that they mentioned for withdrawing from the use of orthoses was the considerable time that they needed to spend on putting on and taking off the orthosis . although the results of the afore - mentioned investigations can not support the effects of walking and standing with orthosis on physiological health of the sci individuals , it is difficult to ignore the positive influences of orthosis . it is recommended to undertake further studies with a sufficient number of participants , and follow the subjects for a long time . moreover , the performance of the subjects in using the orthoses as well as the impact of the orthoses on the health status of the subjects must be measured according to the standard methods discussed in this article . a number of publication have emphasized that walking with orthosis is associated some benefits for individuals with sci , such as improving bmd , improving the functions of cardiovascular , digestive and respiratory systems , decreasing muscles spasm , and joint contractions . however , the findings of various studies have shown that the effects of using orthosis on physiological health are not as much as they are supposed to be . there is no any strong evidence that the use of orthosis can decrease bone osteoporosis , muscle spasm , and improve general health . it can be concluded that in order to have any influences on the health status of sci patients , the use the orthosis for standing and walking must be long - life . moreover , orthoses must be worn four to five sessions of at least one hour every week . a variety of orthoses have been designed to enable sci individuals to stand and walk . they use different mechanisms to stabilize the paralyzed joints , and to move the limbs forward during walking . different sources of power such as pneumatic pumps , hydraulic pumps , muscular force resulting from electrical stimulation , and electrical motors have been attempted for walking . however , the results of different studies have shown that the performance of sci individuals during walking with the mechanical orthosis is very low , and the patients experience a lot of problems in using the orthoses . the patients reported some problems such as high demand for the energy expenditure and mechanical work during walking with orthoses , poor cosmesis of the orthoses , especially the hip guidance orthosis , needing considerable time and sometimes assistance for donning and doffing , and problems related to the fear of falling . it is recommended that to have any influences on physiological health of the sci subjects , orthosis must be used for a long time . therefore , the design of the orthosis must allow easy donning and doffing of the orthosis regularly . it is recommended to design a new orthosis with attachable components , which allow the subjects to wear it independently . the use of some sources of external power in orthoses may improve the performance of the subjects during walking .
, there is a loss of electron and formation of ferric molecule , which in turn results in formation of methemoglobin . in healthy individuals too , there is a small proportion of methemoglobin ( 2 - 3% ) . this disorder is typically seen in three settings : endemic methemoglobinemia ; in individuals of consanguineous unions ; or in compound heterozygous cytochrome b5 reductase deficiency , which is primarily seen in sporadic cases . in hereditary form , there is a deficiency of enzyme - cytochrome b5 reductase ( deficiency of reduced nicotinamide adenine dinucleotide [ nadh ] ) . in normal individuals it leads to a reduction of iron to the ferrous form . in the deficiency of this enzyme acquired methemoglobinemia is known to occur due to reduction of hemoglobin to methemoglobin and is commoner than the congenital variety . it may occur due to drugs such as benzocaine , lidocaine , flutamide , dapsone , nitrites , phenazopyridine , amiodarone , amyl nitrite , primaquine , chloroquine , quinones , sulfonamides acetaminophen , hydrogen peroxide , disulfiram , ibuprofen , and metoclopramide . this may be due to aniline ring observed in drugs such as acetaminophen and sulfa drugs . chemicals such as aniline dyes , gasoline , and food items , for example , spinach can produce similar reactions . cases have been reported from karnataka in infants < 6 months old due to high nitrite content in drinking water and a case from north india in a child who worked with malaria , in aniline dye factory . this article describes methemoglobinemia in a child after parenteral administration of quinine - an anti - malarial drug , causing a severe acute kidney injury ( aki ) in a young child . a 4-year - old male child hailing from north india , and born out of nonconsanguineous marriage presented to emergency with chief complaints of high grade fever with chills and rigors since 8 days , vomiting since 3 days , and dark red colored urine since last 2 days . prior to hospitalization , the child was treated in a private clinic where injection quinine was given for suspected malaria ( though there was no laboratory evidence of malaria ) . child was conscious , alert , febrile ( temperature 101f ) , pulse was 120/min , respiratory rate was 34/min , and blood pressure was 122/94 mm hg . there was no evidence of icterus , edema , cyanosis , clubbing , and lymphadenopathy . complete blood count showed hemoglobin 8.2 g / dl , hematocrit 33.1% , platelet count 405 10/l with methemoglobinemia ( 9.6% ) . pulse oximetry showed oxygen saturation of 80% with an arterial po2334 mm hg , with a chocolate brown blood on exposed to air . renal functions were deranged with blood urea 183 mg / dl ; serum creatinine level 4.4 mg / dl . there was evidence of hepatic dysfunction ( serum bilirubin 4.2 mg / dl ; serum glutamic oxaloacetic transaminase 694 u / l , serum glutamate pyruvic transaminase 113 u / l ) . the child received supportive packed red blood cells , amlodipine for hypertension and antihyperkalemic measures . methemoglobin level gradually decreased , and at a time of discharge its level was 2.9% [ table 1 ] . laboratory parameters at the time of last follow - up , 2 months after discharge , the child has normal renal and hepatic functions with a serum creatinine of 0.6 mg / dl . clues that methemoglobinemia is present include the development of cyanosis in the presence of a normal arterial po2or the presence of chocolate brown blood in the videoscopic field . in acquired methemoglobinema , this stops the ongoing formation of methemoglobin . in lesser degrees of methemoglobinemia ( i.e. , an asymptomatic patient with a methemoglobin level < 20% ) , no therapy other than discontinuation of the offending agent ( s ) may be required . if the patient is symptomatic , or if the methemoglobin level is > 20% , which is often the case in deliberate or accidental overdoses or toxin ingestion , specific therapy with methylene blue is indicated . we did not give methylene blue in our case , since he was improving on removing the offending quinine . quinine has a wide range of adverse events including mild events such as tinnitus , dizziness , disorientation , nausea , hypoglycemia , visual changes , and auditory deficits to life - threatening complications such as renal failures , cardiac arrhythmias , and black water fever . there have been very few publications , which mention hemolysis and severe methemoglobinemia due to quinine . we still can not rule out an underlying genetic etiology in this child , as a risk factor for acute acquired methemoglobinemia is the asymptomatic heterozygous state for cytochrome b5 reductase deficiency . the classic description of acute toxic methemoglobinemia in united states military personnel receiving malarial prophylaxis in vietnam demonstrated for the first time that heterozygotes for this autosomal recessive disease can , under certain conditions , develop a disease state that is more clinically significant than their asymptomatic homozygous peers . this is the first case in a young child , with a severe form of aki following quinine ingestion and acquired methemoglobinemia . this is a learning case emphasizing greater awareness of potential adverse events of antimalarial drugs ; especially in the pediatric population .
most symptomatic patients present with postprandial fullness and gastrointestinal hemorrhage ; however , other patients present with an incidental submucosal filling defect on upper gastrointestinal contrast studies or a smooth effacement of gastric mucosa overlying a submucosal mass ( schindler 's sign ) on endoscopy . although leiomyoma is classically a submucosal lesion , intraluminal or extramural growth has been noted . open celiotomy with gastric wedge resection , partial gastrectomy , enucleation , and extended gastrectomy with en bloc resection of adjacent organs are various methods of treatment for gastric leiomyoma before the use of laparoscopy . with the progression of laparoscopic surgery beyond laparoscopic cholecystectomy , below , we describe a case of laparoscopic wedge resection and review the various laparoscopic techniques used to treat gastric leiomyoma . an upper endoscopy demonstrated a 2-cm lesion on the greater curvature of the stomach 6 cm from the gastroesophageal junction . the patient remained hemodynamically stable without any changes in her hematocrit . a contrast upper gastrointestinal series displayed a well - circumscribed mass in the region of the upper gastric body ( figure 1 ) . digital spot film from an upper gastrointestinal examination demonstrating the gastric mass located in the proximal stomach . the long white arrows show the bulging contour deformity on the external surface of the stomach . intraoperatively , a small , white , smooth , well - encapsulated nodule was noted on the greater curvature lying among the short gastric vessels . the short gastric vessels were divided by using the harmonic scalpel ( ultracision ethicon endo - surgery , cincinnati , ohio ) to completely free and visualize the mass ( figure 2 ) . a wedge resection of the mass with a rim of normal gastric tissue was performed with a laparoscopic stapling device ( figure 3 ) . postoperatively , the patient did well , was discharged the next day with oral pain medications , and was seen in the clinic in follow - up without any problems . the first gastric stromal tumor was noted at autopsy in 1762 , however , the first successful resection was performed over 100 years later . gastric stromal tumors are divided into 3 groups based on malignant potential : leiomyoma , leiomyosarcoma , and leiomyoblastomas . these methods have been applied to other benign gastric tumors ( eg , submucosal gastric lipoma ) as well as leiomyosarcoma . gastric resection without lymph node dissection in the treatment of malignant neoplasm ( such as leiomyosarcoma ) may be controversial although studies have demonstrated no benefit from the addition of systemic lymph node dissection or more extensive resection in the treatment of leiomyosarcoma . these results are important when considering the laparoscopic technique for any gastric stromal tumor resection because often the diagnosis of leiomyosarcoma versus leiomyoma may not be evident until after resection . in fact , biopsies from endoscopy yield the diagnosis in less than 60% of the time . although some authors feel that more extensive resections may be necessary for high - grade leiomyosarcoma , others believe that a wedge resection is sufficient for leiomyosarcomas . the choice of technique should be based both on size and location of the lesion . for easily accessible anterior lesions , a simple wedge resection is possible via an endostapling device although some have suggested the use of laparoscopic suturing as a more cost - effective method . we , like others , prefer the use of an endostapling device to reduce operative time as well as to avoid contamination from a gastrotomy . simple wedge resection has also been described for the use of posterior lesions via access through the lesser sac . because the lesion in our patient was on the greater curvature and easily accessible after dissection of the short gastric vessels , a wedge resection was the method we chose to use . lesions on the posterior wall , close to the gastroesophageal junction , or close to the pylorus may be difficult or impractical for a simple wedge resection . also , resection of a tumor close to the gastroesophageal junction or the pylorus may cause significant postoperative stenosis . thus , laparoscopic resection via an anterior gastrotomy has been described . with this technique , an anterior gastrotomy is first performed ; then , the mass is removed either with all layers of the posterior gastric wall or removed leaving the posterior gastric wall partially intact . closure of the defect can be performed via the anterior gastrotomy via laparoscopic stapling or suturing techniques . another method described is the combined use of laparoscopic , endoscopic , and intragastric techniques for the resection . this technique is ideal for lesions that are difficult to excise with a simple wedge resection . utilizing this method , the minilaparoscopic or needlescopic ( 2-mm ) ports do not usually require closure , which offers a specific advantage over standard laparoscopic ports . resection of the leiomyoma leaving the serosa intact is performed with visualization via the endoscope . some authors close the intragastric defect with intragastric suturing , while others leave the defect open . the seromuscular layers are dissected from the gastric mucosa to allow for enucleation of the leiomyoma . the utilization of this technique has significant drawbacks . because preoperatively , the distinction between leiomyoma and leiomyosarcoma is not always known , rupture of the tumor mass or incomplete resection would be inappropriate treatment . no matter which technique is used , intraoperative endoscopy is a useful adjunct especially when trying to localize small tumors . frozen section should always be considered in order to ( 1 ) exclude other possible diagnoses and ( 2 ) to assure clear margins . differentiating from high - grade and low - grade lesions by frozen section is not accurate however . postoperative recovery is short as demonstrated by our case with a 1-day postoperative hospital stay . this shorter stay should translate into a reduction in pulmonary and infectious complications related to extended hospital stays . another advantage is smaller incisions ( better cosmesis ) and thus subsequent decreased postoperative pain and narcotic requirement . laparoscopic surgery for gastric leiomyoma offers a safe and effective approach compared with open laparotomy .
external tooth bleaching is a conservative and well - accepted technique for whitening teeth with surface and intrinsic staining , which is opening up new avenues and becoming much more prevalent in esthetic dentistry . the idea is that using this technique can effectively make stained teeth whiter through direct application of bleaching agents . bleaching agents contain peroxide , which bleaches the enamel to change the color of the teeth . to date , various bleaching agents , in different concentrations , have been used in dental clinics to treat tooth discoloration ; however , the most commonly used one is 10% carbamide peroxide due to its superior characteristics as an external bleaching agent , i.e. it is effective , works quickly , and has relatively few side effects while achieving desirable results when used to treat tooth discoloration . therefore , 10% carbamide peroxide was chosen as the bleaching agent in this study . however , 10% carbamide peroxide does have some controversial side effects . numerous studies have shown that the marginal sealing of composite resin restorations decreased whether the fillings were done before or after bleaching using 10% carbamide peroxide . also , bleaching may harm the dental pulp , resulting in pulpal reactions such as increased tooth hypersensitivity . as we know , the active mechanism of bleaching agents mainly depends on a complex oxidation reaction , which releases oxygen and other free radicals . the oxygen and free radicals establish their primary mechanism of action in tooth bleaching by penetrating through the porosities of the enamel prism to the dentin . increased microleakage of composite resin restorations placed after bleaching may be due to the presence of residual peroxide and residual oxygen that inhibits resin polymerization by interfering with the adhesion of restorative materials . several methods have been proposed to decrease microleakage due to bleaching , including removal of the superficial enamel layer , pretreatment of the bleached enamel with alcohol , use of adhesives containing organic solvents , cleansing of the cavity with antioxidants , and a post - bleaching period ranging from 24 h to 3 weeks . it has been shown that removal of the superficial enamel layer is ineffective because bonding weakens both superficially and internally . one method that is recommended clinically is to delay bonding until 2 - 3 weeks after bleaching . studies have shown that residual peroxide and oxygen slowly dissipate with time , eventually eliminating any reduction in composite resin bond strength associated with the bleaching , but this is time consuming for practitioners and patients . several antioxidant agents have been introduced , such as sodium ascorbate , ascorbic acid , butylhydroxyanisole , catalase , ethanol , acetone , glutathione , peroxide , -tocopherol , and sodium bicarbonate , in order to better control restoration microleakage ; however , only a few of them were found to be effective . we hypothesized that treating bleached enamel with an antioxidant before bonding might reverse the increase in microleakage and might be a viable alternative to delayed restoration after bleaching . recently , a biocompatible and neutral antioxidant , 10% sodium ascorbate , was shown to be able to remove the residual peroxide and oxygen , so that compromised bonding to bleached tooth structures , such as dentin or enamel , could be reversed . furthermore , moosavi et al . demonstrated that the addition of surfactant ( 0.2% tween 80 ) to a sodium ascorbate formulation could significantly reduce the microleakage after nonvital bleaching . to our knowledge , only one study has investigated the use of catalase in improving the composite resin bond strength after tooth bleaching . in another study , it was reported that catalase could be used as an adjunct to effectively eliminate residual hydrogen peroxide from the pulp chamber and the surrounding periodontal tissues following intracoronal bleaching of nonvital teeth . in the present study , we examine the effect of catalase on improving adhesion between composite resin and externally bleached teeth and on reduction of microleakage after external tooth bleaching . the aim of this study is to investigate the effects of three antioxidants ( sodium ascorbate , sodium ascorbate combined with a surfactant , and catalase ) on the microleakage of composite resin restorations after external tooth bleaching with 10% carbamide peroxide . the ethics committee of ninth people 's hospital , medical college , shanghai jiao tong university , china approved this study . teeth with caries , hypoplastic areas , or cracks and teeth that had a history of treatment were excluded . after extraction , the soft tissues , dental calculus , and stains were immediately removed from selected surfaces of the teeth . the saline containing the selected teeth was changed every week for 1 month in order to avoid bacterial growth . the stored teeth were randomly divided into five experimental groups ( n = 10 per group ) after bleaching with 10% carbamide peroxide . an unbleached group ( n = 10 ) was used as the control group . prior to bleaching , tooth surfaces were polished with oil- and fluoride - free fine pumice and water using a brush and a slow - speed hand - piece . unbleached teeth were immersed in artificial saliva , which was composed of 1 g sodium carboxymethylcellulose , 4.3 g xylitol , 0.1 g potassium chloride , 0.1 g sodium chloride , 0.02 mg sodium fluoride , 5 mg calcium chloride , 5 mg magnesium chloride , 5 mg calcium chloride , 40 mg potassium phosphate , 1 mg potassium thiocyanate , and 100 g distilled deionized water , at 37c for 10 days . box - shaped class v cavities were made on the buccal surface , around the cementoenamel junction , using a round diamond bur in a high - speed hand - piece under air and water - cooling . occlusal margins and gingival margins of the prepared cavities were located in the enamel and the root , respectively . the prepared cavities were approximately 2 mm in height , 3 mm in the mesio - distal direction , and 2 mm in depth . adper easy one self - etching bonding agent ( 3m / espe , st paul , mn , usa ) was used for bonding of the composite resin , according to the manufacturer 's instructions . this was spread gently with air for 5 s and then cured for 10 s using a pen - style , high - power led curing light ( dentsply , milford , de , usa ) with an intensity exceeding 950 mw cm . following this , composite resin ( filtek z350 , shade a3 , 3m / espe ) was placed into cavities and polymerized in 1.5 - 2-mm - thick layers for 20 s. the composite resin was then finished by bur , and the teeth were preserved at 37c in normal saline that was changed daily . the teeth were first bleached by the following method and then immediately filled with composite resin . the teeth were placed with the occlusal edges positioned downward in a plastic holder containing 10% carbamide peroxide ( opalescence ; ultradent products , south jordan , ut , usa ) for 8 h a day for 10 days . after completion of each daily bleaching procedure , the teeth were rinsed with flowing water and then washed using a soft toothbrush in order to remove the remaining bleaching gel . after the final bleaching , washing , and overnight storage in saliva , the cavities were immediately prepared and restored in the bleached teeth as described above for group 1 . in group 3 , the teeth were bleached as described above and then , after the final washing , immersed in artificial saliva at 37c . after 3 weeks , cavities were prepared and composite resin restorations were placed as in group 1 . in group 4 , the teeth were bleached and the cavity prepared immediately as described above for group 2 . cavities were then treated with 10% sodium ascorbate using a cotton pellet prior to filling with composite resin . briefly , 10% sodium ascorbate was smeared on the bonding surface for 10 s using a cotton pellet that was then placed into the cavity for 1 min . after removal of the cotton pellet , the cavity was rinsed with distilled water for 2 min and dried gently with air to prepare it for composite resin restoration . after bleaching , a novel antioxidant agent composed of 10% sodium ascorbate and 0.2% tween 80 was applied to cavities in the bleached teeth , as described above for group 4 . after restoration , all teeth were immersed in distilled water at 37c for 24 h and then thermocycled from 5c to 55c for 2000 cycles using a 45 s dwell time at each temperature . after thermocycling , the specimens were laid aside and dried at room temperature for 24 h. all surfaces of the teeth , except for a 1-mm zone surrounding the restorations margins , were covered with three coats of nail polish and modeling wax to provide an impermeable barrier to the test fluid ( dye ) . the specimens were then immersed in 2% methylene blue for 24 h at 37c . after staining , the teeth were washed with tap water and the nail polish and modeling wax were removed with a scalpel . the teeth were sectioned longitudinally , from buccal to lingual , through the center of the restorations , using a low - speed diamond saw under water - cooling . the microleakage was assessed by two calibrated examiners , who were blind to the treatment groups , using a stereomicroscope ( stereo discovery.v12 ; zeiss , oberkochen , germany ) at 50 magnification . each examiner independently judged the depth of the stain according to standard ranking as shown below . microleakage scores : 1 = dye penetration occurred up to half of the cavity wall depth 2 = dye penetration greater than half of the cavity wall depth 3 = dye penetration involving the base of the cavity . the occlusal wall and the ethics committee of ninth people 's hospital , medical college , shanghai jiao tong university , china approved this study . teeth with caries , hypoplastic areas , or cracks and teeth that had a history of treatment were excluded . after extraction , the soft tissues , dental calculus , and stains were immediately removed from selected surfaces of the teeth . the saline containing the selected teeth was changed every week for 1 month in order to avoid bacterial growth . the stored teeth were randomly divided into five experimental groups ( n = 10 per group ) after bleaching with 10% carbamide peroxide . an unbleached group ( n = 10 ) was used as the control group . prior to bleaching , tooth surfaces were polished with oil- and fluoride - free fine pumice and water using a brush and a slow - speed hand - piece . unbleached teeth were immersed in artificial saliva , which was composed of 1 g sodium carboxymethylcellulose , 4.3 g xylitol , 0.1 g potassium chloride , 0.1 g sodium chloride , 0.02 mg sodium fluoride , 5 mg calcium chloride , 5 mg magnesium chloride , 5 mg calcium chloride , 40 mg potassium phosphate , 1 mg potassium thiocyanate , and 100 g distilled deionized water , at 37c for 10 days . box - shaped class v cavities were made on the buccal surface , around the cementoenamel junction , using a round diamond bur in a high - speed hand - piece under air and water - cooling . occlusal margins and gingival margins of the prepared cavities were located in the enamel and the root , respectively . the prepared cavities were approximately 2 mm in height , 3 mm in the mesio - distal direction , and 2 mm in depth . adper easy one self - etching bonding agent ( 3m / espe , st paul , mn , usa ) was used for bonding of the composite resin , according to the manufacturer 's instructions . this was spread gently with air for 5 s and then cured for 10 s using a pen - style , high - power led curing light ( dentsply , milford , de , usa ) with an intensity exceeding 950 mw cm . following this , composite resin ( filtek z350 , shade a3 , 3m / espe ) was placed into cavities and polymerized in 1.5 - 2-mm - thick layers for 20 s. the composite resin was then finished by bur , and the teeth were preserved at 37c in normal saline that was changed daily . the teeth were first bleached by the following method and then immediately filled with composite resin . the teeth were placed with the occlusal edges positioned downward in a plastic holder containing 10% carbamide peroxide ( opalescence ; ultradent products , south jordan , ut , usa ) for 8 h a day for 10 days . after completion of each daily bleaching procedure , the teeth were rinsed with flowing water and then washed using a soft toothbrush in order to remove the remaining bleaching gel . after the final bleaching , washing , and overnight storage in saliva , the cavities were immediately prepared and restored in the bleached teeth as described above for group 1 . in group 3 , the teeth were bleached as described above and then , after the final washing , immersed in artificial saliva at 37c . after 3 weeks , cavities were prepared and composite resin restorations were placed as in group 1 . in group 4 , the teeth were bleached and the cavity prepared immediately as described above for group 2 . cavities were then treated with 10% sodium ascorbate using a cotton pellet prior to filling with composite resin . briefly , 10% sodium ascorbate was smeared on the bonding surface for 10 s using a cotton pellet that was then placed into the cavity for 1 min . after removal of the cotton pellet , the cavity was rinsed with distilled water for 2 min and dried gently with air to prepare it for composite resin restoration . after bleaching , a novel antioxidant agent composed of 10% sodium ascorbate and 0.2% tween 80 was applied to cavities in the bleached teeth , as described above for group 4 . unbleached teeth were immersed in artificial saliva , which was composed of 1 g sodium carboxymethylcellulose , 4.3 g xylitol , 0.1 g potassium chloride , 0.1 g sodium chloride , 0.02 mg sodium fluoride , 5 mg calcium chloride , 5 mg magnesium chloride , 5 mg calcium chloride , 40 mg potassium phosphate , 1 mg potassium thiocyanate , and 100 g distilled deionized water , at 37c for 10 days . box - shaped class v cavities were made on the buccal surface , around the cementoenamel junction , using a round diamond bur in a high - speed hand - piece under air and water - cooling . occlusal margins and gingival margins of the prepared cavities were located in the enamel and the root , respectively . the prepared cavities were approximately 2 mm in height , 3 mm in the mesio - distal direction , and 2 mm in depth . adper easy one self - etching bonding agent ( 3m / espe , st paul , mn , usa ) was used for bonding of the composite resin , according to the manufacturer 's instructions . this was spread gently with air for 5 s and then cured for 10 s using a pen - style , high - power led curing light ( dentsply , milford , de , usa ) with an intensity exceeding 950 mw cm . following this , composite resin ( filtek z350 , shade a3 , 3m / espe ) was placed into cavities and polymerized in 1.5 - 2-mm - thick layers for 20 s. the composite resin was then finished by bur , and the teeth were preserved at 37c in normal saline that was changed daily . in group 2 , the teeth were first bleached by the following method and then immediately filled with composite resin . the teeth were placed with the occlusal edges positioned downward in a plastic holder containing 10% carbamide peroxide ( opalescence ; ultradent products , south jordan , ut , usa ) for 8 h a day for 10 days . after completion of each daily bleaching procedure , the teeth were rinsed with flowing water and then washed using a soft toothbrush in order to remove the remaining bleaching gel . after the final bleaching , washing , and overnight storage in saliva , the cavities were immediately prepared and restored in the bleached teeth as described above for group 1 . in group 3 , the teeth were bleached as described above and then , after the final washing , immersed in artificial saliva at 37c . after 3 weeks in group 4 , the teeth were bleached and the cavity prepared immediately as described above for group 2 . cavities were then treated with 10% sodium ascorbate using a cotton pellet prior to filling with composite resin . briefly , 10% sodium ascorbate was smeared on the bonding surface for 10 s using a cotton pellet that was then placed into the cavity for 1 min . after removal of the cotton pellet , the cavity was rinsed with distilled water for 2 min and dried gently with air to prepare it for composite resin restoration . after bleaching , a novel antioxidant agent composed of 10% sodium ascorbate and 0.2% tween 80 was applied to cavities in the bleached teeth , as described above for group 4 . after restoration , all teeth were immersed in distilled water at 37c for 24 h and then thermocycled from 5c to 55c for 2000 cycles using a 45 s dwell time at each temperature . after thermocycling , the specimens were laid aside and dried at room temperature for 24 h. all surfaces of the teeth , except for a 1-mm zone surrounding the restorations margins , were covered with three coats of nail polish and modeling wax to provide an impermeable barrier to the test fluid ( dye ) . the specimens were then immersed in 2% methylene blue for 24 h at 37c . after staining , the teeth were washed with tap water and the nail polish and modeling wax were removed with a scalpel . the teeth were sectioned longitudinally , from buccal to lingual , through the center of the restorations , using a low - speed diamond saw under water - cooling . the microleakage was assessed by two calibrated examiners , who were blind to the treatment groups , using a stereomicroscope ( stereo discovery.v12 ; zeiss , oberkochen , germany ) at 50 magnification . each examiner independently judged the depth of the stain according to standard ranking as shown below . microleakage scores : 1 = dye penetration occurred up to half of the cavity wall depth 2 = dye penetration greater than half of the cavity wall depth 3 = dye penetration involving the base of the cavity . the kruskal wallis test was used to assess differences in microleakage among the different groups . representative photographic images of microleakage made under a stereomicroscope ( 50 ) for each of the six groups are shown in figure 1a f . photographs of six groups under a stereomicroscope ( 50 ) : ( a ) unbleached group ( group 1 ) ; ( b ) group in which the filling was done immediately after bleaching ( group 2 ) ; ( c ) delayed filling group ( group 3 ) ; ( d ) sodium ascorbate treated group ( group 4 ) ; ( e ) sodium ascorbate combined with surfactant treated group ( group 5 ) ; ( f ) catalase - treated group ( group 6 ) edge microleakage degree among the different groups the results of the kruskal wallis test demonstrated a statistically significant difference in microleakage among groups ( p < 0.01 ) . differences were observed for both the occlusal as well as the gingival wall [ table 1 ] . bleaching with 10% carbamide peroxide ( groups 2 - 6 ) significantly increased microleakage values of restorations compared to the unbleached group ( group 1 ) . the unbleached group 1 displayed the least amount of microleakage , while the group in which the filling was done immediately after bleaching ( group 2 ) showed the greatest amount of microleakage [ table 1 and figure 2 ] . group 1 displayed the least amount of microleakage , and showed significant differences with groups 2 - 6 ( * p < 0.01 ) , group 2 showed the greatest amount of microleakage , groups 3 and 4 behaved similar to group 2 ( not significantly different ) , while the microleakage in groups 5 and 6 decreased significantly ( p < 0.05 ) compared to group 2 * : p < 0.01 compared to group 1 , : p < 0.05 compared to group 2 the delayed filling group 3 and the sodium ascorbate treated group 4 also had a large amount of microleakage that was similar to that in group 2 where the filling was done immediately after bleaching . there was no significant difference between the delayed filling and sodium ascorbate treated groups ( p > 0.05 ) . microleakage in group 5 treated with sodium ascorbate combined with surfactant , and also in the catalase - treated group 6 , was significantly ( p < 0.05 ) lower compared to group 2 in which the filling was done immediately after bleaching . however , microleakage in these two groups was still significantly greater ( p < 0.05 ) than in the unbleached group . a large number of studies on the interactions between bleaching agents and the bond strength of composite resins to enamel have shown that there is a significant decrease in the bond strength when the composite resin is bonded to bleached enamel compared with unbleached enamel . however , to date , there have been relatively few studies on the increase in the microleakage of composite resin restorations after tooth bleaching , especially external tooth bleaching . therefore , the aim of this study was to investigative the effects of three antioxidants on the microleakage of composite resin restorations after external tooth bleaching with 10% carbamide peroxide . our results showed that bleaching with 10% carbamide peroxide ( groups 2 - 6 ) significantly increased the microleakage values of restorations compared to the unbleached group ( group 1 ) . this may be due to the presence of residual peroxide and oxygen , which has been shown to interfere with resin attachment and inhibit resin polymerization . the appearance of the hybrid layer in bleached enamel is less regular and distinct than that in unbleached enamel , and these enamel irregularities may also play a role in the increased microleakage . reported that the residual oxygen left after bleaching may be removed from the enamel surface during the immersion process . in a subsequent study , they showed that immersion of specimens in artificial saliva for 2 weeks could improve the bond strength of composite resin to enamel after bleaching . this finding was not supported by the results of our current study , where the delayed filling group accumulated microleakage that was not significantly different from microleakage in the immediate restoration group . in this study , we found significant differences when comparing microleakage in the bleached control group to that of groups that had antioxidant treatments ( groups 5 and 6 ) . as in previous studies , these results suggest that antioxidants play an important role in decreasing microleakage when restorations are done immediately after bleaching . moreover , in our study , except for the unbleached group , the lowest microleakage values were observed in samples treated with new antioxidants , such as sodium ascorbate combined with tween 80 ( group 5 ) , or with catalase ( group 6 ) . ascorbic acid and its salts are well - known antioxidants that can reduce a variety of oxidative compounds , especially free radicals . moreover , ascorbic acid ( vitamin c ) and its salts are non - toxic and have been widely used in the food industry . thus , they have no adverse biological effects or clinical hazards when used in the oral cavity . lai et al . suggested that sodium ascorbate allowed free radical polymerization of the adhesive to proceed without premature termination by restoring the altered redox potential of the oxidized bonding substrate and eventually reversing the compromised bonding . sodium ascorbate was used in solution form in the present study and in previous studies . however , sodium ascorbate solution is more difficult for practitioners and patients to manipulate than the hydrogel form . the hydrogel form of sodium ascorbate can be placed in the bleaching tray before bonding . some studies have shown that there is no significant difference in microleakage between the two forms of sodium ascorbate ( hydrogel and solution ) , but other studies have reported that the hydrogel form is better . there have been few studies researching sodium ascorbate hydrogel , and it could be a novel research area for future development of a factory - made antioxidant . notably , there were no significant differences found in microleakage between the group treated with only sodium ascorbate ( group 4 ) and the delayed filling group ( group 3 ) . one reason may be that the application time of the antioxidants was too short . in a study carried out by bulut et al . , sodium ascorbate solution was used for 10 min in order to neutralize the oxidizing effect of carbamide peroxide . used sodium ascorbate solution for 3 h and suggested that sodium ascorbate should be used for at least one - third of the amount of time that the oxidizing bleaching agent was applied ( e.g. bleaching gel was generally applied for 8 h a day ) . however , it is time consuming and impractical to apply the antioxidants for this proposed length of time prior to composite resin restoration . as an example , the sodium ascorbate hydrogel would need to be placed in the bleaching tray for 3 h by the patients themselves before bonding . in the present study , the antioxidants were used in group 4 for only 1 min in order to correspond with the catalase group . as with a previous study , the sodium ascorbate , by itself , had no significant effect on microleakage . thus , in future studies , the application time will be extended to 10 min . proposed that a bleached surface in the access cavity may reduce the surface tension and contact angle , as well as the cohesive force between the sodium ascorbate molecules after treatment with 10% sodium ascorbate gel in combination with 0.2% tween 80 . first , 0.2% tween 80 is a nonionic surfactant that can allow the antioxidant agent to easily penetrate the dentin , improving the efficacy in the dentinal tubules and enamel . second , 0.2% tween 80 's detergent effect on the surface can improve the sealing of composite resin by increasing the free surface energy of the tooth . accordingly , the 3-h length of time for antioxidant treatment could be decreased when the antioxidant is combined with this surfactant , and this was supported by our study . our results showed that when sodium ascorbate combined with 0.2% tween 80 was used for 1 min on the cavity , microleakage decreased significantly . rotstein et al . reported that catalase can effectively eliminate residual hydrogen peroxide when used as an adjunct following intracoronal bleaching of nonvital teeth . kum et al . also found that pretreatment of a bleached surface with catalase prior to bonding significantly improved the composite enamel bond strength . similarly , our results suggested that catalase should be recommended for clinical use since it significantly reversed the adverse effects of peroxide and oxygen on microleakage associated with composite resin restorations after external tooth bleaching , and it was effective after a short application time . however , further studies need to be done to optimize the efficacy of catalase for this purpose . studies on the effects of other antioxidant agents for neutralizing the adverse effects of bleaching materials on composite resin restorations have not been done . two other antioxidants , 10% ascorbic acid and vite , have displayed high antioxidant activity among the substances tested by dpph ( 1,1-diphenyl-2-picrylhydrazine ) guided fractionation assay . however , 10% ascorbic acid is inappropriate for clinical use due to its very low ph value ( about 1.8 ) . it is difficult to store it for a long period of time because the solution gradually oxidizes over time during storage to become less reductive . vite , a lipid - soluble antioxidant ( ph 6.8 ) , has recently been shown to have good antioxidant activity in dentin and enamel , similar to ascorbic acid . this may be due to the presence of alcohol in the composition of the vite , as previous studies have reported that applying alcohol to the bleached enamel increases the bond strength , although not to the level of the non - bleached teeth . the depth of dye penetration at the interface between the wall of the cavities and the restorations is adopted as a criterion by most studies on microleakage , but ordinally ranked observational data is only semi - quantitative and incorporates subjective variability . further studies are needed in which the depth of the dye penetration in the cavity is measured directly on all specimens using a stereomicroscope , thus providing continuous data and more precision . in studying microleakage caused by bleaching agents , the scanning electron microscope ( sem ) may be useful to observe the ultrastructure of specimens with microleakage . several authors have used sem and reported that increased porosity of enamel is manifested by an over - etched appearance with loss of prismatic structure . in the study of trkn and kaya , the interface between resin and bleached enamel , observed on sem , displayed a granular and porous aspect with a bubbly appearance that may have been the result of oxidized peroxide that was trapped in the subsurface layer of the enamel . in the future , teeth that have been treated with our experimental antioxidant formulation further , it should be determined whether the higher microleakage rates that were observed in vitro subsequent to bleaching translate to significant deterioration of restorations in actual clinical situations and whether this deterioration can be reduced in the clinic using an effective antioxidant formulation . in addition , determining the minimum time required for antioxidant treatment after external tooth bleaching will be necessary . therefore , further studies are needed to better understand these issues and address practical concerns in terms of clinical applications . microleakage increased significantly after external bleaching with 10% carbamide peroxide , and decreased when the bleached teeth were treated with sodium ascorbate combined with tween 80 , or with catalase . catalase was more effective at decreasing microleakage , while delayed filling or treatment with sodium ascorbate alone did not effectively decrease the microleakage of composite resin restorations .
although clinically recognized metastatic thyroid cancer is rare , data from autopsy show that thyroid metastasis findings range from 1.9% to 24.2% , with the common sources of primary cancer being skin , lung , and breast.1,2 however , of the clinically recognized metastases to the thyroid , more than 50% of the time the primary cancer is renal cell carcinoma ( rcc).3 rcc represents 3% of all adult malignancies.4 the presentation and behavior of thyroid metastases from renal cancer are variable.5 here , we report a very unusual case of metastatic rcc to the thyroid gland in a patient who had undergone right nephrectomy for rcc 14 years earlier . a 77-year - old man was referred to our department from another hospital for thyroid masses detected about 1 month previously . he had no subjective symptoms , such as stridor , dyspnea , hoarseness , or dysphagia . his medical history was remarkable in that he had undergone right nephrectomy for stage i rcc 14 years previously . the pathologic type of rcc was revealed as clear cell . he had type 2 diabetes mellitus and essential hypertension for the past 5 years . medications for chronic diseases as prescribed by his primary care physician were as follows : felodipine 5 mg , losartan 50 mg / hydrochlorothiazide 12.5 mg , glimepiride 2 mg , and metformin 750 mg , each daily . a soft and painless 7.05.0 cm sized mass was palpable on the left thyroid , and a smaller 3.5 cm soft mass was palpable on the right thyroid . fine needle aspiration ( fna ) cytology showed a few atypical follicular cells with nuclear atypia , suggesting papillary thyroid carcinoma . a computed tomography ( ct ) image of the neck showed two large cystic and degenerating thyroid masses : a 7.25.5 cm sized mass on the left thyroid and a 3.83.8 cm sized mass on the right thyroid without lymph node ( ln ) metastasis ( fig . no apparent extrathyroidal invasion or enlarged lns were observed during the operation . on gross inspection of the resected specimen , two large , well - circumscribed , mixed cystic and solid masses were found , one in each thyroid lobe , with hemorrhagic changes in the left mass ( fig . the histopathologic findings of the thyroid masses were small nests of clear cells separated by a prominent sinusoidal network ( fig . 2b ) with abundant optically empty cytoplasm , sharply outlined boundaries , and moderately atypical nuclei ( fig . these microscopic findings were similar to those of the previous renal cell carcinoma , clear cell type ( fig . immunohistochemical ( ihc ) studies were performed for thyroglobulin ( tg ) , thyroid transcription factor-1 ( ttf-1 ) , cd10 , cytokeratin , vimentin , and epithelial membrane antigen ( ema ) . the clear cells displayed strong immunoreactivity for cytokeratin , vimentin , cd10 , and ema and were negative for tg and ttf-1 ( fig . , these microscopic and ihc findings confirmed that the thyroid masses were metastatic rcc , clear cell type . an abdominal ct scan revealed a 3.7 cm sized mass on the right nephrectomy site with a normal left kidney . metastatic carcinomas to the thyroid gland are rarely found in clinical practice.5 rcc is well - known for its highly variable clinical presentations and courses , being called the " internist 's tumor".6 rarely , a thyroid mass can be an initial clinical sign of relapse of rcc after a surgical treatment or can even appear as an initial clinical presentation of rcc before the detection of primary cancer.5 possible reasons for the low frequency of clinical metastatic thyroid carcinoma are not known , although some explanations have been proposed : rich blood supply , high oxygen tension , and high iodine content in the thyroid.7 in the absence of a detailed clinical history , the initial presentation of a thyroid nodule in an otherwise healthy subject makes the diagnosis of metastatic disease challenging . this is complicated further by histologic similarities between metastatic foci and primary thyroid tumors.5 there are no specific clinical features attributable to metastases to the thyroid.3,5 our patient had no subjective symptoms associated with the thyroid masses , and his thyroid function was normal . also , the initial fna cytology suggested primary papillary carcinoma of the thyroid . the true metastatic nature of the tumor is usually recognized after tumor sampling with pathologic assessment . distinguishing primary from metastatic thyroid tumors the use of negative ttf-1 staining or positive periodic acid - schiff staining in conjunction with negative tg staining can accurately lead to the diagnosis of metastatic disease.8,9 the majority of rccs are immunoreactive to keratin and ema.5,7,10 the microscopic examinations of the resected lesions in our patient showed small nests of clear cells separated by a prominent sinusoidal network with abundant optically empty cytoplasm . the clear cells were immunohistochemically positive for cytokeratin , vimentin , cd10 , and ema and were negative for tg and ttf-1 . these microscopic and ihc findings were consistent with those of metastatic rcc to the thyroid in our patient . about 20% to 30% of cases of rcc patients experience cancer relapse distantly after curative nephrectomy.6 furthermore , unusual sites of metastases are characteristic of renal cancer , and virtually any organ site can be involved , including the thyroid.6 although there have been reports of rcc metastasis to the thyroid , and although it is well - known that rcc has variable natural histories , this was a very unusual case of metastatic rcc in the thyroid gland detected 14 years after curative nephrectomy . the mean time before a relapse in the thyroid gland after initial nephrectomy was previously reported to be 9.4 years.5 in conclusion , our case experience and literature review suggest that long - term follow - up including physical examination of the thyroid gland is required after initial nephrectomy for patients with rcc .
the arenaviridae family comprises only one genus formed of 25 species of arenaviruses , as categorized by the ictv , and listed in ( table 1 ) . a further classification is based on the geography of origin and distribution of the viruses and their hosts : old world arenaviruses ( owa ) and new world arenaviruses ( nwa ) . the nwa group harbors all arenaviruses from the american continent and the owa group includes the only arenaviral species present in all continents ( lymphocytic choriomeningitis virus lcmv ) as well as the african arenaviruses . to date , there are no reports of any native viral species from europe or australia . the precise geographic distribution pattern has its origin in a very narrow host range that is characteristic of all arenaviruses . the principal hosts for the nwa are several specific members of the rodent family cricetidae , subfamilies sigmodontinae and neotominae . in the case of lcmv , the rodent reservoir is the worldwide distributed mus musculus , which explains the unusual ubiquity of this virus . one nwa , the tacaribe virus ( tacv ) , was isolated from bats , in what was thought to be an atypical occurrence for an arenavirus . however , a recent discovery of new arenaviral species in boas opens up new avenues of inquiry regarding the viral host range and biodiversity . this viral family is of medical interest because 6 of the 25 members have been consistently associated with clinical symptoms and described as the etiological agents of human diseases . all pathogenic nwa produce hemorrhagic fevers , with very similar physiopathology ( table 2 ) . the phases of the illness are well characterized and progress from prodromal to neurologic - hemorrhagic to convalescent . there is a short incubation period before the onset of symptoms . in the case of argentine hemorrhagic fever ( ahf ) , the syndrome is characterized by renal , neurological , vascular , immunological and hematological alterations . if it remains untreated , the mortality rate can be as high as 15 - 30% . since it was described , ahf has been reported to cause annual outbreaks during the autumn - winter season . however , the number of actual cases in each outbreak has significantly diminished since the application of the vaccine to the risk population within the endemic area . for the 1992 - 2000 period the development of the vaccine against ahf was the product of a collaboration between the us and argentine governments . a greatly attenuated strain of junn virus ( junv ) , candid#1 , was extensively tested in rhesus monkeys and human volunteers with good results . afterwards , a comprehensive clinical trial was conducted in the ahf endemic area [ 8 , 9 ] . a diagram with the passage history that led to the attenuated junv candid#1 strain the intermediate strains were named xj # , in reference to the original strain and the number of passages in mouse brain . the vaccine was originally produced in the usamriid ( united states army medical research institute for infectious diseases ) facilities . in 2005 , the national institute of human viral disease ( inevh ) located in pergamino , argentina , finished the certification process and took over the vaccine production for local use . significant advances were made to develop an effective protection against lassa fever , which is a disease endemic to the west - african region with 3 - 5x10 cases reported annually . its apparently expanding territory , together with the imported cases reported yearly all over the world , make this virus the biggest health threat of the arenavirus family . several different approaches were used to try to achieve protective coverage with a lassa virus vaccine . however , the necessity of a single - dose treatment and the great diversity among the many viral strains converge to increase the difficulty of this task . arenaviruses are enveloped viruses with a bipartite , single stranded rna genome [ 14 , 15 ] . the virion contains three structural proteins ( l , n and z ) while the lipid envelope carries the three - piece glycoprotein ( ssp , g1 and g2 ) . the larger genomic segment ( l rna , ~7 kb ) encodes the rna dependent rna polymerase ( l protein ) , which is the minor component of the virion , as well as z , a zinc finger - protein of 11 kda that could be the arenaviral counterpart of a matrix protein . the other genomic segment ( s rna , ~3,5 kb ) codes for the nucleocapsid protein n and the glycoprotein precursor ( gpc ) , which is processed by cellular proteases into three parts . the arrangement of the two open reading frames in opposite orientations , separated by the stably structured , non - coding intergenic region , gave rise to the term ambisense coding . the first 19 nucleotides at each end of one segment are complementary , which allows a base - pairing that forms a panhandle structure highly conserved among arenaviruses . in order to identify and characterize changes directly related with the attenuated phenotype of the junv vaccine strain , the complete nucleotide sequence of all available intermediate strains ( xj13 , xj17 , xj34 , xj39 , xj#44 , xj48 and candid#1 ) from the vaccine genealogy were analyzed , along with a small number of field strains [ 19 , 20 ] . a comparison of the sequence of the vaccine genealogy strains revealed a set of differences that might be associated with the decrease in virulence ( fig . an alignment of the s rna - derived coding sequences disclosed eleven individual changes which translated into amino acid substitutions . the first of these changes was located in the stable signal peptide ( xj13 i35 xj17 ixj34 ixj39 ixj#44 v35 xj48 icandid#1 v ) , the following three were in the mid - part of g1 ( t168aaaaaa ; e186eeeeeg and s206sssssp ) , two more were found at the carboxyl terminus of g2 ( f427fffffi and t446tsstss ) , and the last five were at the amino half of n ( v47vvvvve ; r59kk krkr ; i158iiiviv ; e268eeee ed and t322tttiti ) . the same analysis , performed on the coding sequences derived from the l gene , revealed only nine nucleotide changes implied in amino acid substitutions . two of these are reversions ( xj13 r881xj#44 gcandid#1 r ; s921gs ) , while the other seven might be associated with the attenuated phenotype ( h76yy ; v415va ; d462nn ; l936lp ; r1156kk ; s1698sf and i1883iv ) . this analysis was extended to include two other completely sequenced junv strains , romero and mc2 , as well as a partial sequence from the cba - iv4454 strain ( s rna and z gene ) . the first strain is classified as of high virulence and was originally isolated from an ahf patient [ 21 , 22 ] . in the genebank the second strain ( mc2 ) is of intermediate virulence and was isolated from a rodent captured in the ahf endemic area [ 23 - 25 ] . the cba - iv4454 strain was also obtained from a patient and is considered of intermediate virulence . the analysis was performed on deduced amino acid sequences for the six aforementioned junv strains . type 1 mutations are those where only one of the junv field strains ( xj13 , romero , iv4454 , and mc2 ) differs from all others . type 2 mutations refer to the positions with changes among the vaccine - related strains ( xj13 , xj#44 and candid#1 ) . this last type allows identifying key positions that might be involved in the attenuation process , while the type 1 mutations take into account the naturally occurring variations . an overlap of these two types of changes uncovered several mutations with probably little significance for the viral virulence . n158 , n268 and n322 all had changes among the six sequences that varied between two particular amino acids , regardless of the strain s origin . something similar happened with positions l881 and l921 , where the only difference was found in xj#44 . a mutation frequency index was calculated , defined as the number of mutations per amino acid in an overlapping - window strategy , with the resulting value assigned to the central residue . the analysis was made for both types of mutations for the complete junv genome sequence , using in - house software . this global plotting approach gives an overview of the distribution of the mutations along the genome . several possible hot - spots were identified , that is , regions of the sequence with a marked tendency towards natural variation . also , a small number of type 2 mutations were found outside the hot - spots , namely gpc168 , gpc427 , gpc446 and n47 for the s rna and positions l76 , l936 , l1156 for the l rna . a further search for type 2 mutations was performed on a set of field samples , isolated from human cases or rodents , captured in the endemic area during the period of 1963 - 91 . out of the genome , four regions were selected for this analysis : i ) g fragment , comprising positions 303 to 941 within the gpc coding sequence ; ii ) n fragment , spanning nucleotides 1632 to 2095 of the nucleoprotein coding sequence and two regions within the open reading frame of the rna dependent rna polymerase . afterwards , the sequences were translated in silico , aligned and scanned for positions with differences between strains . a part of the type 2 mutations obtained for the g fragment are shown in ( table 3 ) . the selected region comprises most of g1 , the consensus site for the arenavirus glycoprotein processing and the beginning of g2 . some of the variants present among the field samples ( from both humans and rodents ) were also found among the attenuated junv strains . consequently , the importance of these positions in the attenuation process should be re - evaluated . the development of a reverse genetics system made it possible to directly test the role of the different variable positions in viral fitness and virulence . used chimeric genomes , baring a combination of xj13 and candid#1-derived sequences , to assess the importance of each portion of gpc . however , a number of variable positions remain to be evaluated by this experimental method . the polymerase domain was mapped to the region iii , with the presence of four conserved motifs : a , b , c and d . the variation analyses showed only one change in this region , at position 1156 ( rkk ) . albeit the mutation has a conservative character , it is probably related to the attenuation process because there are no natural variants in this region . the other changes in the l orf are located between regions ii and iii ( l936 , llp ) and in region i ( l76 , hyy ) . the modification of a leucine for a proline brings about a mayor structural change and is present only in the candid#1 strain , in spite of being immersed in a region of high type 2 mutations index . the change at position l76 is near a predicted atp / gtp binding site ( www.expasy.org ) . even though these results are preliminary , they suggest an important role for the polymerase in the virulence attenuation , in accord with reports for other viruses [ 29 - 31 ] . also , some of the mutations in the structural proteins ( nucleoprotein and the two glycoproteins ) could be associated with the attenuation of virulence . the n protein has a highly conserved carboxyl - terminus , containing a zinc binding domain . the mutation at n47 ( vve ) lies outside that region , but could be related to other characteristics of the protein , such as the oligomerization capability , possibly interfering with one or both of n s functions in the viral life cycle . in the genome replication , n promotes the synthesis of antigenomic , full - length copies of the s rna . in the assembly of the virion , the protein binds the genomic rna , forming the nucleocapsid . the other structural protein , gpc , sported a mutation at the carboxyl end of g1 ( gpc168 , taa ) , directly affecting the conserved sequence ( n166r167t168k169 ) for a predicted n - glycosylation site ( netglyc 1.0 ) . first , the outer domain , located at the carboxyl end , which is outside the virion and interacts with g1 ; second , the transmembrane domain , which interacts with the stable signal peptide , and last , the inner domain , situated at the amino end , which could interact with the nucleocapsid protein or the z protein , on the inside of the virion [ 26 , 34 ] . the changes within g2 are in the transmembrane domain ( gpc427 , ffi ) or in the cytoplasmic tail ( gpc446 , tts ) and , in both cases , could affect these important interactions . the potential attenuation markers were found to be unevenly distributed in the genomic segments , with a higher degree of conservation in the l rna , which indicated a faster evolution rate for the polypeptides encoded in the s rna . in a comparison of genomes of virulent and avirulent strains of pichinde arenavirus , lan and collaborators found that attenuation related mutations mapped to comparable genomic regions , but were fewer in number . if the comparison is limited to only the field strains of junn virus ( xj13 , romero , mc2 and iv4454 ) the mutation distribution pattern became markedly different for the large and small genomic segments . in a protein sequence analysis of the field strains , 45 divergent sites were found for the s rna derived orfs and 48 sites for the l rna derived orfs . interestingly , for the l rna , 46 out of the 48 sites were xj13-specific changes , while for the s rna , this was only the case in 4 of the 45 sites . as stated before , the g fragment included the sequence for the cleavage site between g1 and g2 . the recognition site sequence has been published as qlprrslk251aff with the cleavage occurring between k251a or lk251 . most of the analyzed samples did nt have mutations in that region . only isolate h_fha5054 presented changes at positions 244 and 245 ( qh and lf respectively ) , but a portion of the variation observed for the g fragment , namely the part corresponding to g1 , could be associated with the host jump of a rodent strain to humans ( receptor affinity differences ) . they could therefore be directly linked with changes in the strains virulence ( increase or decrease ) through a cellular tropism change . another key feature of this region was the n - glycosylation target sequence n166r167t168k169 , as determined by netglyc 1.0 . this precise sequence is only present in the 65% of the field strains , while in the remaining 35% there is an alanine residue ( a168 ) . complementary bioinformatics studies performed on this region predicted only slight changes to the protein properties . on the other hand , the importance of two positions , strongly related to the n - glycosylation of junv and macv was established . also a high conservation degree of the residues involved , along with their environ was found to be necessary . in recent years , an analysis of lcmv proved the implication of each n - linked glycan of the glycoprotein with gpc expression , fusion activity and infectivity . the hypothesis was that mutations present in field strains ( natural variants ) would be of minor importance in virulence attenuation . in fact , none of the type two mutation analyzed here were absent in field samples . therefore , the data presented in ( table 3 ) could signify that the attenuated phenotype is not influenced by mutations in the g1 region . that result concurs with albario and collaborators , who mapped the essential attenuation - related mutations to g2 . previous studies suggested that sequence changes within the intergenic region could play a role in the attenuation processes of arenaviruses . conversely , our analysis of the intergenic regions from candid#1 , xj48 , xj#44 , xj39 , xj34 , xj17 and xj13 revealed a total conservation among the strains for both genomic rnas . if there is a low tolerance for nucleotide changes in the region , it would suggest an evolutionary constraint , probably related to the calculated secondary structure and the associated transcription regulation function [ 32 , 41 ] . also , there was a high degree of conservation of the non - coding regions at the genomic ends of the analyzed strains , with nucleotide homology values ranging from 93% to 97% . however , when viral rnas obtained from infected cells were analyzed , a great variability could be observed at the 3 and 5 non - coding regions of rnas derived from the same parental strain . a number of 5 non - templated bases were found for both candid#1 genomic segments . when comparing the 5 end of the genomic rnas with the 3 end of the antigenomic rnas ( which are the template for the former ) , at least one additional guanine was present at all 5 ends of genomic clones with additional bases , as has been described for other arenaviruses . on the contrary , the 3 race of genomic forms yielded several clones with short deletions . a calculation of rna secondary structure from candid#1 s and l rnas predicted the formation of a panhandle between the 5 and 3 ends of each genomic segment . the observed 3 deletions all mapped to regions within the panhandle ( fig . , these results support the employ of cell - derived rnas as primers for the synthesis of viral rna , as well as 5 terminal sequences from virus - derived rnas into incomplete panhandle structures as templates for the completion of the 3 terminal sequences . the resulting heterogeneity may well be a consequence of the action of different rna editing mechanisms , as reported for other arenaviruses . still , the role of these regions in the process of attenuation remains to be established . interestingly , a comparison of the sequence of the ends ( 3 and 5 ) of both genomic segments revealed several positions that were highly conserved and could be part of the minimal promoter . the untranslated sequences on the extremes of the s rna , as well as the 5 end of the l rna , have an approximate length of 80 nucleotides . this is not the case for the 3 end of l rna , which has only 30 nucleotides . considering that the genomic forms serve as templates for the antigenomic forms and vice versa , all non - coding terminal regions have to include the promoter sequence that is the recognition site of the viral polymerase , in order to achieve the completion of the replicative cycle . since one of these regions only has a size of 30 nucleotides , it is reasonable to conclude that the minimal promoter sequence is contained in that range . in support of this , if a sequence comparison is made of the 80 nucleotides of the ends of s and l rnas , the homology value is 60% for the 5 ends and only 50% for the 3 ends . but , if the analysis is reduced to only 30 nucleotides , the value of the 3 region ascends to 71% . in fact , there is a 19 nucleotide sequence motif highly conserved among arenaviruses ( arena region ) that can be found in all junv genomic ends . the remaining 11 bases of the 3 end of the l rna ( gctcaagtgcc ) have an elevated homology degree with two regions within the s rna 3 end . a comprehensive analysis of the genomic sequence of sublcade b1 new world arenaviruses showed that the element that mapped to positions 38 - 46 ( gcucaagug for junv l rna and gcucagug for s rna ) was conserved within the group . therefore , it could be possible to identify a sequence motif at the 3 end of both genomic rnas of most arenaviruses . also , in the particular cases of junv , machupo and tacaribe rna s , the motif described as gsyc(a)1 - 2gur , has a relatively high positional conservation within the rna secondary structure when calculated by bioinformatic tools . this works aim was to present a condensed report of the studies about the molecular determinants of viral virulence in junv . a set of putative attenuation markers have been identified within the gpc , z and l orfs of junv vaccine strain , candid#1 . the main reason for this is that these changes were found inside regions with a low natural mutation frequency ( gpc168 , gpc427 , gpc446 , n47 , l76 , l936 and l1156 ) . increasing complete genome sequence information availability , particularly from junv strains , has been a significant contribution for the identification of further putative virulence attenuation markers . in recent years several sequences were published for members of the vaccine strain lineage [ 20 , 27 ] . they were included in previously done informatic analyses , confirming the results observed before , that is , only a few changes are necessary to obtain an attenuated phenotype . to further study the role in the virulence attenuation process of different point mutations , albario and collaborators developed a junv reverse genetics system . their results showed a direct involvement in the attenuated phenotype of certain point mutations within the gpc orf of candid#1 . moreover , in order to analyze the natural mutation distribution , goi and collaborators designed a rt - nested - pcr based technique and applied it to human and rodent derived samples from the argentine hemorrhagic fever endemic area . a comparison was instituted between the gpc derived sequence data ( g fragment ) and the results published by albario and collaborators . , the greater variability appears mainly in the portion of the glycoprotein complex that is exposed to the exterior and therefore comes in contact with the immune system . changes to the sequence and conformation of epitopes could explain an increase in virulence due to escape from the natural host defenses . also , slight variations within the receptor binding region have been shown to confer the capability of infecting human cells as well as the animal host . however , there are certain constraints acting on the outer part of the glycoprotein , related to its capacity of efficiently performing the original function within the viral cycle . studies addressing similar questions for other arenaviruses produced interesting results . zhang and collaborators created reassortants using an attenuated and a virulent picv parental strain . they found that the virulence in guinea pigs could mainly be attributed to the s segment . nevertheless , the virulence level of the parental strain was not attained , pointing to determinants being present in the l segment . similar studies made with lcmv strains mapped the attenuation markers of this virus principally to its l segment . although both studies determine the virulence in an animal model of guinea pigs , the results obtained for each virus are different . . it would be interesting to assay what these mutations are for junv , principally taking into account the available intermediate strains of the vaccine genealogy , and the immense combination probabilities . the sequence information accumulated through the analysis of a number of strains with varying virulence degrees will be the springboard for natural biodiversity studies . the mutations identified as potentially central to the attenuation process are ideal candidates for mutagenesis and reverse genetics assays . however , more complex studies will have to be made to determine the minimal changes that render an attenuated phenotype , since it is likely that many variations act synergically to decrease the observed virulence . also , compensatory changes might arise to make up for structure or activity losses that hinder viral fitness [ 48 , 49 ] . therefore , even if a particular change is found in both field and vaccine strains , it does not necessarily negate its importance in virulence attenuation . conversely , some variants present in natural and vaccine strains might have a cumulative effect upon the activity of a single viral protein or on the capacity of interaction with other viral or cellular proteins . this protein is responsible for the budding of the virus and the correct virion assembly . all of the roles of z within the viral cycle are performed through interaction with proteins . the apparently low tolerance of changes within the z orf suggests that its functions greatly depend on the correct structure / sequence . possibly , the regions of interaction of its partners are also submitted to a similar constraint . it will be interesting to analyze the precise regions once they have been positively identified . a great step towards identifying possible co - variation sites will come with the elucidation of the crystallographic structure of the viral polypeptides and their interaction partners . the beginning has been made with the first glycoprotein and partial nucleoprotein and l [ 51 - 53 ] structures and we are hoping for a steady advance in this area . summing up , this work presented a set of mutations that are probably related with an attenuated virulence phenotype . also , most of the mutations analyzed here ( type 1 and 2 ) were mapped to a few discrete genomic regions . the development of a rt - pcr based method followed by sequencing , was the basis for a broad analysis of junv strains in search of natural genomic variability . this method could furthermore be useful if a program for the monitoring of vaccinated individuals is implemented .
deliberate self - injury or pathological self - mutilation is the deliberate alteration or destruction of the body tissue without conscious suicidal intent . the diverse behaviors that constitute pathological mutilation have been categorized into the following three basic types : major - infrequent acts that result in significant tissue damage ; stereotypic - fixed , rhythmic behavior seemingly devoid of symbolism ; and superficial or moderate behavior such as skin cutting , burning , and scratching . self - injurious behavior is reported among patients with wide range of psychiatric disorders , notable being all types of psychoses , schizophrenia , affective disorders , substance dependence , mental retardation , obsessive compulsive disorder , dissociative disorders , and factitious disorder . we present a case with self - injurious behavior which offered significant diagnostic and management challenge as well as ethical dilemma for the treating team , as responsibility for the restoration of his vision by surgery hinged on psychiatric opinion . a 52-year - old married hindu male shopkeeper from middle socioeconomic status family , educated up to 7 standard , with no past or family history of psychiatric illness , with history of diabetes mellitus for past 2 years , with well - adjusted premorbid personality was referred from department of ophthalmology to seek psychiatric opinion regarding his fitness to undergo cataract surgery in his left eye . he was blind in his right eye due to self - inflicted injury . mera friend maarta hai ( my friend beats me ) while the family members complained of apne aap ko maarte hain ( beats himself ) . nine years back , patient had met with road traffic accident ( rta ) while he was riding pillion on a motorbike with his friend . his friend succumbed to his injuries in the local hospital after 2 days , while he escaped with minor injuries in the form of lacerations and abrasions ; there was no head injury . he missed the time he had shared with the deceased and felt worried about welfare of friend 's family . he did not voice any guilt that his friend had died while he had got only minor injuries . however , he experienced fleeting anxiety during which he felt what would have happened to his family if he had died and thanked god for saving him . at home , he interacted well with family and friends , enjoyed watching tv , took care of self , had normal sleep and appetite , and did not have depressed or anxious mood . within about a month of rta , he gradually resumed his usual activities and started going regularly to his shop . after about 5 months of rta , patient was found walking alone near the railway station by a neighbor . he told the neighbor that his friend had come to his shop and had asked him to follow him . when he was confronted with the fact of his friend 's death later , he told his family that his friend 's appearance , voice , and dress was same as before and there was brightness around him . when asked why his friend was not visible to others , he expressed his inability to explain it . however , he continued to believe that his friend had not died but stayed in the same city . following the recurrence of similar incident after about 2 months , he was stopped from going to shop by his son . two months later , when the patient was alone at home , he was found to be beating his head . when asked , he told that his friend had come , had asked the patient to follow him and on refusal to do so , the friend had started beating him . at that time , he was not anxious or fearful . despite being confronted with the fact that the family members had seen him beating his own head with his hands , he continued to maintain that his friend had beaten him up . following this incident , the family believed initially that such events of his being beaten up by his dead friend were occurring due to the influence of evil spirits , so he was shown to faith healers . also , they shifted to another city far from their native place . however , the patient continued to experience episodes where he would injure himself by beating or scratching . the episodes of going away from home did not recur as he was never left alone by family members . after about 2 years , for the first time , medical consultation was sought at local hospital ; his eeg , ct scan and mri of head did not reveal any abnormality . he continued to have similar episodes 3 to 4 times / year , lasting for about an hour during which he would sustain injuries over different parts of the body which he claimed were caused by his friend . also during these episodes , he would touch and manipulate already existing minor wounds resulting in a large wound . there was no history of stereotyped cadence of his behavior during these episodes . around a year back , he was diagnosed to have cataract of right eye and was operated at the local hospital . they rushed and found that dressing from the operated eye was removed , there were nail marks and bleeding from the operated eye which he claimed were done by his friend . though he was rushed to the hospital immediately , vision in his right eye was completely lost . despite complete loss of vision in the right eye due to self - injury ( injury by the friend as he believed ) , he remained indifferent to his inability to see and the resultant disability . throughout this period , the patient continued to believe that his friend was alive despite evidence to the contrary . however , his sleep , appetite , mood , personal care , social interaction , involvement in recreational activities , and helping in household chores remained normal . there was no history of persistent anxiety , depression or elation , depressive cognitions , guilt feelings , anniversary reactions , or first - rank symptoms . about 2 months back , due to failing vision in the left eye as well , he was advised cataract surgery for the left eye at the local hospital . but in view of the injury to right operated eye and eventual complete loss of vision , doctors there referred him to ophthalmology department , all india institute of medical sciences for the surgery . he was examined by ophthalmologist and due to possibility of repeating the same behavior after the surgery of left eye which would have resulted in his becoming completely blind , he was referred to the psychiatry department for the opinion . on physical examination , multiple healed scar marks over various part of the body were found , he had cataract in left eye and opacity over pupillary area in right eye . rest of the general and systemic examination was within normal limits . on mental status examination , he was cooperative , spoke relevantly and coherently . he was oriented to time , place , and person , attention was aroused and well sustained , had intact comprehension and memory , could do simple and complex calculations , and abstract thinking was intact . his verbal fluency , he could identify his fingers , name objects held in his hands , and identify letters and numbers traced on his hands . he had delusion that his dead friend was alive and blamed his friend for beating him . la belle indifference was noted as he was observed to be indifferent to the fact that he was not working ; he had lost vision in his eye and his life was restricted to his home . he had episodic behavioral disturbances in the form of wandering ( at least in first two episodes and further prevented by the family ) , hallucination - like experiences , self - injurious behavior causing injuries and visual loss , consistent delusional attribution of the behavior to his friend , emotional indifference , relatively intact personal , social and biological functioning despite chronicity , and no features suggestive of organic cause on history , examination , and investigations . over the years , his problems had resulted in not pursuing his occupation ( due to wandering away from his shop ) , him being always accompanied by one of the relatives ( due to hitting himself ) , and more involvement of family members in his care ( due to loss of vision in one eye ) . at the time of presentation , he needed help from family members for activities of daily living because of poor vision . the family had come from a distance of more than 800 km just to seek second opinion regarding the safety of performing surgery on his only eye ( left ) as his other eye was lost due to self - injurious behavior ; they were unwilling to stay for long time due to logistic reasons like the only earning member of the family , his son , was accompanying him . this patient 's presentation could not be explained on the basis of single diagnostic entity , so differential diagnoses were considered ( icd10 classification system ) . persistent delusional disorder ( f22 ) : the patient had single well - systematized delusion , firm false belief of his friend being alive ( during and in between the episodes ) , not shared by others . ( f 68.1 ) : the patient had history of manipulating his wounds and injuring himself on his own and then ascribing it to another person for which he was brought to medical attention on several occasions . he was also relieved of all his responsibilities as the head of the family and was not working for several years . however , the patient himself would never show any active interest in seeking treatment for any of his problems and would accept whatever treatment was offered to him . dissociative disorder ( nos ) ( f 44.9 ) : during the episodes of seeing his friend , there was loss of contact with the surroundings , he would communicate with friend , he was not able to perceive other stimuli around him , and he could only be aroused by a vigorous stimulus like shaking or patting by a family member suggesting a trance - like state . though the patient would not assume any new identity during these episodes , there was some evidence for the loss of identity of self as he would ascribe hitting himself the actions of his friend . however , these episodes would only occur when the patient was alone . also , there was h / o wandering ( at least in first two episodes and further prevented by the family ) . furthermore , he seemed oblivious to his problems and dysfunction and exhibited la belle indifference . localization - related ( focal ) ( partial ) symptomatic epilepsy and epileptic syndromes with complex partial seizures ( g40.2 ) : experiential hallucinations in which both visual and auditory components combine to form one single experience are usually seen in cases of temporal lobe epilepsy . however , there was no evidence of seizures either historically or on eeg . complicated grief ( f 43.21 ) : the patient refused to accept the death of his friend and would see his friend in the same state as he was at the time of death . however , he would not seek out his friend on his own and did not resist the change in place of residence . also , there was no history of mummification of objects belonging to the friend or anniversary reaction . post - traumatic stress disorder ( f 43.1 ) : his symptoms had appeared within 6 months of the initial traumatic experience . however , there was no h / o re - living the traumatic experience and avoidance of travel by similar means . also , there were no features suggestive of either autonomic hyperarousal or emotional numbing anytime during the history . working diagnoses of persistent delusional disorder and factitious disorder were made . due to the short stay , the underlying issues concerning psychopathology in terms of his motive , primary and secondary gains could not be explored and dealt with . as immediate concern of the family was the faltering vision in left eye , it was decided to ascertain the urgency of eye surgery . he was started on 2 mg risperidone which was later increased to 4 mg ; he developed no side effects . he was discharged on 4 mg risperidone and was advised to report after 2 months . it was agreed upon by both the treating teams to keep him under close joint follow - up and supervision , to assess his mental status on subsequent follow - ups , and take decision about performing cataract surgery . in the present case , psychiatrists had come into picture to ascertain patient 's fitness for undergoing eye surgery . though his competency to give consent for the surgery was not in doubt , the manner in which he had injured his operated eye made it essential to deal with his underlying psychopathology before advising for his next surgery . he exhibited persistent self - injurious behavior , overt motive for which had remained unclear and he attributed this injurious behavior to a friend long dead . interestingly , similar symptom of hitting oneself but attributing it to others has been labeled as alien hand syndrome . in this syndrome , there is failure to recognize ownership of one 's limb and the offending limb is recognized as foreign . this same phenomenon was observed in our patient ; he accepted the act of beating , but shifted the blame of his self - injurious behavior on to his dead friend . alien hand syndrome is reported in patients with medial , frontal , or callosal lesions ; however , in our patient , neurological examination and investigations did not suggest any organic involvement . in a review of 41 cases with self - inflicted eye injuries , most patients were reported to be male , and had a diagnosis of schizophrenia , drug or alcohol abuse , depressive disorders , or other psychosis ; 33% of the patients also showed other types of injurious behavior . in our case , diagnoses of persistent delusional disorder and factitious disorder were entertained . for the treating teams , irrespective of the diagnosis , the management of this patient remains difficult . the cataract surgery is definitely required for failing vision in his left eye . risks and benefits of not operating and possibility of self - injury in case of an eye surgery will have to be appreciated by all the stakeholders . the challenge for both the treating teams will be to operate and prevent damage to the operated eye . this case report illustrates a case of self - injurious behavior which posed multiple challenges for the treating team . management of self - injurious behavior in such cases requires close cooperation between psychiatrists and other medical specialists , to ensure quick medical care of the patient , prompt diagnosis and treatment of any injuries , and treatment of the underlying psychopathology that led to this behavior .
percutaneous endoscopic gastrostomy ( peg ) tube placement has been the minimally invasive procedure of choice for enteral access , or decompression , since the early 1980s . herniation through gastrostomy site is considered an extremely rare complication with only four cases reported . we present two unusual cases of gastrostomy site herniation , the surgical management thereof , and a corresponding review of the literature . the first patient is a 65 year old caucasian male who presented to the surgery clinic after a prolonged hospitalization for congestive heart failure exacerbation resulting in the need for gastrostomy tube placement . the patient had the peg tube in place for approximately a month and then it was removed by traction technique after no longer in use . the patient noticed an enlarging defect about 3 weeks after removal . at time of presentation he complained of epigastric pain and a bulge at his previous gastrostomy tube incision site . the hernia was easily reduced with gentle traction , and a gastro - cutaneous attachment resected ( fig . a polyester composite mesh was then used to repair the gastrostomy site ventral wall defect ( fig . 2 ) . the second patient is a 66 year old obese caucasian male who was hospitalized in the intensive care unit for lithium overdose . he required prolonged enteral feeding , and for that purpose he received a percutaneous endoscopic gastrostomy . subsequently he recovered well and had the tube removed by simple traction in the office . five months later he complained of continued pain in the midepigastric region around his gastrostomy site scar . on physical exam he was taken to the operating room and his hernia was repaired by open surgery with a composite mesh . both patients had non - complicated reducible hernias at presentation and no extra imaging or specific diagnostic modalities were required . authordatediagnosisremoval methodinterventionoutcomechuang 2003leakage from around peg tube , bulge with coughingtractionremoval of peg tube via traction method , plan for surgical interventionpneumonia , respiratory failure , and death before surgeryboldo - roda 2005leakage from around peg tube , bulge with physical activitytractionn / aunkkaplan 2006leakage from around peg tube , bulge with physical activitytractionreferral for surgical interventionunkozutemiz 2007bulgingtractionn / aunk peg site herniation has been described previously but without description of operative intervention received . with the increasing numbers of peg tubes being placed every year discussion of preventing this complication was presented by boldo - roda et al . and included avoidance of placement of peg tube through linea alba , as this is an area of potential weakness ; and possibly using cut and push technique rather than traction . it is possible that vigorous traction during removal may create a more permanent cavity than expected . however cut and push technique carries its own innate risks and clinicians should keep this in mind when deciding on the removal technique of choice . while gastrostomy site hernia is an exceedingly rare complication with only four other cases reported in the literature , it is likely , given the number of gastrostomy tubes placed per year , that it is simply underreported . clinicians should stay vigilant when performing physical exams or additional workup in patients with ongoing leakage , bulge or pain at the gastrostomy site ; as this is potentially a surgically correctable entity , and can be safely managed via laparoscopic or open techniques . written informed consent was obtained from the patients for publication of these two case reports and accompanying images . a copy of the written consents is available for review by the editor - in - chief of this journal on request .
this report describes the clinical and parasitological findings in a domestic shorthair cat with isolated thoracic tetrathyridiosis . the cat was a stray from malta that had lived in germany for several years since as an indoor - only cat . it must be considered that the cat had been infected years previously while in malta , and had lived at least 4 years without any clinical signs . it was possible to diagnose this uncommon disease and initiate an effective treatment with fenbendazole , praziquantel and supportive care . tetrathyridiosis is a rare finding in cats , especially in germany , but it seems to be a potential differential diagnosis of pleural effusion . mesocestoides corti , which was the causative parasite in this case , has not previously been isolated in germany . because tetrathyridiosis is only diagnosed post mortem in most cases , little is known about effective therapeutic options . furthermore , clinical signs of this disease can be absent for several years and can potentially be triggered by neoplastic conditions or immunosuppression . tetrathyridiosis seems to be a treatable disease that can be controlled by adequate antiparasitic therapy . mesocestoides species live as adult worms in the small intestine of carnivores as definitive hosts . in europe , mesocestoides lineatus , mesocestoides leptothylacus and mesocestoides litteratus are most prevalent . the life cycle of mesocestoides species is not known in detail but a cycle including two intermediate hosts and one definitive host is generally assumed . wild carnivores , especially red foxes ( vulpes vulpes ) are known as an important worldwide reservoir of mesocestoides species . vertebrates , including mammals , are potential second intermediate hosts , in which the infective larval stages ( tetrathyridia ) develop in serosal body cavities and different organs mammals like dogs and cats , and also humans , are infected by consumption of the organs of these intermediate hosts and therefore act as definitive hosts . thoracic involvement of tetrathyridiosis seems to be an extremely rare finding in dogs and cats . furthermore , tetrathyridiosis is a disease primarily described in southern european countries such as italy , turkey and spain . to our knowledge , there is only one reported case of tetrathyridiosis in a dog in germany . in many cases the diagnosis of tetrathyridiosis following the rare diagnosis of tetrathyridiosis no consistent therapeutic recommendations exist and little is known about potential therapy in dogs and cats . praziquantel and fenbendazole seem to be effective against mesocestoides species infections in dogs and cats . nothing is known about treatment duration and combination therapy , especially with regard to long - term outcome . based on the geographic history of the cat discussed herein , a long asymptomatic period after infection with tetrathyridia must be suspected . another interesting finding is the possible relationship between the tetrathyridiosis and the late diagnosis of concurrent anaplastic lymphoma . a 6-year - old domestic shorthair cat was presented with dyspnoea to the emergency service of the small animal hospital of the veterinary faculty of the university of leipzig . it had been moved from malta to germany about 6 months after neutering . since then furthermore , the right pinna had been partially resected owing to a squamous cell carcinoma 3 years previously . when the cat was hospitalised to resect the carcinoma , chest radiographs showed various subcutaneous bullets distributed over its body . the cat had been vaccinated recently , but dewormed irregularly and the last time was several years previously . the cat had been treated repeatedly for ulcerative dermatitis . immediately before presentation it was treated with ciclosporin ( 6 mg / kg bodyweight q48h po ) because of these dermatological signs . the cat showed laboured breathing with a respiratory rate of 68 breaths per minute during the clinical examination . over the caudal aspects of the lungs after the initial stabilisation of the cat with oxygen supplementation , thoracic radiographs were taken ( figure 1 ) . a large amount of pleural and potentially mediastinal fluid obscured the heart silhouette and other soft tissue structures . because of these findings , neoplasia involving both lung and pleural cavity was a very likely differential diagnosis . a large volume of pleural fluid obscures the heart silhouette and other soft tissue structures . the bullets can be localised in the thoracic wall and the neck despite the potentially guarded prognosis the owner was willing to perform further diagnostics and therapy , and the cat was hospitalised . only 70 ml of the effusion could be evacuated from the chest because the needle was repeatedly blocked with white , filamentous structures . no abnormal findings could be gathered and the pleural effusion was considered to be of extracardiac origin . further analysis of the effusion revealed a reactive inflammatory exudate ( 6850 cells/l , protein content 47 microscopically , the white filamentous structures had a small granular appearance and moved in a meandering pattern . because of the suspected parasitic origin the samples of the pleural effusion , as well as faecal samples , g / l [ reference interval ( ri ) 611 g / l ] ) with lymphocytopenia ( 0.41 g / l [ ri 16 blood testing for feline leukaemia virus via snap - test ( idexx ) and pcr analysis was negative . drops of the pleural fluid were screened at a magnification of 40 ( figure 2 ) . a faecal sample was tested for parasitic stages , especially for cestode proglottids and eggs , by a combined sedimentation flotation method . ( a ) flat contractile tetrathyridium with unsegmented body , filled with dense granula ( g ) , and a smooth clear tegumental border ( t ; 40 magnification ) ; ( b ) tegument ( t ) and parenchyma ( p ) with numerous calcareous corpuscles ( c ; 400 magnification ) the aspirate was centrifuged ( 2000 g , 5 mins ) and the dna extracted from two parasitic tissue samples of the sedimented structures using the qiaamp dna mini kit ( qiagen ) according to the manufacturer s instructions for tissue samples . a cestode - specific pcr was performed based on the mitochondrial 12s rrna gene , as previously described . pcr products were run on a 1.5% agarose gel , stained with ethidium bromide and the bands visualised by ultraviolet light . the pcr products underwent sequencing ( interdisziplinres zentrum fr klinische forschung , university of leipzig , germany ) followed by blastn analysis ( ncbi basic local alignment search tool , nucleotide blast 2.2.26 ) . the faecal samples were tested negative for any parasitic stage , including cestode proglottids and eggs . all dna samples of the pleural effusion and particles tested positive for cestode dna ( product length approximately 370 base pairs ) . sequencing of the samples revealed a high homology to the amplified sequence of the mesocestoides corti 12s ribosomal rna gene ( genbank : hm011122.1 ; homology level : 98% ) . the cat received fenbendazole ( 50 mg / kg q24h po ) and praziquantel ( 5 mg / kg po ) as an antiparasitic treatment . in addition , supportive treatment ( infusions , theophylline [ 4 mg / kg q8h iv ] ) and amoxicillin / clavulanic acid ( 14 mg / kg q8h iv ) were given . shortly after starting the therapy the dyspnoea disappeared . contrast - enhanced computed tomography of the thorax revealed the presence of multiple pulmonary nodules with diameters ranging from 37 mm . the pulmonary nodules did not show enhancement after the administration of iodinated contrast medium ( iomeprol ; 600 mg iodine / kg ) . fenbendazole ( 50 mg / kg q24h po ) was administered continuously for 4 weeks and praziquantel ( 5 mg / kg po ) was repeated three times at an interval of 14 days . two weeks after discharge the cat was presented for follow - up examination . at this time , the cat was in good general condition . radiographs revealed a smaller volume of fluid in the pleural space and a marked widening of the mediastinum . six weeks later ( 9 weeks after initial presentation ) , the cat was re - presented to the emergency service of the hospital because of the sudden onset of anorexia and lethargy . nonetheless , a nodule in the right caudal lung lobe was seen that was not obviously present in the previous radiographs ( figure 3a , b ) . a small amount of pleural fluid can be recognised on the left side ( closed triangle in [ b ] ) . a nodule not previously seen is present in the right caudal lung hemithorax ( arrows in [ b ] ) the most recent blood samples showed pancytopenia with marked leukopenia and marked thrombocytopenia ( leukocytes 0.45 [ ri 611 g / l ] , thrombocytes 3.7 g / l [ ri 150550 g / l ] and haematocrit 22% [ ri 2845% ] ) . because of the pancytopenia a bone marrow aspirate was taken . mesocestoides species infections are quite uncommon in german domestic animals the prevalence of patent infections is < 0.1% in cats and dogs . in other countries the prevalence is distinctly higher ; for example , 7.1% of iranian stray cats harbour adult m lineatus . most case reports of tetrathyridiosis originate from turkey or other southern countries . in this case , owing to the cat originating from malta , there is a possibility that it had been infected with tetrathyridia or adult intestinal mesocestoides species before moving to germany . moreover , the finding of 98% genetic homology of the amplified dna fragment to the m corti 12s ribosomal rna gene suggests the presence of tetrathyridia of a mesocestoides species closely related to m corti ( syn vogae ) . this species is not known to occur in middle europe , which corroborates our import theory . this also corresponds to the statement of the owner that the cat received very irregular antiparasitic treatments . another point supporting the hypothesis of the infection occurring in malta is that the cat lived only indoors since moving to germany several years ago . the faeces of these cats were examined several times and parasites were never been detected . nevertheless , in case of an in - house reservoir the potential first intermediate host remains unclear because all cats lived as indoor - only cats and infection by direct route is not known in mesocestoides species . according to the owner owing to the geographical distribution of m corti , which is not known in middle europe , an infection within germany seems very unlikely . based on the available information there is no relation between the previously diagnosed ulcerative dermatitis and the development of tetrathyridiosis . a typical aetiology of this disease ectoparasites and allergy could be excluded . however , the immunosuppressive treatment with ciclosporin could be a predisposing factor for the development of clinically relevant tetrathyridiosis . these parasites could migrate through the intestinal wall and take residence as tetrathyridia in body cavities avci et al reported a cat with an infected mammary gland without further affected tissues . jabbar et al reported a pleural larval mesocestoidiasis in a cat , which was diagnosed post mortem and was probably the reason for high - grade dyspnoea . previous studies in mice described a good efficacy of mebendazole against mesocestoides species larvae , while praziquantel is known to be effective against all stages of feline cestodes in general . the combination of both drugs was chosen owing to the limited amount of data available and the severity of disease in the present case . the long - term therapy was effective in terms of a significant reduction in the larval burden and resolving the clinical signs , together with the symptomatic treatment . crosbie et al did not experience success when using a combination therapy of albendazole and praziquantel , but described fenbendazole as very effective against tetrathyridiosis in dogs . papini et al , however , observed inefficacy of therapy with fenbendazole alone . repeated treatment with praziquantel was also successful in eliminating peritoneal tetrathyridia in a dog . based on these partially contradictory treatment reports we decided to treat the cat in this report with long - term ( 4 weeks ) fenbendazole in combination with repeated praziquantel administration unfortunately , it is not known if there were larvae left after completion of the therapy . the thoracic nodule , seen in the last follow - up radiographs , might be a sign of recurrent parasitic colonisation . the concurrent anaplastic lymphoma could have promoted the development of clinical tetrathyridiosis . however , via the first cytological evaluation of pleural effusion no evidence of lymphoma was found . additionally , the cat had been immunosuppressed with ciclosporin , which could be a predisposing factor for development of tetrathyridiosis . to our knowledge , neither fenbendazole nor praziquantel , in contrast to ciclosporin , seem to be risk factors for inducing neoplastic conditions such as lymphoma . although very rare , tetrathyridiosis has to be considered as a differential diagnosis in cats presenting with respiratory signs due to pleural effusions and pulmonary nodules . the presence of these radiological findings alone does not prove the diagnosis of neoplastic disease . compared with pleural effusion of neoplastic origin , tetrathyridiosis is a treatable disease , which can be controlled by adequate antiparasitic therapy . furthermore , clinical signs of this disease can be absent for several years and can potentially be triggered by neoplastic conditions or immunosuppression .
patients with cancer cachexia experience loss of weight often accompanied by anorexia and other debilitating symptoms that impact everyday life . two decades of exploratory investigation of the manifestations , meaning and management of cancer cachexia reveal emotional and social impacts for both patients and their family members . this literature provides explanations of why problems are experienced and , as yet unproven , propositions of what can be done about problematic psychosocial effects of cachexia [ 1 , 2 ] . the literature overlooks the potential importance of psychosocial impact and intervention to treatment . since cachexia is multicausal , optimal management of the syndrome is now thought likely to require multimodal treatment . these multimodal treatments can be offered solely for the purpose of reducing cachexia s clinical impact . however , there may be importance in understanding psychosocial effects because of their implications foralleviating cancer cachexia - related sufferingimproving the multimodal treatment of cancer cachexia alleviating cancer cachexia - related suffering improving the multimodal treatment of cancer cachexia this review discusses patient and family impacts , the understanding of what might be done to limit negative impacts and possible implications for research and clinical practice . whilst the implications for the alleviation of cachexia - related suffering have previously been discussed in the literature , the implications for multimodal treatment have not . the clinical impact of cancer cachexia has been the focus of most discussion and research . a brief overview will be given here before considering the patient and family impacts of the syndrome . it manifests itself as involuntary weight loss often accompanied by anorexia and fatigue , all of which have negative clinical impacts , including the deterioration of physical function , quality of life , nutritional status and treatment outcomes . understanding the nature and management of the clinical impact of cancer cachexia for example , fearon et al . provided a consensus definition of cachexia : cachexia is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass . the prominent clinical feature of cachexia is weight loss in adults ( corrected for fluid retention ) or growth failure in children ( excluding endocrine disorders ) . anorexia , inflammation , insulin resistance and increased muscle protein breakdown are frequently associated with wasting disease . wasting disease is distinct from starvation , age - related loss of muscle mass , primary depression , malabsorption and hyperthyroidism and is associated with increased morbidity . cachexia is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass . the prominent clinical feature of cachexia is weight loss in adults ( corrected for fluid retention ) or growth failure in children ( excluding endocrine disorders ) . anorexia , inflammation , insulin resistance and increased muscle protein breakdown are frequently associated with wasting disease . wasting disease is distinct from starvation , age - related loss of muscle mass , primary depression , malabsorption and hyperthyroidism and is associated with increased morbidity . it is important to note that cancer cachexia can be present across the entire course of the disease . the group of experts who developed the definition proposed staging of the syndrome : precachexia ( where there are clinical metabolic changes , but less than 5 % loss of weight ) , cachexia ( more than 5 % weight loss , but sub - groups responsive to different treatment options ) and refractory cachexia ( irreversible catabolic cancer ) . this claim is based on the proposition that early stage cachexia is more likely to be responsive to treatment resulting in meaningful clinical impacts including improved tolerance to cancer treatments . cachexia is often accompanied by malnutrition , which exacerbates the syndrome and contributes to its progression . in addition to the cachectic process , nutritional impact symptoms such as pain and nausea contribute to the progression of the syndrome by compromising nutritional intake , thereby adding a malnutrition component . there is a growing consensus that optimising nutritional intake is an important consideration to treat this malnutrition . future multimodal treatments of cancer cachexia are likely to combine pharmacological treatment of the syndrome with the assessment and management of nutritional impact symptoms and appropriate nutritional support . . however , until effective multimodal treatments have been established in everyday clinical practice , there remains another consideration : the detection and management of cachexia - related psychosocial distress [ 1 , 6 ] , also described as cachexia - related suffering . this distress is caused by the impact of cachexia on both patients and their families . other symptoms are many and experienced by some , but not all , of the cachectic population . common symptoms are anorexia , fatigue , functional loss , early satiety and food aversions . patients can describe change in appearance and loss of physical strength often accompanied by change in eating habits ( amount , type and pattern of food intake ) [ 7 , 8 ] . there is a long - recognised relationship between food , eating and patient experience : eating is a social activity , and food has social and psychological significance for individuals . these nonbiological uses of food can contribute to nutritional disease or nutritional complications of other diseases . conversely , nutritional therapies may disrupt established eating patterns , creating negative psychological and social consequences that can result in noncompliance or even rejection of therapy . eating is a social activity , and food has social and psychological significance for individuals . these nonbiological uses of food can contribute to nutritional disease or nutritional complications of other diseases . conversely , nutritional therapies may disrupt established eating patterns , creating negative psychological and social consequences that can result in noncompliance or even rejection of therapy . there is an extensive literature evidencing the symbolic meanings of food , eating and weight amongst the healthy population . these meanings include sense of identity , personal control , status , independence , health and well - being , dependency , relationship enhancing , relationship constraining , life enhancing and life threatening [ 10 , 11 ] . the meanings of food , eating and weight that emerge in the talk of people who are well are also evident in the lives of those with cancer cachexia . . found 23 % ( 11/48 ) of weight - losing outpatients with advanced cancer to report improved body image . overweight people with cachexia have been found to assume weight loss has health benefits that are identical to the benefits of being of a normal weight when in good health . these observations evidence that weight and physical appearance are important to how we see ourselves and how we think others view us . the positive impact of cancer cachexia is perhaps less obvious , even hidden , compared to the negative emotional and social impacts . mcclement uses the phrase dealing with a body in shambles in an attempt to capture the experience of disease - induced weight loss . she draws on emotive statements made by participants in an interview study to communicate the negative impact of cancer - related weight loss.this bony thing shows up in the mirror every morning , and my eyes fall on this creature on the other side of the mirror.i was five feet from him before he could figure out who it was . this bony thing shows up in the mirror every morning , and my eyes fall on this creature on the other side of the mirror . these quotes illustrate some of the themes identified in other exploratory studies of patient experience of involuntary weight loss or cancer cachexia . the physical change in appearance changes sense of self , which can lead to puzzlement and confusion , and even challenges identity . the body is experienced differently leading to body image concerns including negative body image , with younger people found to have the greatest dissatisfaction with body image . the negative perceptions of body image can be reinforced by the response of others to the change . a sense of being different can prompt feelings of helplessness , loss of control , abandonment and stigmatisation . such experience can precipitate anxiety and distress [ 19 , 20 ] . people with disease - related weight loss have functional loss accompanied by loss of independence , which can cause distress at being a burden to others .i do nt like being weak frustrated , awfy [ awfully ] frustrated about it i depend on others for lots of things now that i never did afore [ before ] . cause i used to be very very independent . would nt let them do anything for me . i do nt like being weak frustrated , awfy [ awfully ] frustrated about it i depend on others for lots of things now that i never did afore [ before ] . cause i used to be very very independent . would nt let them do anything for me . furthermore , uncontrolled weight loss provokes thoughts of imminent death .you do nt necessarily have to get on the scales , you see the bones begin to protrude and feel the end is nigh . you do nt necessarily have to get on the scales , you see the bones begin to protrude and feel the end is nigh . autonomy , control and independence are socially valued and admired . a dominant discourse in western society is of taking control of one s body to sustain health and independence and prolong life . some authors are beginning to identify tumour , social context or disease site - specific causes of distress . for example , chasen and bhargava have found head and neck cancer patients can experience social isolation as a consequence of drooling and other problems associated with their disease and treatment . a study by lvgren et al . found that those living with a partner found appetite loss more problematic than those living alone . argue for culturally sensitive dietary information on the basis of holding a focus group with a canadian chinese cancer support group . the group members were experiencing conflict between the biomedical perspective on food and nutrition and traditional chinese beliefs about food , health and illness . the personal and social impacts of cancer cachexia whether positive or negative have implications for the management of the syndrome . if involuntary weight loss is welcomed amongst the obese , it may inhibit their self - management of diet and physical activity , currently thought important for maintenance of muscle mass and mitigation of progression of the syndrome . conversely , if involuntary weight loss is unwelcome and evokes distress , talking about weight , diet and physical activity may be difficult , presenting an obstacle to both offering psychosocial support and supporting compliance with multimodal treatment . . found 23 % ( 11/48 ) of weight - losing outpatients with advanced cancer to report improved body image . overweight people with cachexia have been found to assume weight loss has health benefits that are identical to the benefits of being of a normal weight when in good health . these observations evidence that weight and physical appearance are important to how we see ourselves and how we think others view us . the positive impact of cancer cachexia is perhaps less obvious , even hidden , compared to the negative emotional and social impacts . mcclement uses the phrase dealing with a body in shambles in an attempt to capture the experience of disease - induced weight loss . she draws on emotive statements made by participants in an interview study to communicate the negative impact of cancer - related weight loss.this bony thing shows up in the mirror every morning , and my eyes fall on this creature on the other side of the mirror.i was five feet from him before he could figure out who it was . this bony thing shows up in the mirror every morning , and my eyes fall on this creature on the other side of the mirror . these quotes illustrate some of the themes identified in other exploratory studies of patient experience of involuntary weight loss or cancer cachexia . the physical change in appearance changes sense of self , which can lead to puzzlement and confusion , and even challenges identity . the body is experienced differently leading to body image concerns including negative body image , with younger people found to have the greatest dissatisfaction with body image . the negative perceptions of body image can be reinforced by the response of others to the change . a sense of being different can prompt feelings of helplessness , loss of control , abandonment and stigmatisation . people with disease - related weight loss have functional loss accompanied by loss of independence , which can cause distress at being a burden to others .i do nt like being weak frustrated , awfy [ awfully ] frustrated about it i depend on others for lots of things now that i never did afore [ before ] . cause i used to be very very independent . would nt let them do anything for me . i do nt like being weak frustrated , awfy [ awfully ] frustrated about it i depend on others for lots of things now that i never did afore [ before ] . cause i used to be very very independent . would nt let them do anything for me furthermore , uncontrolled weight loss provokes thoughts of imminent death .you do nt necessarily have to get on the scales , you see the bones begin to protrude and feel the end is nigh . you do nt necessarily have to get on the scales , you see the bones begin to protrude and feel the end is nigh . a dominant discourse in western society is of taking control of one s body to sustain health and independence and prolong life . some authors are beginning to identify tumour , social context or disease site - specific causes of distress . for example , chasen and bhargava have found head and neck cancer patients can experience social isolation as a consequence of drooling and other problems associated with their disease and treatment . a study by lvgren et al . found that those living with a partner found appetite loss more problematic than those living alone . argue for culturally sensitive dietary information on the basis of holding a focus group with a canadian chinese cancer support group . the group members were experiencing conflict between the biomedical perspective on food and nutrition and traditional chinese beliefs about food , health and illness . the personal and social impacts of cancer cachexia whether positive or negative have implications for the management of the syndrome . if involuntary weight loss is welcomed amongst the obese , it may inhibit their self - management of diet and physical activity , currently thought important for maintenance of muscle mass and mitigation of progression of the syndrome . conversely , if involuntary weight loss is unwelcome and evokes distress , talking about weight , diet and physical activity may be difficult , presenting an obstacle to both offering psychosocial support and supporting compliance with multimodal treatment . because the symptoms of cancer cachexia impact social interactions and relationships , patients families are affected . little attention has been paid to family members of people with cachexia , in spite of their potential to influence both psychosocial support and treatment compliance . unlike patients , family members have not been found to report patients involuntary weight loss being a health benefit , or even as being of no concern . a consistent story emerges from the few studies exploring family experience of cancer cachexia . in england , ireland , switzerland , canada and america , empirical studies have found the symptoms of cachexia distressing for family members [ 15 , 20 ] , to disrupt daily routines , to be experienced as a cascade of losses , to bring about role change and to be accompanied by lost opportunities for interpersonal interaction . all of these impacts are illustrated in the following quote from a work by meares : i cooked a lot , i baked a lot he ate what i made because i made what he liked we always ate together he would nt eat if i did nt eat , so i stopped eating when he stopped eating and dinner hour , i did nt realize until he stopped eating that there was a dinner hour , and then there was none , and it was so difficult to get through the day because what to do you do from 5 to 7 ? i cooked a lot , i baked a lot he ate what i made because i made what he liked so i stopped eating when he stopped eating and dinner hour , i did nt realize until he stopped eating that there was a dinner hour , and then there was none , and it was so difficult to get through the day because what to do you do from 5 to 7 ? such changes in everyday life can precipitate conflict within families over food : his daughter came last november and she was forcing him to eat gelatin , and i said , he does nt want it . dad you have got to eat , you have got to eat . he would become agitated , and he would say , no , and she would still say for him to eat to be strong . at the end , i was watching his daughters trying to coax him , and never giving up . his daughter came last november and she was forcing him to eat gelatin , and i said , he does nt want it . dad you have got to eat , you have got to eat . he would become agitated , and he would say , no , and she would still say for him to eat to be strong . at the end , i was watching his daughters trying to coax him , and never giving up . conflicting opinion in families relating to food and eating may arise from not knowing what to do in a context where it is unusual to initiate discussion about weight and diet with someone who has visible weight loss . moreover , the limited talk that does take place about weight and eating problems can result in feelings of being neglected by health - care professionals [ 25 , 28 ] , feelings of rejection and even social exclusion : at first i thought we were in limbo , nobody cared , that we could nt turn to anybody i thought that someone should have come and spoke to us as a family to tell us what to expect when he was nt feeling well and he was nt eating we did nt know whether to call for a doctor or what or who to turn to . at first i thought we were in limbo , nobody cared , that we could nt turn to anybody i thought that someone should have come and spoke to us as a family to tell us what to expect when he was nt feeling well and he was nt eating we did nt know whether to call for a doctor or what or who to turn to . the importance of considering the impact of cancer cachexia for family members , as with patients , lies in the management of the syndrome . for example , they may reinforce or challenge a patient s response to the symptoms of cancer cachexia . for example , support a decision by an obese patient to consider self - management of diet and physical activity as being unimportant . similarly , if the patient s involuntary weight loss and other symptoms evoke distress and a reluctance to talk about the experience , then this might be a further obstacle to patient uptake and compliance with both multimodal treatment and supportive psycho - educational interventions . unlike patients , family members have not been found to report patients involuntary weight loss being a health benefit , or even as being of no concern . a consistent story emerges from the few studies exploring family experience of cancer cachexia . in england , ireland , switzerland , canada and america , empirical studies have found the symptoms of cachexia distressing for family members [ 15 , 20 ] , to disrupt daily routines , to be experienced as a cascade of losses , to bring about role change and to be accompanied by lost opportunities for interpersonal interaction . all of these impacts are illustrated in the following quote from a work by meares : i cooked a lot , i baked a lot he ate what i made because i made what he liked so i stopped eating when he stopped eating and dinner hour , i did nt realize until he stopped eating that there was a dinner hour , and then there was none , and it was so difficult to get through the day because what to do you do from 5 to 7 ? i cooked a lot , i baked a lot he ate what i made because i made what he liked so i stopped eating when he stopped eating and dinner hour , i did nt realize until he stopped eating that there was a dinner hour , and then there was none , and it was so difficult to get through the day because what to do you do from 5 to 7 ? such changes in everyday life can precipitate conflict within families over food : his daughter came last november and she was forcing him to eat gelatin , and i said , he does nt want it . dad you have got to eat , you have got to eat . he would become agitated , and he would say , no , and she would still say for him to eat to be strong . at the end , i was watching his daughters trying to coax him , and never giving up . his daughter came last november and she was forcing him to eat gelatin , and i said , he does nt want it . dad you have got to eat , you have got to eat . he would become agitated , and he would say , no , and she would still say for him to eat to be strong . at the end , i was watching his daughters trying to coax him , and never giving up . and i said , good lord , leave the poor man alone ! conflicting opinion in families relating to food and eating may arise from not knowing what to do in a context where it is unusual to initiate discussion about weight and diet with someone who has visible weight loss . moreover , the limited talk that does take place about weight and eating problems can result in feelings of being neglected by health - care professionals [ 25 , 28 ] , feelings of rejection and even social exclusion : at first i thought we were in limbo , nobody cared , that we could nt turn to anybody i thought that someone should have come and spoke to us as a family to tell us what to expect when he was nt feeling well and he was nt eating we did nt know whether to call for a doctor or what or who to turn to . at first i thought we were in limbo , nobody cared , that we could nt turn to anybody i thought that someone should have come and spoke to us as a family to tell us what to expect when he was nt feeling well and he was nt eating we did nt know whether to call for a doctor or what or who to turn to . the importance of considering the impact of cancer cachexia for family members , as with patients , lies in the management of the syndrome . for example , they may reinforce or challenge a patient s response to the symptoms of cancer cachexia . for example , support a decision by an obese patient to consider self - management of diet and physical activity as being unimportant . similarly , if the patient s involuntary weight loss and other symptoms evoke distress and a reluctance to talk about the experience , then this might be a further obstacle to patient uptake and compliance with both multimodal treatment and supportive psycho - educational interventions . theoretical explanations of how people feel and behave in response to cancer anorexia that can inform clinical interventions are few . developed a model that categorised patient and caregiver response to anorexia as fighting back , letting nature take its course or these three categories were proposed as different ways of doing what s best when confronted by anorexia . [ 30 , 31 ] , during a longitudinal interview study with 12 patients with cancer cachexia , found that they shift conscious control of eating once the normal desire to eat was lost . he theorises that taking conscious control is important for emotional and social adaptation to anorexia and therefore that patients should be supported in this behaviour . my own work has proposed that there is a weight loss taboo sustained by the helplessness induced by cancer anorexia in patients , caregivers and health - care professionals . this taboo can obstruct communication which might otherwise help patients and their family members seek help for weight- and eating - related distress . the identified solution is to develop communication techniques that can break through the weight loss taboo . the work also has developed a theory of self - management of weight and eating problems informed by both lazarus theory of adaptation and coping and the ways patients and their family members were found to manage life with cancer anorexia . breaking through the weight loss taboo is seen as a pathway to the reinforcement of existing coping resources and raising awareness of alternatives . more recently , reid et al . reported that the emotional consequences of cancer anorexia may be greatest for caregivers and that distress is caused by the resulting conflict between patient and caregiver perspectives on declining food intake . she argues that family members use feeding as a defence against anxiety , and therefore , the solution is to provide information about cancer cachexia . however , it should be noted that the models identified to date are based on empirical studies of people who would mostly fall into the category of refractory cachexia . no empirical work has been conducted to examine the similarity and difference in psychosocial impact according to the stage of cachexia . the empirically based models of the psychosocial effect of cancer cachexia generate complementary propositions explaining the experience of cancer cachexia for patients and their family members . these propositions , which may help identify effective psychosocial interventions to alleviate the negative impact of cancer cachexia , include the following : providing information about cachexia will alleviate anxiety and break the cycle of conflict .aiding conscious control can facilitate adaptation .enhancing personal and environmental coping resources will aid self - management and alleviate cachexia - related suffering [ 2 , 29].the weight loss taboo is an obstacle to the communication necessary to address cachexia - related problems , such as nutritional impact symptoms . providing information enhancing personal and environmental coping resources will aid self - management and alleviate cachexia - related suffering [ 2 , 29 ] . the weight loss taboo is an obstacle to the communication necessary to address cachexia - related problems , such as nutritional impact symptoms . collectively , the models suggest that providing information about cancer cachexia , supporting adaptation and strengthening coping and intervening in the interactional processes between someone with cachexia and others will all have benefit . these interventions have all been theorised to reduce the negative emotional consequences of cancer cachexia . only one approach has been tested , this being one that combined an interactional process for breaking through the weight loss taboo with information provision and support for self - management of cachexia - related problems . the approach was found acceptable and feasible in a small exploratory trial with no negative consequences . for example , a multimodal treatment might combine pharmacology to treat primary causes such as the tumour - induced inflammatory process that contributes to weight loss , nutritional support to counter any malnutrition component of symptom progression and exercise to maintain muscle mass . however , no empirical work has been conducted to investigate the potential for psychosocial intervention to facilitate multimodal treatments . obstacles to effective multimodal treatment may include psychosocial factors , such as attitudes , beliefs and behaviours of patients , carers and health - care professionals . the weight loss taboo may explain why patients and families can report the unmet need for information about weight loss and anorexia . it may also explain why health - care professionals have been found to manage the syndrome reactively . attention to patient - family - health - care professional communication may be an important consideration for the delivery of effective multimodal therapy . there is evidence that carers influence patient response to cancer cachexia . in other conditions , for example , people on cardiac rehabilitation programmes are more likely to attend with support from a family member , family support and caregiver factors effect medicine adherence , conflict in the family has a negative effect on adherence and , in diabetes , family support , in particular the quality of patient - family relationships , is important for adherence to dietary regimes and other treatments , the nature of support determining positive or negative effect on outcomes . psychosocial interventions may thus have utility and relevance for effective multimodal treatment of cancer cachexia . not only do they have the potential to relieve distress and conflict , but in doing so , they may also improve compliance with other treatment components . attention to the assessment of psychosocial , as well as clinical , impacts of cancer cachexia and implications for patient - health professional communication may be important to both quality of life of patients and multimodal treatment of the syndrome . if multimodal interventions are to be offered in early stages of cachexia before disease is refractory to treatment , then they should perhaps become part of treatment regimes and psychosocial support integrated with supportive care in oncology clinics . in this way psychosocial intervention in cachexia should be researched for potential to not only help people affected by cancer cachexia feel better but also for its potential to improve clinical outcomes through better uptake and compliance with multimodal therapy . jane hopkinson has received a consultancy fee from helsinn healthcare to participate as a member of the scientific advisory board in the development of a website for health - care professionals dedicated to cachexia .
hyperglycemia is a risk factor for microvascular complications in patients with type 2 diabetes . apart from hyperglycemia , dyslipidemia also contributes to the risk of developing complications in patients with type 2 diabetes . even the national cholesterol education program ( ncep ) adult treatment panel iii has recommended achieving low - density lipoprotein cholesterol ( ldl - c ) goals of < 100 mg / dl for patients with diabetes as they are at high risk of developing cardiovascular events . thus , new treatment regimens that can improve both glycemic control and lipid management in type 2 diabetes patients will be clinically beneficial . in this regard , colesevelam hydrochloride , the ldl - c lowering medication , which is also indicated for glycemic control seems promising . colesevelam hydrochloride was previously approved as an adjunct to diet and exercise , to reduce elevated ldl - c in patients with primary hyperlipidemia . on january 18 , 2008 , this agent was also approved as an adjunct to diet and exercise , to improve glycemic control in adult patients with type 2 diabetes . colesevelam is a bile acid sequestrant ( bas ) with a high capacity for binding bile acids in the intestine , thereby impeding their re - absorption and enterohepatic circulation . this leads to the upregulation of the hepatic enzyme , cholesterol 7-alpha - hydroxylase , causing an increase in the conversion of cholesterol to bile acids and the activity of the hydroxymethylglutaryl - coenzyme a ( hmg - coa ) reductase ( rate limiting step of cholesterol synthesis ) . the number of hepatic ldl receptors is also increased , thus increasing the clearance of ldl - c from the blood , resulting in decreased serum ldl - c levels . the exact mechanism by which colesevelam improves glycemic control is unknown . the various explanations suggested are colesevelam acts in the gastrointestinal tract , thereby reducing the amount of glucose absorbed or by binding with the bile acids it disrupts the enterohepatic pathway of bile metabolism , which has indirect effects on glucose metabolism . colesevelam is a hydrophilic , water insoluble polymer that is not hydrolyzed by digestive enzymes . pharmacokinetic studies with colesevelam have not shown clinically significant effects on the bioavailability of digoxin , fenofibrate , lovastatin , metoprolol , quinidine , valproic acid , warfarin or statins . reported adverse events from the various clinical trials include flatulence , dyspepsia , and diarrhea . colesevelam should not be used for the treatment of type i diabetes or for the treatment of diabetic ketoacidosis . colesevelam is contraindicated in individuals with bowel obstruction , those with serum triglyceride ( tg ) concentrations of > 500 mg / dl or with a history of hypertriglyceridemia - induced pancreatitis . caution should be exercised when treating patients with tg levels > 300 mg / dl . colesevelam may decrease the absorption of fat - soluble vitamins a , d , e , and k. patients on vitamin supplements should take their vitamins at least four hours prior to colesevelam . caution should be exercised when treating patients with a susceptibility to vitamin k or fat soluble vitamin deficiencies . the efficacy of colesevelam for the improvement of glycemic control was assessed in three double - blind , placebo - controlled trials in which this agent was combined with metformin , sulfonylureas , or insulin . in the first trial , the patients already receiving treatment with metformin alone ( n = 159 ) , or metformin in combination with other oral agents ( n = 157 ) , were randomized to receive either colesevelam 3.8 g / d or placebo as an add - on therapy , for 26 weeks . the addition of colesevelam to metformin alone was associated with a - 0.4% least - squares mean change ( lsmc ) in the glycated hemoglobin ( hba1c ) level from the baseline , versus no change with the addition of placebo ( treatment difference , - 0.5% ; p = .002 ) . the addition of colesevelam to metformin in combination with other oral antidiabetic agents was also associated with a - 0.4% lsmc in hba1c versus a 0.3% lsmc with the addition of placebo ( treatment difference , - 0.6% ; p < 0.001 ) . in another trial , patients reporting inadequate glycemic control with sulfonylurea alone ( n = 156 ) or sulfonylurea plus other oral antidiabetic agents were randomized to receive either colesevelam 3.75 g / d or placebo as an add - on therapy , for 26 weeks . the addition of colesevelam to sulfonylurea alone was associated with a - 0.3% lsmc in the hba 1clevel ; the addition of colesevelam to sulfonylurea plus other oral antidiabetic agents was associated with a - 0.4% lsmc in hba1c . in another 16-week study , involving 287 type 2 diabetes patients being treated with insulin monotherapy or in combination with an oral anti - diabetes agent , addition of colesevelam 3.75 g / dl decreased ldl - c by 12.8% , increased triglycerides by 21.5% , lowered hba1c by 0.5% , and fasting glucose levels by 14.6 mg / dl . colesevelam is indicated as an adjunct to diet and exercise , to reduce elevated ldl - c in patients with primary hyperlipidemia , as a monotherapy or in combination with an hmg - coa reductase inhibitor . colesevelam offers physicians a new treatment option that addresses two cardiovascular risk factors , high ldl - cholesterol and blood glucose in patients with type 2 diabetes . the inclusion of colesevelam may represent a novel therapeutic add - on strategy for improving multiple metabolic parameters in these patients .
colon cancer , however , is generally a disease of the elderly and occurs only rarely during pregnancy . its symptoms typically include nausea , vomiting , abdominal pain , and altered bowel movements , which commonly overlap with symptoms of normal pregnancy . therefore , physicians and patients usually attribute them to the general manifestations of pregnancy without further workup or delay diagnostic tests because of potential fetal risks . a missed diagnosis of colorectal cancer is accordingly associated with development of advanced disease and poorer prognosis . although the evaluation of colon cancer in the general population includes abdominal computed tomography ( ct ) to detect pericolonic extension and intraperitoneal metastases , ct is contraindicated during pregnancy , particularly the first trimester , because of radiation teratogenicity and colonoscopy is not yet an established procedure because of concerns about fetal risk . treatment of colon cancer during pregnancy is also different from therapy for colon cancer in normal population . for example , adjuvant chemotherapy or adjuvant radiotherapy is beneficial to the mother but may be harmful to the fetus . in this paper , we report the case of a pregnant woman that was diagnosed with adenocarcinoma of the descending colon at her 22nd gestational week . a 31-year - old gravida iii para i patient presented at the emergency department in her 22nd gestational week with crampy left sided flank pain , which were first attributed to possible kidney stones . after outpatient treatment with acetaminophen and nsaids she was readmitted 2 days later with nausea , vomiting and obstipation . physical examination revealed a gravid abdomen with left sided abdominal pain on palpation and normal bowel sounds , meeting the criteria of constipation , which is known to be common in pregnant women . despite receiving conservative treatment for her constipation , her symptoms became worse . after 3 days of ineffective treatment of her obstipation a magnetic resonance imaging ( mri ) scan of her abdomen was obtained . the scan showed distended small and large bowel with a sudden change of diameter between the distended descending colon and the collapsed sigmoid colon and rectum ( fig . 1 ) . a subsequent colonoscopy revealed a stenosing carcinoma of the descending colon , which was confirmed by endoscopic biopsies . after consultation with the obstetricians an open left sided hemicolectomy with end - to - side hand - sewn transversosigmoidostomy was performed 10 days after initial presentation . intraoperatively dilated small and large bowel with a palpable tumor mass at the descendo - sigmoidal junction could be identified ( fig . tumor histology confirmed the diagnosis of adenocarcinoma of the descendo - sigmoidal junction with a diameter of 50 mm . the patient developed an anastomotic insufficiency 4 days after hemicolectomy with acute peritonitis . within the same day intraoperatively the anastomosis was found dehiscent involving half of the suture with consecutive fecal peritonitis . unusually the sigmoid colon distal to the anastomosis was dilated due to entrapment of the distal sigmoid colon between the uterus and the pelvis . five days after hartmann 's procedure the patient was diagnosed with dehiscence of the abdominal wall , necessitating a third operation . after discussion at the tumor board conference chemotherapy with 5-fluorouracil was started in the 29th gestational week . despite the eventful and complicated postoperative course the obstetric surveillance of the baby did not show any abnormalities during in - patient treatment . the patient had an uneventful delivery of a healthy baby in her 39th gestational week . it is a rare event and its symptoms ( e.g. constipation , change in bowel habits , nausea and vomiting ) , which can occur due to physiological changes during pregnancy , can create difficulties to establish the diagnosis . a study by the american society of colon and rectal surgeons investigating 41 cases revealed a mean age of 31 years when diagnosis was obtained . majority of tumors were localized in the rectum ( 63% ) and the sigmoid colon ( 20% ) , and dukes stage at presentation was b or higher in all patients . diagnosis of colorectal cancer during pregnancy is often delayed because symptoms are unspecific until the disease is advanced . in one reported case diagnosis although constipation in pregnancy is a common symptom differential diagnosis of a mechanical stenosis should always be contemplated , especially when conservative treatment of constipation fails . the mainstay of diagnostic evaluation in pregnant patients with colorectal cancer includes abdominal imaging and endoscopy . cea can be used as a prognostic factor since elevated values prior to surgery are related to a higher rate of recurrence and disseminated disease . magnetic resonance imaging ( mri ) is the imaging tool of choice as abdominal computed tomography ( ct ) is contraindicated in pregnancy . despite of relative contraindications in pregnant women , endoscopic confirmation should be obtained to gain pathological diagnosis of colorectal carcinoma . surgery is the gold standard of treatment for colon cancer in pregnancy based on maternal cancer prognosis , gestational age and intraoperative findings . the tumor resection should not be delayed owing to significant tumor progression and worsening of the prognosis . in relation to the stage of the disease chemotherapy 5-fu has been shown to increase survival in colorectal cancer and to be safe in pregnant women . in summary , obstructing colorectal cancer can be a rare reason for the common problem of constipation in pregnancy . . clinical examination , mri scan and colonoscopy will reveal the tumor in most cases and should be followed by surgical treatment and chemotherapy according to the stage of disease . 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 . urs von holzen was the treating surgeon and participated in design and coordination and helped to draft the manuscript.key learning pointscolorectal carcinoma is a rare but fatal event during pregnancy.presentation can coincide with the signs and symptoms of pregnancy.diagnoses are often delayed.treatment is not different from those in the general population.special care regarding fetal safety should be considered . colorectal carcinoma is a rare but fatal event during pregnancy.presentation can coincide with the signs and symptoms of pregnancy.diagnoses are often delayed.treatment is not different from those in the general population.special care regarding fetal safety should be considered .
polymyalgia rheumatica ( pmr ) is relatively common among people who are over 50 years of age . the combination of persistent pain over multiple joints and muscles of the body , morning stiffness , and elevated erythrocyte sedimentation rate ( esr ) are characteristics of this condition.1 multiple genetic and environmental factors are considered to influence susceptibility to this illness , but the exact etiology and pathogenesis of pmr remains unknown . although there are reports on the occurrence of pmr after influenza vaccination , we here report the first case in which pmr occurred after influenza infection . institutional review board approval was not sought , as this is a case report that does not require irb approval as per our irb protocol . an 85-year - old woman with a medical history of diabetes mellitus and dyslipidemia presented in march 2015 to an internal medicine outpatient clinic with myalgia , morning stiffness , and difficulty in standing up from a chair lasting for several weeks . she also complained of pain in the shoulders bilaterally , particularly on the right , and in the proximal arms bilaterally . symptomatic treatment of unknown detail did not improve her symptoms , and she presented to our clinic . prior to the onset of her symptoms in february 2015 , she had developed high fever , was diagnosed with influenza b by one of the rapid influenza diagnostic tests at a clinic nearby , and was provided with intravenous peramivir . however , she started to develop myalgia and the other symptoms described on the day of defervescence . medications given by her internist at presentation included insulin , glibenclamide , voglibose , rosuvastatin , neurotrophin , and acetaminophen . on physical examination she was alert and oriented , with blood pressure of 142/85 mmhg , pulse rate of 66/minute , respiratory rate of 13/minute , and body temperature of 36.6c . there was tenderness over the trapezius muscles , elbow joints bilaterally , both thighs , particularly on the lateral side , and both calves . she was able to stand up from a chair without using her arms , but she found it somewhat difficult and had to do it slowly , although she was able to walk in the examining room without difficulty . the rest of her physical examinations , including neurological , were unremarkable . laboratory tests revealed white blood cell counts of 10.310/l with 73% neutrophils , hemoglobin of 10.5 g / dl with mean corpuscular volume of 86.2% , c - reactive protein ( crp ) of 4.79 mg / dl , and esr 56 of mm / hour . iu / l , and rheumatoid factor ( rf ) and anticyclic citrullinated peptide antibody ( anti - ccp ) levels were within normal limits . based on the presence of myalgia , morning stiffness , physical findings suggestive of bursitis in the shoulders and glenohumeral areas , absence of rf and anti - ccp , absence of ck elevation , and elevated crp and esr , her symptoms improved gradually , and she continues to receive a lowered dose of prednisolone as of this writing . even though the etiology of pmr remains unknown , a number of infectious agents have been suggested to cause pmr . these include mycoplasma pneumoniae , chlamydia ( chlamydophila ) pneumoniae , and parvovirus b19.2 in addition , there are reports of pmr after influenza vaccination.38 the exact role of influenza vaccination on the development of pmr remains unknown , but may be associated with specific human leukocyte antigens ( hlas ) , such as hla - drb1 and hla - dqb1.3 whether postvaccination pmr is caused by influenza virus antigen or adjuvants in the vaccine is another unanswered question . the incidence of pmr in japan appears to be correlated with the influenza season.9 however , there has been no single case report of pmr after influenza virus infection from japan or otherwise . this report is therefore the first , as far as we know of , case report of post - influenza virus infection pmr . although we could not demonstrate influenza virus directly from the patient , rapid influenza diagnostic tests are highly specific,10 and her history of influenza , together with her medical history , was fairly plausible , even though the lack of a standard confirmatory test , such as reverse - transcription polymerase - chain reaction , for the diagnosis of influenza infection somewhat lessens the scientific validity of our hypothesis . rheumatoid arthritis can occur in the elderly , and can be difficult to distinguish from pmr clinically . however , both rf and anti - ccp antibody were negative in this case , making this diagnosis less likely . giant - cell arteritis and remitting seronegative symmetric synovitis with pitting edema ( rs3pe ) are considered to be associated with pmr , and they can be very similar to pmr , but the lack of such symptoms as fever , headache , and pitting edema made both diagnoses less likely . diseases with myositis , including those caused by statins and influenza itself , are less likely , since there was no elevation of ck , another characteristic of pmr . influenza per se can cause myalgia , myositis , or even rhabdomyolysis , but these tend to occur in children and the onset is during not after influenza illness,1113 unlike such infections as chikungunya.14 our case met diagnostic criteria by hunter , healey , jones , hazelman , and bird , with typical age , symptoms , elevated esr , and good response to low - dose corticosteroid,15 and the case also met the provisional classification without ultrasound of the european league against rheumatism / american college of rheumatology collaborative initiative,16 making the diagnosis of pmr most likely . the incidence of pmr is common in the us,1 but had been thought to be less common in japan . however , a recent report suggests pmr may be more common in japan , with possible misdiagnosis or underdiagnosis.9 if one suspects pmr more avidly and looks into the preceding episodes of viral infections , such as influenza , we might be able to see more association between such viral infections as influenza and pmr . this single case report like ours merely suggests but does not prove the causality of influenza infection as a cause of pmr , even though chronological immediacy strongly suggests its contributory role in the pathogenesis of pmr . in conclusion , we report a case of pmr following influenza virus b infection . further studies may reveal the true incidence of pmr in japan , its triggering factors , including influenza virus infection , and its etiology and pathogenesis .
spinal artery aneurysms are most commonly associated with high - flow states secondary to either arteriovenous shunting or occlusive disease of larger arteries causing increased flow through spinal arteries recruited as collateral circulation.1)2)4 ) an aneurysm is considered " isolated " when it occurs in the absence of such a high flow state . aneurysms of the spinal vasculature are more common along the anterior axis than the posterior axis , and isolated aneurysms account for only a small fraction of these . histologically , isolated spinal artery aneurysms have features shared with cerebral artery aneurysms,7 ) suggesting that at least some may be the product of a congenital predisposition to aneurysm formation . however , isolated anterior spinal artery and cerebral artery aneurysms have been associated with both central nervous system - specific and systemic vasculitis.5)13)17)20 ) here , we present the first case to our knowledge documenting a posterior spinal artery ( psa ) aneurysm in the context of a hypersensitivity vasculitis ( leukocytoclastic vasculitis ) . a 53-year - old male with a history of hepatitis c , hepatitis b , and poly - substance abuse presented to an outside hospital for bilateral lower - extremity recurrent cellulitis , hypoalbuminemia and acute kidney injury . on the ninth day of admission , the patient developed acute paraplegia associated with a t5/6 sensory level deficit . a thoracic spine magnetic resonance imaging ( mri ) obtained on the day of presentation was read as negative for evidence of compressive cord lesion and the patient was medically managed with broad - spectrum antibiotics and steroids . the patient 's neurological symptoms did not improve with medical management and 15 days later a second thoracic spine mri was obtained . 1a ) with compressive effects at the level of c7-t1 with associated increase in t2 signal intensity in the cord extending from c7-t5 , suggesting edema . thoracic spinal angiography revealed a 1.5 mm ectasia of the right t9 segmental artery 's radiculopial branch , consistent with an aneurysm of the posterior spinal artery ( fig . in addition to the right t9 aneurysm , a segment of the left t5 radiculopial artery revealed vascular pathology along the posterior aspect of the spinal cord suggestive of either an inflammatory process or post - hemorrhagic reactive changes ( fig . vascular bleeding and lumen irregularities consistent with an acute inflammatory process were noted throughout the cervical and thoracic spine , and the angiogram also revealed a 3 mm right middle cerebral artery ( mca ) aneurysm . a coil was placed in the segmental artery supplying the t9 aneurysm for subsequent intra - operative localization during the open resection . the patient underwent a multilevel laminectomy ( c7-t2 and t8 - 10 ) for hematoma evacuation and microsurgical aneurysm resection . the histology was consistent with a dissecting aneurysm with localized regions of lymphocytic infiltration of the intima , and fibrosis and necrotic changes in regions of the tunica media ( fig . samples of the hematoma from the t2-level had histological features consistent with an organizing intradural hematoma . multiple medical issues complicated the postoperative course . neurologically , the patient 's pre - operative deficits remained unchanged for the duration of his hospitalization . renal biopsy demonstrated membranoproliferative glomerulonephritis secondary to hepatitis c virus - related cryoglobulinemic vasculitis and he underwent treatment with rituximab , plasmapheresis and prednisone with improvement in his renal function . the patient subsequently developed a urinary tract infection with multiple - drug resistant klebsiella and was started on imipenem . a 53-year - old male with a history of hepatitis c , hepatitis b , and poly - substance abuse presented to an outside hospital for bilateral lower - extremity recurrent cellulitis , hypoalbuminemia and acute kidney injury . on the ninth day of admission , the patient developed acute paraplegia associated with a t5/6 sensory level deficit . a thoracic spine magnetic resonance imaging ( mri ) obtained on the day of presentation was read as negative for evidence of compressive cord lesion and the patient was medically managed with broad - spectrum antibiotics and steroids . the patient 's neurological symptoms did not improve with medical management and 15 days later a second thoracic spine mri was obtained . 1a ) with compressive effects at the level of c7-t1 with associated increase in t2 signal intensity in the cord extending from c7-t5 , suggesting edema . thoracic spinal angiography revealed a 1.5 mm ectasia of the right t9 segmental artery 's radiculopial branch , consistent with an aneurysm of the posterior spinal artery ( fig . in addition to the right t9 aneurysm , a segment of the left t5 radiculopial artery revealed vascular pathology along the posterior aspect of the spinal cord suggestive of either an inflammatory process or post - hemorrhagic reactive changes ( fig . vascular bleeding and lumen irregularities consistent with an acute inflammatory process were noted throughout the cervical and thoracic spine , and the angiogram also revealed a 3 mm right middle cerebral artery ( mca ) aneurysm . a coil was placed in the segmental artery supplying the t9 aneurysm for subsequent intra - operative localization during the open resection . the patient underwent a multilevel laminectomy ( c7-t2 and t8 - 10 ) for hematoma evacuation and microsurgical aneurysm resection . the histology was consistent with a dissecting aneurysm with localized regions of lymphocytic infiltration of the intima , and fibrosis and necrotic changes in regions of the tunica media ( fig . samples of the hematoma from the t2-level had histological features consistent with an organizing intradural hematoma . . neurologically , the patient 's pre - operative deficits remained unchanged for the duration of his hospitalization . medically , the patient deteriorated over the next 60 days . renal biopsy demonstrated membranoproliferative glomerulonephritis secondary to hepatitis c virus - related cryoglobulinemic vasculitis and he underwent treatment with rituximab , plasmapheresis and prednisone with improvement in his renal function . the patient subsequently developed a urinary tract infection with multiple - drug resistant klebsiella and was started on imipenem . very little is known about the pathophysiology of isolated psa aneurysms . here , we present the case of a ruptured psa aneurysm in the context of a leukocytoclastic vasculitis . spinal angiography demonstrated evidence of diffuse inflammatory process affecting multiple levels , histology confirmed the presence of a leukocytic infiltrate at the site of the resected aneurysm and subsequent skin biopsy confirmed the diagnosis of leukocytoclastic vasculitis . leukocytoclastic vasculitis is a small - vessel hypersensitivity vasculitis resulting from immune complex deposition in the vessel wall , leading to lymphocytic infiltration . this inflammatory process may have predisposed our patient to aneurysm formation , or it may have compromised the wall integrity of a pre - existing psa aneurysm , precipitating rupture . we identified 14 cases of isolated psa aneurysms , which are summarized in table 1 . none occurred in the context of a confirmed vasculitis but it is not clear that vasculitis was ruled out . for the current case , an important question remains whether diagnosis and treatment of the vasculitis could have prevented aneurysm rupture . the natural history of spinal aneurysms likely mirrors that of intracranial aneurysms , although published natural history data of spinal aneurysms is sparse . most presented after an initial rupture and rehemorrhage occurred in 2 of 15 cases ( 13% ) . the average age at presentation was 48.6 ( 95% confidence interval : 37.8 - 59.5 ) years . when cervical and thoracolumbar lesions were considered separately , cervical lesions tended to present at a younger age ( fig . three of 15 patients ( 20% ) exhibited multiple aneurysms ; 2 of the 14 reported cases harbored other spinal artery aneurysms,14 ) while our patient had an additional mca artery aneurysm . this rate is similar to the rate of multiple aneurysms observed in patients with cerebrovascular aneurysms , where 20 - 30% of patients with at least one cerebrovascular aneurysm will have multiple aneurysms.19 ) our analysis of the limited data available is consistent with the assumption that psa aneurysms and cerebral aneurysms share similar epidemiology , natural history and re - hemorrhage risk . eighty - three percent of cervical - level psa aneurysms present with signs and symptoms of intracranial sah , including severe headache , nuchal rigidity , and nausea and vomiting in the absence of myelopathy . for infra - cervical psa aneurysms , the presentation is more suggestive of spinal sah ( myelopathy , localized back pain , and radicular pain ) . cervical lesions are more likely to present suddenly than infra - cervical lesions ( 66% vs. 33% , respectively ) . in summary , ruptured cervical psa aneurysms are more likely to present in younger patients with symptoms of intracranial sah ( including blood in the basal cisterns ) without any recent history of progressive symptom . in contrast , ruptured infra - cervical psa aneurysms presented in older patients with symptoms of spinal sah and a one - week history of lower back or abdominal pain . once identified , psa aneurysms are amenable to resection , endovascular intervention , or watchful waiting . for ruptured psa aneurysms , our literature analysis suggests that more aggressive management should be considered for cervical psa aneurysms , as the only reported deaths have followed re - hemorrhage in these lesions prior to intervention . indeed , the mortality rate of cervical lesions is higher than that of thoracolumbar lesions ( 33% vs. 0% mortality ; fig . while newer microcatheters have made it possible to embolize these lesions , surgeons have historically chosen resection because the operation is curative and the superficial nature of the psa aneurysm facilitates an open surgical approach . in addition , surgical decompression with a laminectomy can relieve compression caused by a hematoma . however , outcome is independent of approach ( endovascular vs. open ) . of patients who underwent intervention , those presenting with pain or meningitic symptoms were significantly more likely to have a positive outcome than those who presented with symptoms of cord compression ( 100% vs. 40% improvement ; fig . thus , outcome now appears more dependent on symptoms at presentation , leaving the physican to choose intervention based on case - specific factors and personal preference . for the present case , endovascular intervention was not pursued because occlusion of the proximal segmental vessel of the t9 aneurysm would not provide definitive cure , and there was concern that direct liquid embolic injection could result in additional spinal cord infarction . since the patient required an evacuation of his hematoma to decompress his spinal cord , we felt that the aneurysm could be resected surgically at the same time and would provide a tissue sample to help establish a diagnosis for his suspected systemic vasculitis . however , the patient 's postoperative course was complicated by medical factors that resulted in the death of the patient after a protracted hospital course . in general , isolated psa aneurysms are difficult to diagnose but are amenable to surgical intervention with good results . cervical lesions have a higher mortality than thoracolumbar lesions , providing justification for more aggressive management . patients who present with pain or meningitic symptoms are more likely to recover than those who present with symptoms of cord compression , a trend consistent with the results of the presented case . once diagnosed , both open and endovascular approaches have been used successfully with high cure rates and low morbidity . consequently , the choice of management strategy will ultimately depend on the specific anatomic pathology , the medical status of the patient , the patient 's personal preference and the surgeon .
spinal location of a lymphomatous lesion is commonly seen as a metastasis in patients known to have systemic lymphoma . many authors have described the entity of primary spinal epidural lymphomas ( psel ) , where there is no other evidence of lymphoma in the body other than a solitary lesion in the epidural space . here we have defined the term primary spinal intradural extramedullary lymphoma ( psiel ) and report the second case of psiel in the literature . this 11-year - old boy presented with 3 days history of progressively increasing radicular pain over the sacrogluteal region bilaterally ; left side more than the right side , along with lower limb weakness and sensory impairment in both lower limbs . he also developed urinary retention and was catheterized elsewhere 1 day prior to his presentation . his sensory examination also revealed an asymmetrical sensory impairment in the lower limbs with the upper limit being d12 dermatome . both knee jerk and ankle jerk were absent with plantars not being elicitable . he did not have any neurocutaneous markers , palpable lymph nodes or spinal deformity . with this presentation and neurological examination , an emergency magnetic resonance imaging ( mri ) of the lumbosacral spine with screening was performed , which showed a well - defined sausage shaped intradural lesion at l2 , l3 level , without evidence of any other central nervous system ( cns ) lymphomas . the lesion was iso - intense in t1- and t2-weighted images [ figure 1 ] . pre - operative magnetic resonance imaging of the spine showing a sausage shaped intradural extramedullay lesion at l1 and l2 spinal levels . ( a ) t1-weighted sagittal , ( b ) t2-weighted sagittal , ( c ) t1-weighted axial , ( d ) t2-weighted axial , and ( e ) mr myelogram post - operatively he had total relief of pain and his motor power improved by one medical research council grade and sensations were slowly improving . histoplathological examination of the tumor revealed a dense infiltration of round cells admixed with cells with a large eosinophilic cytoplasm and vesicular nucleus with prominent nucleolus , which was consistent with non - hodgkin 's lymphoma [ figure 2 ] . detailed investigation was carried out to stage the disease , which included complete hematological examination , including bone marrow aspiration and computerized tomography of the chest and abdomen . histopathological examination of the tumor showing a dense infi ltration of round cells admixed with cells with large eosinophilic cytoplasm and vesicular nucleus with prominent nucleolus , consistent with non - hodgkin 's lymphoma on follow - up after 1 month he had received radiotherapy to the local area of 32 gy in 20 fractions and was on chemotherapy ; cyclophosphamide , hydroxydaunomycin , oncovin - vincristine , and prednisone regimen . post - operative mri showed total excision of the lesion [ figure 3 ] . at 1 year follow - up , he was having near normal power , normal sensations , and normal bladder control . ( a ) t1-weighted sagittal and ( b ) t2-weighted sagittal , ( c ) clinical photograph at 1 year the most common occurrence is a metastatic spinal lymphoma in patients known to have systemic lymphoma . in the literature , there are many cases of primary spinal epidural lymphomas ( psel ) reported , where there is no other evidence of lymphoma in the body other than a solitary lesion in the epidural space . the present case is an unique from these cases reported in literature that it has a purely intradural extramedullay origin . tumors with a characteristic histopathological picture of a lymphoma that are seen purely in the spinal intradural extramedullay compartment , with an accompanying negative diagnostic work - up for lymphoma at other sites are termed psiel . they operated on a cervical intradural extramedullary lesion which they thought initially as meningioma . after performing a laminectomy , the biopsy of the lesion was in favor of hodgkins lymphoma ; hence , the surgery was abandoned and the patient subsequently received chemotherapy . on further investigations , mediastinal , and the current patient presented classically like any other tumor in the cauda equina except for the sub - acute onset of symptoms and early bladder involvement . there was no radiological evidence of extradural infiltration or bony involvement . keeping in mind the age of the patient and location , a pre - operative diagnosis of ependymoma other differentials for a tumor in this location include neurofibroma , meningioma , paraganglioma , and rarely hemangioblastoma or an astrocytoma of the filum terminale . there was no evidence of the systemic lymphoma as confirmed by whole body contrast computed tomography evaluation done post - operatively . earlier reports have suggested positron emission tomography to be highly sensitive in picking up lymphoma . the mean suvmax ( mean + standard deviation ) for non - hodgkins lymphoma is 3.2 - 43.0 . intraoperatively the lesion was grey in color , firm in consistency , and relatively avascular . after laminectomy , on opening the dura the tumor was filling the dural sac and causing severe pressure on the cauda equina . there was an attachment to the dura anteriorly , which was coagulated . in the previous reported case by yamashita et al . , there was an attachment to a cervical spinal root and the adjoining dura as well . pathogenesis of this tumor is thought to be similar to that of primary cns lymphomas , where a clone of malignant systemic lymphocytes having specific adhesion molecules might have given rise to the tumor . marcotte et al . have described a case with intradural lymphoid hyperplasia in the cervical region as an immunoreaction to an unidentified antigen . lymphatic cell rests in these lymphatic spaces giving rise to lymphoma might be another probable hypothesis . intra - operative evidence of the dural attachment might be due to its origin from these lymphatic spaces . this being the second case of primary spinal intradural extramedullay lymphoma , there are no guidelines available in literature for optimal management . this entity can be presumed to have a similar pathogenesis and clinical profile like psel . most authors prefer to administer chemotherapy in conjunction to radiotherapy ( rt ) and not alone . chemotherapy has been given before rt , after rt , sandwiched between , or administered concomitantly with rt . various combinations of chemotherapeutic agents have been recommended , cyclophosphamide , vincristine , and prednisone being an integral part of all the combinations . combined modality treatment , including rt and chemotherapy , is the most efficient treatment and has a better outcome in terms of systemic control , local control , and 5-year and long - term survival . chemotherapy in the management of systemic and primary cns lymphomas is an established treatment modality . psiel being a rare entity , a pre - operative diagnosis is almost never thought off . if psiel is suspected pre - operatively , a limited laminectomy with biopsy and frozen section is to be carried out to diagnose lymphoma . if frozen section is in favor of lymphoma , surgery can be abandoned and chemoradiation would be more appropriate . if a patient presents with a cauda equina syndrome or acute bladder involvement , like in the current case , then early surgical resection should be preferred followed by chemoradiation . primary spinal intradural lymphomas form the rare differential diagnosis for spinal intradural extramedullary lesions and for cauda equina syndrome .
enterococci , an important cause of clinical infections , have emerged as an increasingly important cause of nosocomial infections in the last decades , being now the third to fourth - most prevalent nosocomial pathogen worldwide . the emergence and spread of resistance to vancomycin as well as other glycopeptide agents like teicoplanin among enterococcus species greatly reduces the number of treatment options . in addition , spread of vancomycin - resistant enterococci ( vre ) represents an immediate threat to public health [ 2 , 3 ] . since few active compounds are available for vre infections , rapid identification of these isolates are essential for implementation of control measures in order to restrict the emerging trouble of these strains . a variety of typing methods have been used to examine clonal relatedness among human vre isolates but little is known about the epidemiology of vancomycinresistant enterococcus faecalis ( vref ) . comparisons with respect to the epidemiological concordance and the overlap in the data for random amplification of polymorphic dna ( rapd ) versus pulsed field gel electrophoresis ( pfge ) suggested that rapd analysis is well - suited for epidemiological typing of enterococci . in our previous study , high frequency of vref as high as 16% was reported among hospitalized patients at children 's medical center from august 2009 to june 2010 . in the absence of any report on the genetic relatedness of e. faecalis especially vancomycin resistant e. faecalis ( vref ) isolates in iran , we undertook this study to characterize these isolates using rapd - pcr genotyping method . the case definition for inclusion in this study was admission to the children 's medical center hospital ( cmc ) between august 2009 and june 2010 , and a culture positive for e. faecalis at least 48 hours after hospital admission . the kirby - bauer disk diffusion method was used to determine the antimicrobial susceptibility of e. faecalis isolates to vancomycin and teicoplanin according to the clinical and laboratory standards institute . isolates with potential vancomycin and teicoplanin resistance by this method were confirmed by e - test method ( ab biodisk , solna , sweden ) according to the manufacturer 's specification . dna was extracted from vref isolates using qiaamp dna mini kit ( qiagen ) according to the manufacturer 's instruction . the genetic similarity of the strains was investigated by amplification of random amplified polymorphic dna ( rapd ) . rapd - pcr analysis was performed using primer ( 5'- acg cgc cct-3 ' ) . the reaction mixture ( final volume 25 l ) consisted of 19 l h2o , 2.5 l 10x reaction buffer , 0.75 l mgcl2 ( 100 mm ) , 0.6 l dntp mixtures ( 10 mm ) , 0.2 l taq - polymerase ( 5u/ l ) , 1 l primer ( 10 mm ) , 1l of the dna . rapd reactions were performed in a thermocycler by using the following temperature profile : initial denaturation at 94c for 5 min followed by 36c , 5 min ; 72 c , 5 min ; repeat it 4 time then 94c , 1 min ; 36 c , 1 min ; 72c , 2 min for 30 cycles . amplification products were analyzed by electrophoresis in 2% agarose gel with tbe buffer and stained with ethidium bromide 0.1% . the electrophoretic profiles were scored according to the presence ( 1 ) or absence ( 0 ) of a particular band , similarity of all pair - wise combinations of the numerical profiles was determined by dice 's coefficient and clustered by unweighted pair - group analysis using arithmetical averages ( upgma ) using the freetree program ( 0.9.1.50 version ) . dna was extracted from vref isolates using qiaamp dna mini kit ( qiagen ) according to the manufacturer 's instruction . the genetic similarity of the strains was investigated by amplification of random amplified polymorphic dna ( rapd ) . rapd - pcr analysis was performed using primer ( 5'- acg cgc cct-3 ' ) . the reaction mixture ( final volume 25 l ) consisted of 19 l h2o , 2.5 l 10x reaction buffer , 0.75 l mgcl2 ( 100 mm ) , 0.6 l dntp mixtures ( 10 mm ) , 0.2 l taq - polymerase ( 5u/ l ) , 1 l primer ( 10 mm ) , 1l of the dna . rapd reactions were performed in a thermocycler by using the following temperature profile : initial denaturation at 94c for 5 min followed by 36c , 5 min ; 72 c , 5 min ; repeat it 4 time then 94c , 1 min ; 36 c , 1 min ; 72c , 2 min for 30 cycles . amplification products were analyzed by electrophoresis in 2% agarose gel with tbe buffer and stained with ethidium bromide 0.1% . the electrophoretic profiles were scored according to the presence ( 1 ) or absence ( 0 ) of a particular band , similarity of all pair - wise combinations of the numerical profiles was determined by dice 's coefficient and clustered by unweighted pair - group analysis using arithmetical averages ( upgma ) using the freetree program ( 0.9.1.50 version ) . during the study period , 91 children aged < 1 month to 12 years old were colonised or infected with e. faecalis . patients were hospitalized for a mean of 24 days ( range 1 - 105 days ) . twenty - one of the patients were hospitalized in urology ward , whereas the others were distributed in infectious ward ( n = 14 ) , surgical ward ( n = 12 ) , gastroenterology ward ( n = 11 ) , nephrology ward ( n = 11 ) , neonatal intensive care unit ( nicu ) ( n = 7 ) , cardiovascular intensive care unit ( cicu ) ( n = 7 ) , oncology ( n = 4 ) and pediatric intensive care unit ( picu ) ( n = 4 ) . the mics to vancomycin and teicoplanin were 32 g / ml in these isolates . fingerprints of dna fragments by rapd - pcr were recorded . figure 1 demonstrated the similarity relationships between strains in 4 distinct clusters ( a - d ) . cluster a contained the majority of the isolates ( n = 37 , 41% ) , while cluster b contained 17 ( 19% ) , cluster c contained 14 ( 15% ) , and cluster d contained 23 ( 25% ) of the isolates . phylogenetic tree among 91 e. faecalis isolates , constructed by free tree " software " and distance coefficient ( dice ) , showing relationships , by the upgma method . surprisingly , all vref isolates belonged to cluster a. patients that were colonized / infected with vref strains were identified in gastroenterology ward ( n= 4 ) , infectious ward ( n = 3 ) , urology ward ( n = 3 ) , cicu ( n = 3 ) and nicu ( n = 2 ) . in the current study , we have investigated the genotypic characteristics of vref isolates by rapd method . although the epidemiology of e. faecium is well described , little is known about the epidemiology of vancomycinresistant isolates of e. faecalis . data on the prevalence of these strains are scarce in iran , and to the best of our knowledge there is no published information concerning the genetic relatedness of vref isolates . the similarity phylogram built for theses strains , demonstrate the presence of four distinct clusters ( a , b , c , d ) . in these clusters , some isolates were allocated in groups of higher or lower similarity and most strains were discriminated . clusters a contained the majority of the isolates ( n = 37 , 41% ) that were identical and forming one real clone . it is of interest to note that 100% of vref isolates belongs to cluster a , indicating that there may have occurred horizontal transmission of the same strain between these patients . in our hospital , patients may be admitted briefly to a ward that does not match the medical care needed due to lack of bed on the appropriate wards . in addition , they might move to other wards when the level of care required changes . therefore , patient movements may result in the dissemination of bacteria around the hospital , especially if proper infection control procedures are not instituted . predominance of one clone suggests frequent transfer of patients from one ward to another ward . in addition both person - to - person transmission and selective antibiotic pressure can be probable mode of spread [ 10 , 11 ] . patients can remain colonized for prolonged period of time from months to years . in contrast to gram - negative bacteria , vre broadly contaminates the environment and can remain for a long time ; therefore , medical devices are commonly positive on wards with vre patients and patients might acquire nosocomial vre from heavily contaminated environment . in cluster b , 10 of 17 isolates had 100% similarity , forming four real clones . cluster d with 23 isolates had three real clones , which was formed by seven isolates and presented the biggest genetic distance among all . due to the genetic distance observed especially in cluster b and d , it is probable that the non - vref isolates in this study had diverse origins that may have been due to constant cross transmission of enterococal strains . our results are in agreement with those of studies of hospital - acquired vre which indicated clonal dissemination as a major mechanism for the spread of isolates [ 6 , 13 , 14 ] . typing of vref isolates in the three studied hospitals in spain by pfge exhibited indistinguishable or closely related patterns . analysis of our results similar to other studies indicate vana gene as common determinant for glycopeptide resistance in enterococcus spp . identification of the source and route of dissemination of vre is helpful for controlling outbreaks . clonal dissemination of vana gene encoded vre have been reported from other parts of world [ 19 - 22 ] . a study from argentina another molecular typing of vre strains from uk revealed cross transmission of predominance vre pulsotype with 92% containing vana gene . in conclusion , rapid spread of vref from a clonal origin calls for implementation of careful isolation and infection control measures . therefore , environmental control by routine disinfection of patient area as well as screening of high risk patients and isolation of colonized patients should be imposed in order to diminish risk of acquiring nosocomial vre .
we present a case of postpartum female with hemolysis , elevated hepatic enzymes , low platelets ( hellp ) with deranged renal function , and cerebral venous thrombosis , without a history of hypertension and proteinuria . plasma exchange therapy was successfully used in reversal of organ dysfunction and resolution of hemolysis in this patient . a 20-year - old primigravida ( 36 weeks gestation ) woman was admitted in the semi - urban private hospital in north india with generalized tonic clonic seizure ( gtcs ) . magnesium sulfate iv 4 g and 0.5 g / hour infusion . an emergency lower segment cesarean section ( lscs ) was done and she delivered a 2.5 kg healthy male baby . after 6 hours of surgery , she became drowsy , blood pressure ( bp ) dropped to 70/38 mm hg and had decreased urine output ( 20 ml for the last 2 hours ) . she was started on dopamine infusion and transferred to a higher level tertiary care hospital , 36 hours after the lscs . in our emergency department , she got repeat gtcs , was given 4 mg lorazepam and loaded with inj . her bp was 100/74 mm of hg on vasopressors ( dopamine 12 g / kg / min and norepinephrine 15 g / min ) and she had anuria for 6 hours . her abdomen was distended , tense to palpation and there was per vaginal blood - stained discharge . the records of antenatal visits to urban health center were reviewed and there was no evidence of hypertension or proteinuria . her arterial blood gas ( abg ) showed severe metabolic acidosis , peripheral smear showed 3 + fragmented red blood cells ( rbcs ) , reticulocyte count 3.4% , direct and indirect coomb 's tests were negative besides other investigations [ table 1 ] . eclampsia with differential diagnosis of thrombotic thrombocytopenic purpura ( ttp ) versus hellp syndrome was made . laboratory tests results she was given four packed rbcs and platelets in view of active bleeding , 10 mg i.v . her abdominal distention further increased and her hemoglobin ( hb ) was 5.2 g / dl , platelet count ( pc ) was 53 109/l , peripheral smear showed 3 + fragmented rbc and lactate dehydrogenase ( ldh ) was 4580 u / l . in view of persistent hemolysis and worsening platelets and multiorgan dysfunction , therapeutic plasma exchange was started with 100% volume replacement with fresh frozen plasma ( ffp ) . on the next day , she was disoriented and not moving her limbs after stopping sedation . her non - contrast computerized tomography ( ncct ) scan head revealed bilateral parasaggital region and centrum semiovale ischemic infarct . she was continued on plasmapheresis with 80% volume replacement with ffp for five cycles over 5 days . on day 8 , her magnetic resonance imaging ( mri ) brain showed bilateral parasaggital , occipitoparietal , high frontal infarcts and magnetic resonance venogram ( mrv ) revealed cerebral venous thrombosis including inferior saggital , left transverse , sigmoid sinuses and cortical veins thrombosis [ figures 13 ] . in view of altered mental status and anticipated prolonged ventilation , percutaneous tracheostomy was done on day 9 . her renal functions improved and she was started on enoxaparin 60 mg subcutaneous ( s.c . ) brain ( t2 flair ) magnetic resonance venogram ( mrv ) neck and brain patient 's neurological condition gradually improved ; she started responding to verbal commands . she was then switched to warfarin on day 19 to target international normalized ratio ( inr ) 2.02.5 and enoxaparin was stopped . her tracheostomy was decannulated on day 28 and she was discharged on the next day . on follow - up after 28 days , her weakness had further improved and she was ambulated with support and warfarin was continued for 1 year to target inr 2.03.0.the patient and her family were counseled regarding the possibility of recurrence of symptoms during pregnancy . the hellp syndrome is usually associated with hypertension and proteinuria ; however , it can present without preeclampsia in 1020% patients . there are two main diagnostic criteria [ table 2 ] depending on the platelet counts , ldh and aspartate aminotransferase ( ast ) levels . the differential diagnosis of hellp syndrome includes idiopathic thrombocytopenic purpura ( itp ) , acute fatty liver of pregnancy ( aflp ) , and ttp . the management of these life - threatening microangiopathic disorders differs ; therefore , an accurate diagnosis is required . diagnostic criteria for hellp syndrome ttp shares pathophysiological characteristics of the hellp syndrome and differentiation between the two is sometimes difficult . we started dexamethasone as recommended by sibai et al . , but the patient 's condition continued to deteriorate with conservative measures . there are few case reports of use of plasmapheresis or plasma exchange in postpartum hellp syndrome . in our case , there was evidence of persistent hemolysis ( fragmented rbcs , ldh > 600 u / l and anemia ) , thrombocytopenia and deranged ast / alanine aminotransferase ( alt ) . our case thus fulfills the diagnostic criteria of hellp syndrome , but due to the absence of clear history of hypertension and proteinuria and non availability of adamts 13 test , we had difficulty deciding whether it was hellp or ttp . life - threatening neurological complications of the hellp syndrome are rare ; there are few case reports of cerebral infarction after delivery . this is the first case to our knowledge , where we found cerebral venous thrombosis with non - hemorrhagic cerebral venous infarct . in our case , we used low molecular weight heparin , enoxaparin after resolution of hemolysis and thrombocytopenia , and switched her on warfarin to target inr 2.03.0 for 12 months as recommended by american college of chest physician ( accp ) 2008 guidelines . the distinction between preeclampsia - eclampsia , hellp and ttp may not always be possible due to the various overlapping clinical and laboratory findings . the therapeutic plasma exchange therapy should be considered in persistent , life - threatening microangiopathy that is refractory to conservative measures .
the chronic prostatitis syndromes and symptomatic benign prostatic hyperplasia ( bph ) are common among aged men and women . the etiology of prostatitis syndrome remain elusive , it does appear to be associated with a high incidence of voiding dysfunction . lower urinary tract syndromes ( luts ) are sometimes associated with enlarged prostate , commonly referred to as bph . the bph sometimes causes blocks of bladder outflow which cause pain and inflammation of the urinary tract . if untreated they can cause impair urinary frequency , nocturia , incomplete emptying , and urinary hesitancy . in the past , the treatment of luts , associated with clinical bph , was restricted to surgical interventions , such as transurethral resection of the prostate or open nucleation of the enlarged adenoma . in the last decade , however , minimally invasive treatment as well as noninvasive treatment options have been explored and developed , many of them based on the administration of heat to the enlarging adenoma and administration of the drug therapy including alpha - blockers and 5 alpha - reductage inhibitors . selective alpha - adrenergic receptor antagonists ( aaras ) such as prazosin , terazosin , doxazosin , and tamsulosin are important in the treatment of symptomatic benign prostatic hyperplasia ( bph ) [ 4 , 5 ] . among these aaras only tamsulosin is uroselective which selectively blocked alpha-1a receptor ( alpha-1a predominate in the urinary tract and 1b in the vasculature ) . therefore all aaras are suspected for the cardiovascular side effects as they all blocked the alpha-1b . alfuzosin is the most recently approved aaras , with limited cardiac toxicity in the united states for symptomatic treatment of bph . alfuzosin , selective alpha-1a blockers differs from other aaras by the absence of a piperidine moiety and the presence of a diaminopropyl spacer , which confers alfuzosin with specific biochemical properties . alfuzosin is currently marketed throughout europe , asia , and latin america exclusively for the treatment of symptomatic bph . there are 2 bioequivalent formulations available : an immediate release form ( 2.5 mg , 3 times daily ) and a sustained - release form ( 5 mg , 2 times daily and 10 mg once daily ) . the efficacy of both formulations has been demonstrated in well - designed placebo - controlled studies [ 9 , 10 ] . the onset of action of alfuzosin is rapid from the first dose and it maintains symptom relief for up to 3 years . alfuzosin is highly water soluble and after fasted conditions its oral bioavailability was increased by 40% in consumption of 25% food more than the normal food intake and cmax by 50% . a once - daily formulation , which delivers alfuzosin via a novel prolonged - release system , has been developed to improve the convenience of dosing and to provide optimal pharmacokinetic coverage over 24 h [ 14 , 15 ] . since the drug is highly water soluble ( bcs class 1 drug ) , controlling its release from the dosage forms is the major challenge to fabricate controlled release formulation . in order to control its release from the dosage forms , present marketed reference listed drug ( rld ) exploited many pharmaceutical rate controlling polymers , namely , hydroxy propyl ethyl cellulose , microcrystalline cellulose , ethylcellulose , and hydroxy propyl methyl cellulose to control release of highly soluble alfuzosin . therefore the aim of the present formulation development was to prepare control release alfuzosin 10 mg tablets by hot - melt extrusion process with the use of mono- and diglycerol as a rate controlling membrane . the other aim of the study was to match the in vitro characteristics and in vivo performance of the formulation using bioequivalence study in the healthy volunteers to establish bioequivalence with respect to rld uroxatral . alfuzosin hydrochloride was obtained from the dr . reddys laboratory , hyderabad , india . marketed product was obtained from the local pharmacy shop . mono- and diglycerides nf ( lmwitor 900 ) were donated from hetero lab , hyderabad , india . the purified talc usp ( luzenac corp . ) and magnesium stearate nf were purchased from signet chemical corporation pvt . ltd , mumbai , india and colloidal silica ( aerosol 200 ) was purchased from evonik degussa india pvt . k3edta containers , disposable syringe , and ria vial were supplied by bd , india . excipients compatibility screeningthe excipients were selected based on the results of compatibility study which were further confirmed by comparing the excipients used by reference listed drug . the compatibility study was conducted at 40c/75% rh for 4 weeks . the closed glass vial containing physical mixture of alfuzosin hydrochloride and excipients in a particular ratio were subjected at 40c/75% rh for 4 weeks to determine the change of related substances as compared to the original api ( active pharmaceutical agents ) . if total impurity of api was increased to more than 1% , then it was concluded to have interaction of those excipients with the drug . the excipients were selected based on the results of compatibility study which were further confirmed by comparing the excipients used by reference listed drug . the closed glass vial containing physical mixture of alfuzosin hydrochloride and excipients in a particular ratio were subjected at 40c/75% rh for 4 weeks to determine the change of related substances as compared to the original api ( active pharmaceutical agents ) . if total impurity of api was increased to more than 1% , then it was concluded to have interaction of those excipients with the drug . selection of excipients and their gradesthe grade of mono- and diglycerides used was of nf grade ( imwitor 900 , sasol ) . the quantity of mono- and diglycerides was used in concentrations up to 45% as a rate controlling agent . trials were carried out using the excipients in concentrations of 10.0% , 20.0% , 30.0% , 40.0% , 50.0% , and 60.0% in hot melt granulation process to get the desired release profile with some quantity being added intragranularly and the other quantity added extragranularly . the grade of colloidal silicon dioxide used was of pharmaceutical grade ( aerosil 200 ) with concentrations up to 3.0% as a lubricant . the grade of talc and magnesium stearate used was of pharmaceutical grade and their quantities were chosen based on requirements . the grade of mono- and diglycerides used was of nf grade ( imwitor 900 , sasol ) . the quantity of mono- and diglycerides was used in concentrations up to 45% as a rate controlling agent . trials were carried out using the excipients in concentrations of 10.0% , 20.0% , 30.0% , 40.0% , 50.0% , and 60.0% in hot melt granulation process to get the desired release profile with some quantity being added intragranularly and the other quantity added extragranularly . the grade of colloidal silicon dioxide used was of pharmaceutical grade ( aerosil 200 ) with concentrations up to 3.0% as a lubricant . the grade of talc and magnesium stearate used was of pharmaceutical grade and their quantities were chosen based on requirements . formulation developmentsin order to establish the robustness of the proposed formulation , and to optimize the rate controlling polymer concentration , the following ranges around the target formulation were investigated in the experiments . the level of various rate controlling membranes at different proportion is given in table 1 . in order to establish the robustness of the proposed formulation , and to optimize the rate controlling polymer concentration , the level of various rate controlling membranes at different proportion is given in table 1 . 20 mesh and blended together in jacketed rapid mixer granulator ( rmg ) for 10.0 min with intermittent raking of the mixture . the hot - melt granulation of the blend was carried out by passing the steam or hot water through jacket of the rmg until the temperature of the blend reached to 70c and the entire dry blend converted into molten mass [ 18 , 19 ] . the molten mass was cooled at room temperature by passing cold water through jacket and the dried granules were sifted with colloidal silicon dioxide through 1.2 mm screen . blended granules were lubricated by no . 40 meshes passed talc for 15.0 min and then lubricated with magnesium separate no . the lubricated blend was taken for compression by 10/32 beveled edge sc punches , plain on both sides to the target weight of 225 mg . the details of manufacturing process and in process quality control used for the product development are given in figure s1 ( see supplementary material available online at doi:10.5402/2012/813836 ) . six different concentrations of rate controlling agents were formulated in six different tablets formulations of same hardness and dissolution of all formulations were carried out using usp dissolution apparatus type ii under nonsink conditions in 900 ml hcl ( 0.1 n ) . similarly , different hardness of 6 formulations were prepared and the dissolution of the drugs were tested using usp type ii dissolution apparatus in 900 ml hcl ( 0.1 n ) . 20 tablets were grinded using a mortar and pestle and the mixture equivalent to 10 mg drug was weighted . mixture was then transferred to 100 ml volumetric flask and dissolved with small quantity of water followed by 10 min settling . 10 ml of the solution was diluted further to 100 ml in a 100 ml volumetric flask . scale up of alfuzosin hydrochloride mr tabletsscale up in the jacketed rmg and 27 station compression machines were successfully accomplished for the drug product placebo . additionally based upon the design of experiments on laboratory scale batches , acceptable ranges for critical process parameters for melting , mixing , and compression were determined . this process knowledge was used to successfully scale up from the laboratory scale to pilot scale in the production of the pivotal batch [ 20 , 21 ] . the blend for the same was carried out in 25 liter jacketed rmg involving granulation , cooling , sifting and milling followed by blending and lubrication in 1 lot . the lubricated blend was compressed to 13300 tablets . scale up in the jacketed rmg and 27 station compression machines were successfully accomplished for the drug product placebo . additionally based upon the design of experiments on laboratory scale batches , acceptable ranges for critical process parameters for melting , mixing , and compression were determined . this process knowledge was used to successfully scale up from the laboratory scale to pilot scale in the production of the pivotal batch [ 20 , 21 ] . the blend for the same was carried out in 25 liter jacketed rmg involving granulation , cooling , sifting and milling followed by blending and lubrication in 1 lot . the molten masses was then chopped with fast chopper speed and slow mixing for 2.0 min . milling of the resultant granules was done through oscillating granulator with a screen size of 0.8 mm nearly 9 kg blends . finally extragranular ingredients were blended through a conta blender ( 30/60/120 l capacity ) for 20 min . with a speed of the motor the blends were taken out at 10 , 15 , and 20 min for analysis of blend to establish blend uniformity . all the above parameters were kept constant of the pilot and pivotal batches of the formulations except screen size was increased to 1.2 mm instead of 0.8 mm ( table s1 in supplementary material ) . in process testing procedure1 g of blend in a clean dry petridis was taken and observed visually against black background . uv absorption spectrum of sample preparation exhibited maxima and minima , which were at the same wavelengths as that of standard preparation obtained in the assay . standard preparation and sample preparation were stable at room temperature up to 24 h. blend analysis was conducted by sampling the mixture in the intermediate bulk containers ( ibcs ) that is , in stainless steel bunkers ( containers ) . 1 g of blend in a clean dry petridis was taken and observed visually against black background . uv absorption spectrum of sample preparation exhibited maxima and minima , which were at the same wavelengths as that of standard preparation obtained in the assay . standard preparation and sample preparation were stable at room temperature up to 24 h. blend analysis was conducted by sampling the mixture in the intermediate bulk containers ( ibcs ) that is , in stainless steel bunkers ( containers ) . sample preparationthe alfuzosin hydrochloride was weighed accurately in a 100 ml volumetric flask containing 10 ml alcohol and was sonicated for 30 min with occasional shaking . the volume was made up to the mark with methanol diluents and diluted to a known concentration of about 0.005 mg per ml of alfuzosin hydrochloride . the alfuzosin hydrochloride was weighed accurately in a 100 ml volumetric flask containing 10 ml alcohol and was sonicated for 30 min with occasional shaking . the volume was made up to the mark with methanol diluents and diluted to a known concentration of about 0.005 mg per ml of alfuzosin hydrochloride . procedurethe absorbance of standard preparation and sample preparation was measured at 244 nm against diluents as blank ( methanol ) [ 25 , 26 ] . the method was validated if the absolute difference of the standard reading at the initial and end of the run was not more than 2.0% . the quantity of alfuzosin hydrochloride ( in % ) for each location was calculated using the following formula 1 as ( ati / as)(ws/100)(2/100)(dti / w3i)(p/100)(ave . wt./10 ) 100% , where ati = absorbance of sample preparation ( i = 1 to 10 ) ; as = absorbance of standard preparation ; ws = weight of working standard taken in mg ; w3i = weight of sample taken in mg ( i = 1 to 10 ) ; dti = dilution of sample preparation ( i = 1 to 10 ) ; p = percentage purity of working standard . the absorbance of standard preparation and sample preparation was measured at 244 nm against diluents as blank ( methanol ) [ 25 , 26 ] . the method was validated if the absolute difference of the standard reading at the initial and end of the run was not more than 2.0% . the quantity of alfuzosin hydrochloride ( in % ) for each location was calculated using the following formula 1 as ( ati / as)(ws/100)(2/100)(dti / w3i)(p/100)(ave . wt./10 ) 100% , where ati = absorbance of sample preparation ( i = 1 to 10 ) ; as = absorbance of standard preparation ; ws = weight of working standard taken in mg ; w3i = weight of sample taken in mg ( i = 1 to 10 ) ; dti = dilution of sample preparation ( i = 1 to 10 ) ; p = percentage purity of working standard . the results of 10 locations were taken for calculation of mean and rsd . standard preparation25 mg of alfuzosin hydrochloride working standard was transferred to a 100 ml volumetric flask and dissolved in methanol by sonication . then 2.0 ml of this solution was diluted to 100.0 ml with diluents ( methanol ) for analysis by spectrophotometer with a max of 244 nm . the blend equivalent to 30 mg of alfuzosin hydrochloride was dissolved in 500 ml volumetric flask using methanol by sonication with occasional shaking for about 30 min , filtered through 0.45 m millipore pvdf filters and 4.0 ml of this filtrate was diluted to 50.0 ml with methanol for analysis . the quantity of alfuzosin hydrochloride ( in % ) per blend for both sample preparations was measured using the formula 2 as ( ati / as)(ws/100)(2/100)(500/wti)(50/4)(p/100)(ave . wt./10 ) 100 , where , ati = absorbance of sample preparation ( i = 1 and 2 ) ; as = absorbance of standard preparation ; ws = weight of working standard taken in mg ; wti = weight of sample taken in mg ( i = 1 and 2 ) ; p = percentage purity of working standard . 25 mg of alfuzosin hydrochloride working standard was transferred to a 100 ml volumetric flask and dissolved in methanol by sonication . then 2.0 ml of this solution was diluted to 100.0 ml with diluents ( methanol ) for analysis by spectrophotometer with a max of 244 nm . the blend equivalent to 30 mg of alfuzosin hydrochloride was dissolved in 500 ml volumetric flask using methanol by sonication with occasional shaking for about 30 min , filtered through 0.45 m millipore pvdf filters and 4.0 ml of this filtrate was diluted to 50.0 ml with methanol for analysis . the quantity of alfuzosin hydrochloride ( in % ) per blend for both sample preparations was measured using the formula 2 as ( ati / as)(ws/100)(2/100)(500/wti)(50/4)(p/100)(ave . wt./10 ) 100 , where , ati = absorbance of sample preparation ( i = 1 and 2 ) ; as = absorbance of standard preparation ; ws = weight of working standard taken in mg ; wti = weight of sample taken in mg ( i = 1 and 2 ) ; p = percentage purity of working standard . pivotal batch of alfuzosin hydrochloride tabletsthe pivotal batch of alfuzosin hydrochloride er tablets 10 mg was planned to make 130,000 tablets . the hot - melt granulation was carried out in 25 l jacketed rapid mixer granulator involving granulation in 3 lots , cooling in 3 lots , sifting , milling followed by blending and lubrication in 1 lot [ 27 , 28 ] . the lubricated blend , after release was compressed to 130,000 tablets . the target hardness , thickness , friability , diameter , and machine speed were kept 4.5 kb , 4.5 0.5 mm , nmt 1.0%w / w , 7.9 0.2 mm , and 20 10 rpm , respectively . the pivotal batch of alfuzosin hydrochloride er tablets 10 mg was planned to make 130,000 tablets . the hot - melt granulation was carried out in 25 l jacketed rapid mixer granulator involving granulation in 3 lots , cooling in 3 lots , sifting , milling followed by blending and lubrication in 1 lot [ 27 , 28 ] . the lubricated blend , after release was compressed to 130,000 tablets . the target hardness , thickness , friability , diameter , and machine speed were kept 4.5 kb , 4.5 0.5 mm , nmt 1.0%w / w , 7.9 0.2 mm , and 20 10 rpm , respectively . preparation of standard drug samples20 mg of alfuzosin hydrochloride working standard was dissolved in 75 ml of mobile phase ( mixture of buffer ( ph 3.5 ) : acetonitrile at a ratio of 4 : 1 ) using sonication . 10.0 ml of this solution was mixed with 50.0 ml mobile phase ( mixture of buffer ( ph 3.5 ) : acetonitrile at a ratio of 4 : 1 ) . 20 mg of alfuzosin hydrochloride working standard was dissolved in 75 ml of mobile phase ( mixture of buffer ( ph 3.5 ) : acetonitrile at a ratio of 4 : 1 ) using sonication . 10.0 ml of this solution was mixed with 50.0 ml mobile phase ( mixture of buffer ( ph 3.5 ) : acetonitrile at a ratio of 4 : 1 ) . the mixture equivalent to 20 mg of the drug was transferred into 100 ml volumetric flask containing 75 ml of mobile phase and sonicated to dissolve . then 10.0 ml of this solution was diluted to 50.0 ml with diluents and mixed . the buffer solution was prepared after mixing 5 ml of 70% perchloric acid with 1000 ml deionized water and finally ph was adjusted to 3.5 0.05 with 10(n ) sodium hydroxide . the mixture equivalent to 20 mg of the drug was transferred into 100 ml volumetric flask containing 75 ml of mobile phase and sonicated to dissolve . then 10.0 ml of this solution was diluted to 50.0 ml with diluents and mixed . the buffer solution was prepared after mixing 5 ml of 70% perchloric acid with 1000 ml deionized water and finally ph was adjusted to 3.5 0.05 with 10(n ) sodium hydroxide . preparation of content uniformity sampleseach tablet was transferred to 100 ml volumetric flask and dissolved in mobile phase after 20 min sonication . then 20 ml of the above solution each tablet was transferred to 100 ml volumetric flask and dissolved in mobile phase after 20 min sonication . then 20 ml of the above solution procedureseparately 20 l mobile phase , standard preparation , and sample preparation were injected into waters hplc system equipped with kromasil cl8 ( ll ) ( 150 mm 4.6 mm , 5 micron ) column at 25c and run for 20 minutes at a flow rate of 1.5 ml / min and max of 254 nm using uv detector to collect response . the percentage assay ( assay% ) on anhydrous basis for both the sample preparations was calculated using the following formula 3 as ( ati / as)(ws/100)(10/50)(100/wti)(p/100)(100/[100 % water ] ) 100 , where ati = peak area of sample injection ( i = 1 and 2 ) ; as = peak area of standard injection ; ws = weight of working standard taken in mg ; wti = weight of sample taken in mg ( i = 1 and 2 ) ; p = percentage purity of working standard . separately 20 l mobile phase , standard preparation , and sample preparation were injected into waters hplc system equipped with kromasil cl8 ( ll ) ( 150 mm 4.6 mm , 5 micron ) column at 25c and run for 20 minutes at a flow rate of 1.5 ml / min and max of 254 nm using uv detector to collect response . the percentage assay ( assay% ) on anhydrous basis for both the sample preparations was calculated using the following formula 3 as ( ati / as)(ws/100)(10/50)(100/wti)(p/100)(100/[100 % water ] ) 100 , where ati = peak area of sample injection ( i = 1 and 2 ) ; as = peak area of standard injection ; ws = weight of working standard taken in mg ; wti = weight of sample taken in mg ( i = 1 and 2 ) ; p = percentage purity of working standard . resolution solution20 mg of alfuzosin hydrochloride working standard and 20 mg of impurity a standard was mixed with 75 ml diluents and made to 100 ml after sonication . 1.0 ml of this solution was diluted to 100.0 ml and injected to hplc . impurity solutions were prepared by dissolving 15 mg of each impurity in 100 ml diluents and 10 ml of it was diluted to 50 ml for analysis . composite impurity solution was prepared by mixing 1 ml of each impurity solution to 100 ml standard solution containing 20 mg alfuzosin hydrochloride ( afh ) . system suitability was evaluated using sensitivity solution as follows : signal to noise ratio ( snr ) be more than 10 ; resolution between afh and impurity a be more than 3 ; column efficiency for analyte peak be more than 2000 theoretical plates ; the tailing factor for analyte peak be less than 2 and relative standard deviation for 5 replicate standards be less than 1.0% . the area of the peak for each impurity was calculated and finally estimated the % of known impurity present in the formulation by the formula 4 . ( at / as)(ws/100)(10/50)(100/wt)(p/100 ) 100 rrf , where at = peak area of corresponding unknown impurity in sample injection ; as = peak area of alfuzosin hydrochloride in standard injection ; ws = weight of working standard taken in mg ; wt = weight of sample taken in mg ; p = percentage purity of working standard and % of total impurities = sum of % all impurities ( known + unknown ) . 20 mg of alfuzosin hydrochloride working standard and 20 mg of impurity a standard was mixed with 75 ml diluents and made to 100 ml after sonication . 1.0 ml of this solution was diluted to 100.0 ml and injected to hplc . impurity solutions were prepared by dissolving 15 mg of each impurity in 100 ml diluents and 10 ml of it was diluted to 50 ml for analysis . composite impurity solution was prepared by mixing 1 ml of each impurity solution to 100 ml standard solution containing 20 mg alfuzosin hydrochloride ( afh ) . system suitability was evaluated using sensitivity solution as follows : signal to noise ratio ( snr ) be more than 10 ; resolution between afh and impurity a be more than 3 ; column efficiency for analyte peak be more than 2000 theoretical plates ; the tailing factor for analyte peak be less than 2 and relative standard deviation for 5 replicate standards be less than 1.0% . the area of the peak for each impurity was calculated and finally estimated the % of known impurity present in the formulation by the formula 4 . ( at / as)(ws/100)(10/50)(100/wt)(p/100 ) 100 rrf , where at = peak area of corresponding unknown impurity in sample injection ; as = peak area of alfuzosin hydrochloride in standard injection ; ws = weight of working standard taken in mg ; wt = weight of sample taken in mg ; p = percentage purity of working standard and % of total impurities = sum of % all impurities ( known + unknown ) . determination of residual solvents in dosage formsdimethyl sulfoxide as a diluents was used to determine residual solvents like methanol , ethanol , diethyl ether , acetone , isopropyl alcohol , dichloromethane , ethyl acetate , tetrahydrofuran in the finished dosage forms using method described elsewhere . stock solution a was prepared by mixing 30 mg methanol , 50 mg ethanol , 50 mg diethyl ether , 50 mg acetone , 50 mg isopropyl alcohol , and 50 mg ethyl acetate standard into a 100 ml volumetric flask containing 25 ml of diluents . stock solution b was prepared by mixing 60 mg dichloromethane and 72 mg tetrahydrofuran standard into a 100 ml volumetric flask containing about 25 ml of diluents . stock solutions a and b were mixed in a ratio of 5 : 1 ( v / v ) in diluents for combined standard . in brief 50 mg of sample was taken in a 20 ml head space vial with 5.0 ml of diluents . supelco capillary column ( 30 m 0.53 mm , 3 m ) was used at nitrogen carrier gas flow rate of 2.0 psi . the initial column temperature was kept for 35c and increased 25c / minute thereafter to reach the final column temperature of 225c while the injection port temperature and detector temperature were kept 230c and 250c , respectively . system suitability was considered to have relative standard deviation for six replicate standard injections not more than 15.0% against each solvent . the residual solvent content in the dosage forms ( in ppm ) was calculated using the equation 5 as ( at / as)(ws/100)(10/100)(5/wt)(p/100 ) 10 , where at = peak area of corresponding solvent in sample injection ; as = peak area of corresponding solvent in standard injection ; ws = weight of corresponding solvent standard taken in mg ; wt = weight of sample taken in mg ; p = percentage purity of corresponding solvent standard . dimethyl sulfoxide as a diluents was used to determine residual solvents like methanol , ethanol , diethyl ether , acetone , isopropyl alcohol , dichloromethane , ethyl acetate , tetrahydrofuran in the finished dosage forms using method described elsewhere . stock solution a was prepared by mixing 30 mg methanol , 50 mg ethanol , 50 mg diethyl ether , 50 mg acetone , 50 mg isopropyl alcohol , and 50 mg ethyl acetate standard into a 100 ml volumetric flask containing 25 ml of diluents . stock solution b was prepared by mixing 60 mg dichloromethane and 72 mg tetrahydrofuran standard into a 100 ml volumetric flask containing about 25 ml of diluents . stock solutions a and b were mixed in a ratio of 5 : 1 ( v / v ) in diluents for combined standard . in brief , the working procedure is as follows . 50 mg of sample was taken in a 20 ml head space vial with 5.0 ml of diluents . supelco capillary column ( 30 m 0.53 mm , 3 m ) was used at nitrogen carrier gas flow rate of 2.0 psi . the initial column temperature was kept for 35c and increased 25c / minute thereafter to reach the final column temperature of 225c while the injection port temperature and detector temperature were kept 230c and 250c , respectively . system suitability was considered to have relative standard deviation for six replicate standard injections not more than 15.0% against each solvent . the residual solvent content in the dosage forms ( in ppm ) was calculated using the equation 5 as ( at / as)(ws/100)(10/100)(5/wt)(p/100 ) 10 , where at = peak area of corresponding solvent in sample injection ; as = peak area of corresponding solvent in standard injection ; ws = weight of corresponding solvent standard taken in mg ; wt = weight of sample taken in mg ; p = percentage purity of corresponding solvent standard . in order to establish release patterns of the drug from the tablets formulation , a dissolution study was conducted for a period of 22 h using usp dissolution apparatus ii ( paddle ) under nonsink condition [ 30 , 31 ] equipped with autosamplers . the dissolution media was 500 ml of 0.1 n hcl , for comparing the release rate among the trial lab scaled batches and to establish the reproducibility of the release rate from the formulation . the formulation which showed reproducible dissolution behavior was considered as final formulation and taken for the further scale up study . in addition 900 ml of dissolution media each of 0.1 n hcl ( ph 1.2 ) , acetate buffer ( ph 4.5 ) , and phosphate buffer ( ph 6.8 ) were used to compare final scale up formulation with innovator formulation . during dissolution , the dissolution media were maintained at 37 0.5c and paddle speed was 100 rpm at ph 1.2 and 4.5 whereas 50 rpm at ph 6.8 . samples through a 40 m filter were injected automatically at each sampling time point . 22 mg of alfuzosin hydrochloride working standard was dissolved in 5 ml methanol and made the volume up to 200 ml with dissolution medium . the quantity of alfuzosin hydrochloride ( in % ) released was quantified by using formula x as ( at / as)(ws/200)(900/10)(10/5)(p/100 ) 100 at 1 h. net / cumulative drug dissolution was calculated at each time point after adding drug present in the volume of sample taken out as correction factor . forty six healthy male indian volunteers aged between 18 and 45 years with bmi ( body mass index ) within 19 to 29.4 kg / m enrolled for the study after health clearance by general physical examination and clinical lab evaluation ( including ecg and chest x - ray ) . the normal value ranges of the following laboratory tests : albumin , alkaline phosphatase , ast , alt , blood glucose , creatinine , -gt , total bilirubin , total protein , triglyceride , total cholesterol , haemoglobin , hematocrit , total and differential white cell counts , routine urinalysis , and negative serology for hiv , hbv , and hcv were determined before enrolling them in the present study . all the subjects gave written informed consent and madras ethics committee , chennai , tamil nadu , india approved the clinical study protocol . the study was conducted in accordance with ich gcp , indian gcp , and icmr guideline with the provisions of the declaration of helsinki ( seoul 2008 ) . the study was an open labeled , randomized , two sequence , two periods , single - dose , two - way crossover design with 14 days washout period between the doses [ 13 , 33 ] . during each period , the volunteers were housed in a clinical pharmacology unit of huclin research limited , chennai , tamil nadu , india on the eve of the dosing day . following over night fasting of about 10 h a single dose of alfuzosin ( 10 mg cr tablets of either test or reference formulation ) was administered orally to each of the volunteers as per randomization code list at sitting posture with the aid of 240 ml of water in a staggered manner in order to easy sample collection . a mouth check following drug administration with the help of a tongue depressor was performed for each of the volunteers to ensure the compliance of the dosing activities . all the subjects were restricted to 2 h after drug administration of water intake and toileting , while at other time water was given ad libitum . a standard meal was provided to all the volunteers at 4 , 8 , and 12 h after drug administration . vitals were recorded at 2 , 4 , 6 , 8 , 12 , and 24 h after drug administration for each volunteer to assess health related complication if any . serial blood samples were collected from each of the volunteers at every period at predose and at 1.00 , 2.00 , 3.00 , 4.00 , 5.00 , 5.500 , 6.00 , 8.00 , 10.00 , 12.00 , 16.00 , 24.00 , 36.00 , 48.00 , and 72.00 h post drug administration . collected blood samples were centrifuged at 3500 rpm for 10 minutes at temperature 10c to collect plasma . a validated lc - ms / ms method was used to estimate the concentration at each time point for all the subjects . the subjects ' data were subjected to noncompartmental analysis to determine the pharmacokinetic properties using winnonlin v5.3 . log transformed pharmacokinetic data were subjected to multivariate anova analysis to construct point estimate ( geometric mean ratio between test and reference ) and 90% ci using two one - sided test to establish bioequivalence . as per bioequivalence guideline of generic product , 90% ci should have a predefined range of 80 to 125% with respect to reference in order to establish bioequivalence with a nominal power of at least 80% . a method for determining alfuzosin in human plasma was validated using an api 3000 lc / msims system with detection in the range of 0.05 to 30.00 ng / ml and aquacil c18 column ( 100 2.1 mm , 5 m ) was attached for separation [ 34 , 35 ] . the interface used with the api 3000 each 500 l aliquot of standard and qc samples were mixed with 25.0 l of deionized water . 0.2 ml of internal standard ( is ) working solution ( 20.0 g / ml ) was added to the entire sample collected from volunteers except blank samples . the sample was applied to prewashed spe cartridges ( waters oasis , 1 ml ) with 1.0 ml of methanol followed by 1.0 ml of deionized water and centrifuged for l min at 3000 rpm at each step . each cartridge was washed with 1.0 ml of deionized water by centrifugation for 2 min at 3000 rpm . the sample was eluted from oasis cartridge by adding 1.0 ml of methanol and centrifugation for 2 min at 3000 rpm . the residue was reconstituted in 300 l of reconstitution solution and 5.0 l was injected onto a lc - ms / ms system . the pharmacokinetic parameters were determined from the plasma concentration and time data using a noncompartmental analysis by winnonlin professional software ( version : 5.3 ; pharsight corporation , usa ) . the log transformed pharmacokinetic data were subjected to anova and two one side t - test analysis to estimate geometric mean ratio , 90% ci and power by the use of sas v 9.3 . as per bioequivalence guideline , [ 33 , 36 ] 90% ci will be constructed , which will be predefined values of 80 to 125% of the reference drug in log scale to establish generic equivalence . iviv link model was used to build correlation ; in vitro fraction of drug released at each time points was subjected to weibul model fit options in ivivc tool kit of winnonlin v 5.3 . the individual subject in vivo data for both test and reference were fitted to wagner nelson model to calculate the relative fraction of the drug absorbed in vivo . unit impulse response of in vivo data followed by deconvolution of the data was used to build correlation with in vitro dissolution data . the correlation coefficient , prediction error of cmax and auct were calculated across all the dissolution media to conclude the discriminative dissolution media for the drug . drug excipients compatibility study was carried out to find out status of impurities present in the formulation ( table s2 in supplementary material ) . there was no increase in impurities , no change in the physical appearance of the formulation . hence it was concluded that mono- and diglycerides , lactose monohydrate , colloidal silicon dioxide , magnesium stearate , and talc were compatible with alfuzosin hydrochloride in the formulation . the generic alfuzosin 10 mg er tablets were prepared by hot - melt granulation using mono- and diglycerides at various proportions used as a rate controlling membrane ( table s3 in supplementary material ) . the list of other ingredients used in the development of the formulation is given in table 1 . various pilot batches were trialed to optimize drug release , hardness and friability ( table 2 ) , and the optimized formula ( f025 ) was chosen based on in vitro dissolution study which corresponds to the reference dissolution among pilot formulations . the blend uniformity was studied as part of in - process test during the manufacturing of 10 mg alfuzosin hydrochloride extended release tablets . blend analysis was conducted by sampling the mixture in the intermediate bulk containers ( ibcs ) that is , stainless steel bunkers ( containers ) . results showed uniform distribution of the drug among top , middle and lower portion of the blend with a insignificant departure of 0.23% ( tables s1 and s4 in supplementary material ) . the assay of 6 individual tablets passed the usp limit of 90 to 110% of drug ( table s4 in supplementary material ) with a relative standard deviation of 0.023 . the hardness of the pivotal and commercial batch was kept for 4.5 kp ( kilo pascal ) . the observed hardness ( mean sd kp , n = 20 ) of the pivotal batch was 4.52 0.23 and that of commercial batch was 4.53 0.31 ( table s4 in supplementary material ) . the friability of the all batches was found to be less than 1% ( 0.8% for biobatch and 0.84% for commercial production batch ) ( table s4 in supplementary material ) . the drug release from the biobatch was identical for the first few hours and got difference in the last hours of the cumulative drug release with respect to reference formulation . the cumulative percentage drug release in 0.01 n hcl ( ph 1.2 ) ( figure 1(a ) ) , acetate buffer ( ph 4.7 ) ( figure 1(b ) ) and phosphate buffer ( ph 6.8 ) ( figure 1(c ) ) in 900 ml media for both biobatch and reference formulation . the cumulative percent drug release of the biobatch decreased to 95% against 96.99% at 24 h in 0.1 n hcl ( ph 1.2 ) ( tables s5s10 in supplementary material ) . 35 out of 42 subjects completed both the periods of the fasting study and there were 7 discontinued subjects . 2 subjects were prematurely withdrew their consent without any reason , 3 subject discontinued due to severe headache and other two subjects did not report for the second period to the facility . there were no clinically significant changes in vital signs , clinical laboratory variables , ecg , x - ray and general physical examination for fasting study . linear regression , with l / x weighting , was used to obtain the best fit of the data for the calibration curves ( figure not shown ) . the lower limit of quantitation ( lloq ) was 0.05 ng / ml and the upper limit of quantitation ( uloq ) was 30.00 ng / ml . quality control samples ( six sets ) at concentrations of low 0.1500 ng / ml ( lqc ) , medium 2.000 ng / ml ( mqc ) , and high 25.00 ng / ml ( hqc ) prepared in human plasma , were analyzed with each assay validation run to ensure acceptable assay precision and accuracy . also six sets of lloq ( 0.050 ng / ml ) and uloq ( 30.00 ng / ml ) samples included in each batch were run . in addition , the stability of alfuzosin during freeze thaws cycles , extracted samples in the refrigerator and on bench top , in biological matrix , stock solution stability at room temperature and 4c 6c and intermediate stock solution stability for alfuzosin at 4c 6c was studied . the overall interday precision ( % cv ) and accuracy for the standards and quality control samples were 1.4 to 6.0% and 96.1 to 104% , respectively . the interday precision and accuracy for the lloq for alfuzosin were 9.7% and 102% , respectively , and for the uloq were 5.1% and 103% , respectively ( table 3 ) . method did not show any matrix effects since the accuracy of the method was 103% at highest calibration concentration . the highest accuracy was also obtained from the diluted standard which was 96.9% ( table 3 ) , bench top stability and stock solution stability ( table s11 in supplementary material ) and freeze thaw stability ( table s11 in supplementary material ) and room temperature stability ( table s11 in supplementary material ) were also in acceptable limit which signified the validity of the method . the linear plasma concentration ( mean sd ) versus time curves of 2 alfuzosin formulations applied to 35 subjects under fasting conditions are given in figure 2 . the mean cmax ( mean sd ng / ml ) of test and reference formulation were 10.212 2.23 and 11.422 2.23 , respectively , under fasting conditions . the mean auct ( mean sd ng / ml hr ) of test and reference were 196.172 12.01 and 193.172 12.01 while mean auci ( mean sd ng / ml hr ) of test and reference formulations were 211.468 13.51 and 196.468 13.51 , respectively . the mean tmax ( mean sd h ) , kel ( mean sd h ) and thalf ( mean sd h ) of test and reference formulations of alfuzosin were 6.00 0.49 and 6.00 0.69 , 0.28 0.1 and 0.27 0.1 , and 7.89 0.87 and 7.89 0.87 , respectively , under fasting conditions . the least square mean of the primary pk parameters calculated from the anova , the ratio and 95% confidence interval of primary pk parameters cmax , auct ( auclast ) , and auci ( auc total ) assuming equal variance between the groups were 109% ( 99.03% to 122.78% ) , 104% ( 92.94% to 116.71% ) , and 110% ( 98.17% to 124.01% ) , respectively , under fasting conditions ( tables 4 and 5 ) . the mean recovery of impurities a , b , d , and e ( mean rsd ) were 83.19 1.89 , 87.86 3.26 , 102.56 0.86 , and 87.97 4.35 , respectively , ( table s2 in supplementary material ) . assay validationthe assay procedure was validated by analyzing three single standard curve sets per day for a total of three days . the standard curve concentrations range from 0.050 to 30.0 ng / ml . on each day of validation , five sets of qc samples at 0.1500 ng / ml ( low ) , 2.000 ng / ml ( medium ) , and 25.00 ng / ml ( high ) were assayed for a total of twelve sets of qc samples for all six days . an integrator was used to determine the chromatographic peak heights of alfuzosin and internal standard . the peak height ratios of alfuzosin to internal standard were used for the calculation of unknown concentrations . the assay procedure was validated by analyzing three single standard curve sets per day for a total of three days . the standard curve concentrations range from 0.050 to 30.0 ng / ml . on each day of validation , five sets of qc samples at 0.1500 ng / ml ( low ) , 2.000 ng / ml ( medium ) , and 25.00 ng / ml ( high ) were assayed for a total of twelve sets of qc samples for all six days . an integrator was used to determine the chromatographic peak heights of alfuzosin and internal standard . the peak height ratios of alfuzosin to internal standard were used for the calculation of unknown concentrations . the graph of fraction of drug dissolved in vitro versus fraction of drug absorb in vivo along with correlation coefficients at each dissolution media was separately constructed ( figures 2(a)2(c ) ) . the prediction error of the cmax and auc of the correlation were found to be 2.62% and 22.02% at ph 1.2 of dissolution media , 2.32% and 17.68% at ph 6.8 of dissolution media , and 2.38% and 18.74 at ph 4.7 of dissolution media , respectively ( table 6 ) . to develop alfuzosin 10 mg extended release tablets , active pharmaceutical ingredient ( api ) was characterized to meet the fda regulation and showed comparative physicochemical and spectral characteristics with respect to the reference . the structural elucidation of finished test formulation was carried out by using ir , nmr ( both proton and carbon ) , mass , dsc , and xrd . the prominent ir peak for aromatic amine ( n h stretching ) , aromatic ( = c h stretching ) , secondary amide ( c = o and c = h stretching ) between test , and reference were similar , which confirmed the structural similarity of functional groups between test and reference drug . the proton nmr spectra between test and reference showed prominent peak at 11.9 ppm , 8.89 ppm , 7.7 ppm , and 3.34 ppm indicating similar chemical environment which was further confirmed by 13c nmr . the chemical shift of proton and carbon nmr between two drugs were similar and hence the structure of these two molecules was considered to be similar . furthermore , molecular weight of both test and reference was 390 ( tables s12s15 ) from mass spectrum for test and reference . similar single pure peak at 339.33c was obtained from the dsc of test and reference drug . the diffraction pattern between two drugs had similar 2 angle peak which further confirmed the similar structure of the molecule between two dosage forms . finally elemental analysis of test and reference showed no change in c , h , and n contents of the drugs implied the requirement as per compendial limit . these studies showed comparable spectral characteristics which signified sameness of test and reference dosage forms . a compatibility study of the drug with the selected impurities was conducted for the period of 30 days at 50c . the selected inactive ingredients were compatible with the study drugs and hence can be used for the development of the formulation since the content of the impurities was within the quality limit ( < 0.11% ) . the generic alfuzosin 10 mg er tablets were prepared by hot - melt granulation using mono- and diglycerides as a rate controlling membrane . six different formulations ( table s3 in supplementary material ) were prepared at different label of lactose , mono- and diglycerides and evaluated in vitro hardness , friability , and dissolution rate , color , texture , diameter , and width of the tablets for optimization of formulation [ 3840 ] . afterwards ten formulations were processed based on the in vitro parameters as mentioned in the reference drug . a batch formulation of the drug was also generated for further evaluations in the commercial scale . finally a pivotal batch ( table 1 ) of alfuzosin hydrochloride er tablets 10 mg was planned to make 130,000 tablets . the recovery of the impurities and relative retention factor ( table s2 in supplementary material ) were less than 2% which indicated that the tablets met regulatory requirement of stability at the finished product . the residual solvent content ( of methanol , ethanol , diethyl ether , acetone , isopropyl alcohol , dichloromethane , ethyl acetate , tetrahydrofuran ) in the finished product was less than 1% which further satisfied the compendial requirement [ 29 , 41 ] . the physicochemical characteristics of the finished products like hardness , assay , dissolution , and friability all are in the limit of the usp ( united state pharmacopeia standard ) . the in vitro dissolution of the test drug after 20 h was 87% as compare to the innovator product which was 103% showing slow controlled release of the drug from the dosage forms . alfuzosin was released from the matrix tablets by diffusion and erosion mechanisms in all of the formulations . assay of finished tablets were between 80 to 120% and rsd was below 10% which indicated that the product was equivalent to usp reference standard ( table s3 in supplementary material ) . hplc method validation for estimation of alfuzosin in human plasma was adequate , since method did not show any matrix effects and the accuracy of the method was 103% at the highest calibration concentration . the highest accuracy was also obtained from the diluted standard which was 96.9% ( table s11 in supplementary material ) , bench top stability and stock solution stability and freeze thaw stability ( table s11 in supplementary material ) , and room temperature stability ( table s11 in supplementary material ) were also within acceptable regulatory limit which signifies the validity of the method . the generic alfuzosin hcl 10 mg er tablets was prepared using mono- and diglyceride as oppose to the innovator formulation containing hpmc and ethyl cellulose . the difference in the formulation between test and innovator drug is presented in table 7 . so it falls under major changes , and hence to get marketing approval product needs to show bioequivalent to the innovator product in therapeutic outcome . hence a bioequivalence study was conducted as per usfda regulatory guideline for the generic products under fasting conditions in a two way cross over design . cross - over design was chosen to establish bioequivalence , since cross over design reduced variability by using same subject in two different periods where genetic changes were no longer a factor to increase variability of the drug and also reduced the bias of selecting the pharmacogenomic unequal subjects between test and reference drugs . hence the cross over design requires minimum subjects to establish bioequivalence of the generic drug . the bias of selecting the subjects in the equivalence trial was further removed by using randomization schedule ( data not shown ) . a single dose two period , 2 sequence , 2 treatment cross - over study with a minimum wash - out period of 7 days was carried out in fasting conditions for alfuzosin formulation ( fda guidelines , 2005 ) . in the present cross - over study , 35 and subjects were restricted to toilet and heavy physical exercise for first 2 h following administration of the drug in order to maintain similar environment . all the subjects were supplied a standard meal that had 950 to 1000 kcal ( kilo calorie ) at 4.00 , 8.00 , and 12.00 h after drug administration . since food may affect the absorption and dissolution of the drug and which in turn increased the variability of the pk parameters . in order to avoid such circumstances food with uniform calorie content a test drug is considered to be pharmacokinetic equivalent in turn bioequivalence to a reference drug product if the 90% ci of the test and reference geometric mean ratios of the aucs and cmax fall within 0.80 to 1.25 . all subjects were monitored for vital sign , blood pressure and pulse rate to document any adverse events . scheduled blood sample was taken from each subject in order to measure the drug in blood at each time point so that a concentration versus time graph can be constructed to describe rate and extend of absorption of the drug . a hplc method was validated to determine the concentration of the drug at each scheduled time points . the linearity range ( 0.050 to 30.0 ng / ml ) was fixed based upon the results of 6 trial batches . the heterosedasticity of various concentrations was eliminated by using appropriate weighing factor to construct the linear calibration curve . the validity of the method was tested by studying matrix effects , dilution integrity stock solution stability at various temperatures ( table s11 in supplementary material ) , ruggedness , % recovery of the drug as well as internal standard , intra- and interday precession and accuracy ( table 3 ) . the stability of the drug was tested against room temperature , freezing temperature , at bench top , reinjection , short - term and long - term stability of the drug ( table s11 in supplementary material ) . the descriptive statistics of the individual subject concentration at each time points was used to construct a linear mean graph ( figure 3 ) and the significance of the linear mean graph was to compare pk profile between test and reference alfuzosin to conclude bioequivalence without statistical testing . in addition , semilog graph was constructed to show the parametric distribution of alfuzosin content in blood plasma and population therapeutic effects ( table 8) . the individual subject 's pk parameters and descriptive statistics of the untransformed pk parameters ( tables 4 and 5 ) were tabulated for visual comparison between test and reference under fasting conditions before statistical analysis . log transformation was done to bring the nonparametric data to parametric forms so that two one sided test and anova can able to estimate the ratio and 90% confidence interval . further sequence , period , and treatment effects were also calculated by the help of generalized linear model in proc glm in sas v 9.2 . in the present model sequence effects were tested at 10% level of significance whereas other effects were tested at 5% level of significance . in addition , subject nested within sequence as an error term used during modeling , were not shown in the data . significance of primary pk parameters was to draw conclusion whether a generic product can be given marketing authorization within the limit of efficacy safety and tolerability of an innovator drug . in the present study single dose pharmacokinetics study is similar to steady - state pharmacokinetics and healthy subjects ' pharmacokinetics is proportional to the patients ' pharmacokinetics . american pharmaceutical association ( 2003 ) has approved drug therapeutic equivalence information ( 2003 ) . the significance of all primary pk parameters are to access the rate and extend of drug absorption which corresponds to onset of action ( cmax , rate of drug absorption ) and duration of action ( auct and auci ) that is , total amount of drug absorbed . if the variability of these three parameters between test and reference are very narrow that is , about within 20% , there will not be any chance to under action ( sub therapeutic effects ) or over action ( adverse reaction ) . so , statistical equivalence of cmax , auct and auci between test and reference drug would bring a substituted generic which can switch over innovator . the primary pk parameters from this study , at 90% cis were within the predefined bioequivalence criteria of 80 to 125% for the study under fasting conditions ( table 8) . the pk study results revealed that the two formulations of alfuzosin were similar in pk characteristics among these healthy indian subjects under fasting conditions . the 90% confidence intervals of log transformed pk metrics for the ratio of cmax , auct and auci were 99.03% to 122.78% , 92.94% to 116.71% , and 98.17% to 124.01% funder fasting conditions ( table 8) and met alfuzosin bioequivalence criteria as per emea regulation ( fda , guideline of orally administered drug 's bioequivalence study ) . furthermore , the mean thalf ( 7.89 h ) and tmax ( 6.00 h ) obtained from the test drug was comparable with reference drug 's thalf ( 7.89 h ) and tmax ( 6.00 h ) ( tables 4 and 5 ) . the safety and tolerability of both the formulations was comparable . alfuzosin was well tolerated by all the volunteers with no clinically significant adverse events ( aes ) such as increased neutrophil count , vomiting abdominal blotting and headache in the healthy subject . since safety of the test formulation was better than the reference in the study , proposed changes in the formulation are acceptable and inactive ingredients used for the development of the formulations were safe . the inactive ingredients of the proposed generic formulation can be further changed to cheaper ingredients to bring down the production cost of the generic product and hence health care cost which warrants further research works . a in vivo in vitro correlation ( ivivc ) was tried to establish in order to select suitable in vitro dissolution media . to establish reasonable correlation , the nonlinearity of observed values was adjusted with the variation by the link mode to generate level - a correlation . since only linear correlation is accepted for the waiver approval , hence other nonlinear regression was not applied for better correlation . based on the ivivc analysis acetate buffer offered highest level a correlation of 0.97 ( figure 2(c ) ) and hence selected as a compendial media for future waiver of higher strength of same formulation without undergoing costly be study . the rate controlling polymer mono- and diglycerides had no significant impact on the impurity profile of the drug . hence these polymers were considered as safe for the development of the extended release formulation as a rate controlling membrane . since assay results of formulations and impurities were found within the acceptance criteria , the hplc method was precise for quantification of impurities in alfuzosin hydrochloride api . in vivo study concluded that the test product is bioequivalence to the reference product in fasting conditions . the significant correlation between in vitro and in vivo parameters indicated that the ivivc was excellent in predicting auct , but not acceptable in predicting cmax because of high variability observed in maximum plasma concentrations among the healthy volunteers which is probably due to first pass metabolism . it is also observed that the least prediction errors of auct was 2.32 at ph 6.8 , hence phosphate buffer ph 6.8 can be consider as a discriminative media for in vitro dissolution study . in final conclusion , the results suggested that the scale - up of highly permeable and highly soluble drugs did not significantly affect either in vitro dissolution or in vivo performance . it was further concluded based on the in vivo study that the test product was bioequivalence to the reference product in the present study .
ubiquitous computing , following the vision of weiser , aims to embed small computer devices into every day objects augmenting them with new functionality , building an environment full of distributed computers . scenarios for major applications include homecare monitoring [ 4 - 6 ] and assistance for health professionals [ 7 - 9 ] . in the ubiquitous healthcare environment , health data are transferred to a remote healthcare server by a wearable system or mobile computer . collected health data can then be managed and analyzed in the server computer to generate case - specific advice . despite the fact that ubiquitous healthcare computing generates massive amounts of data , healthcare enterprises are knowledge poor because this data is rarely transformed into strategic decision - support resources . an intelligent active knowledge system for health data management is necessary to support clinical decisions by health professionals . dual model architecture is gaining relevance for the development of an electronic health records system for ubiquitous healthcare . this architecture , which takes into account the dynamic nature of the healthcare environment , is based on two modeling levels : information and knowledge . the information level is provided by the reference model and the knowledge level by the archetype model . the first is the separation of concepts into two levels , one defining the reference model and another , formed by formal models of domain concepts , defining different clinical concepts . the second rule is that computing systems are based on the reference model , and valid healthcare records extracts are instances of the reference model . this methodology is currently used by the major standards for representing electronic health records - openehr , cen , and health level seven ( hl7 ) . based on dual model architecture , two major technological approaches for semantic interoperability are the ontology - based and the archetype models . the ontology - based modelof clinical information is designed to make health information systems properly interoperable and safely computable . the openehr archetype model is an open standard specification that describes the management , storage , retrieval , and exchange of health data from electronic health records . we review here these two technological approaches for semantically interoperable electronic health records to construct an intelligent active knowledge system for ubiquitous healthcare data management . over the past decades , ontologies have become key components of information systems and have found various applications includingnatural language processing , software engineering , and knowledge management in the semantic web and healthcare . ontology engineering and management in healthcare have a long tradition , starting with controlled vocabularies with restricted lists of terms such as catalogs , unstructured glossaries , and structured arrangements of words . nowadays weed 's " problem - oriented medical record ( pomr ) " methodology formally linked a model of process of care to the information gathered during that care . einstein 's " hypothetico - deductive " model of clinical reasoning mainly accounted for the cognitive aspects of clinical care during diagnosis . the danish " general electronic patient journal ( g - epj ) " included a conceptual model of the iterative problem - solving process and categories of information , implementing both process and information based on rational problem - solving , but proved too rigid in clinical practice . various clinical modeling efforts from the riche project to the present hl7 version 3 standard have based their models on an " act management " paradigm , in which all aspects of healthcare are represented as " acts " , enabling " everything that is done " to be recorded . currently , the most widely - used ontology editor is protg . protg is an open source ontology development and knowledge acquisition environment developed by stanford medical informatics . as a java tool , it provides an extensible architecture for the creation of customized knowledge - based tools and assists users in the construction of large electronic knowledge bases . protg provides two main ways of modeling ontologies : 1 ) protg - frames editor and 2 ) protg - owl editor . in protg - frames , the knowledge model is compatible with the open knowledge base connectivity protocol ( okbc ) . protg supports the construction of domain ontology , the design of knowledge - acquisition forms , and entering domain knowledge . it provides a platform which can be extended with graphical widgets for tables , diagrams , and animation components to access other knowledge - based system embedded applications . while they offer a stable , language - independent vocabulary that helps standardize and explain the meaning of domain terms , the use of ontology editors such as protg is complex and therefore less suited for application to clinical practice . weed 's " problem - oriented medical record ( pomr ) " methodology formally linked a model of process of care to the information gathered during that care . einstein 's " hypothetico - deductive " model of clinical reasoning mainly accounted for the cognitive aspects of clinical care during diagnosis . the danish " general electronic patient journal ( g - epj ) " included a conceptual model of the iterative problem - solving process and categories of information , implementing both process and information based on rational problem - solving , but proved too rigid in clinical practice . various clinical modeling efforts from the riche project to the present hl7 version 3 standard have based their models on an " act management " paradigm , in which all aspects of healthcare are represented as " acts " , enabling " everything that is done " to be recorded . protg is an open source ontology development and knowledge acquisition environment developed by stanford medical informatics . as a java tool , it provides an extensible architecture for the creation of customized knowledge - based tools and assists users in the construction of large electronic knowledge bases . protg provides two main ways of modeling ontologies : 1 ) protg - frames editor and 2 ) protg - owl editor . in protg - frames , the knowledge model is compatible with the open knowledge base connectivity protocol ( okbc ) . protg supports the construction of domain ontology , the design of knowledge - acquisition forms , and entering domain knowledge . it provides a platform which can be extended with graphical widgets for tables , diagrams , and animation components to access other knowledge - based system embedded applications . while they offer a stable , language - independent vocabulary that helps standardize and explain the meaning of domain terms , the use of ontology editors such as protg is complex and therefore less suited for application to clinical practice . openehr archetype models , commonly referred to as archetypes are clinical data models that conform to the openehr reference model . the openehr foundation , an international , on - line community whose aim is to promote and facilitate progress towards electronic healthcare records of high quality , to support the needs of patients and clinicians anywhere , is the originator . the openehr 's information model is the archetype , a re - usable formal model of a domain concept . information models are templates for the acquisition of clinical data which provide semantic interoperability within the bounds of the given information model but not between different information models . archetypes are usually built by domain experts and are computable expressions of a domain content model of medical records , defining the particular configuration or desired composition of instances of clinical concepts . expression is in the form of structured constraint statements , inherited from the reference model . this model describes the health record itself , and is composed of packages , defining openehr specification documents , and information models . the ehr information model is organized in folders and compositions ( figure 1 ) . the package is the top level structure of the ehr and contains the entry and navigation packages . in general , archetypes are defined for wide use ; however , they can be specialized to include local particularities and in healthcare , an archetype can model concepts . an archetype is divided into 3 main parts : descriptive = a unique identifier , machine - readable code describing the clinical concepts modeled by the archetype and various metadata , definition = the main part describing the architecture , content , or restrictions of the archetype , andontology = defines the vocabulary and may contain language translations of code and meanings of codes used within the archetypes and tied to external vocabularies such as snomed or loinc . descriptive = a unique identifier , machine - readable code describing the clinical concepts modeled by the archetype and various metadata , definition = the main part describing the architecture , content , or restrictions of the archetype , and ontology = defines the vocabulary and may contain language translations of code and meanings of codes used within the archetypes and tied to external vocabularies such as snomed or loinc . this structure constrains the cardinality and content of information model instances complying with the archetype . codes representing the meanings of nodes and constraints on text or terms binding to terminologies such as snomed or loinc are stated in the ontology section . the formal language for expressing archetype is archetype definition language ( adl ) , a knowledge description language . adl uses three other syntaxes to describe constraints on data which are instances of some information model - cadl ( constraint from adl ) , dadl ( data definition from adl ) , and a version of first - order predicate logic ( fopl ) . archetypes are represented in web ontology language ( owl ) by mapping each adl construct to its owl counterpart . the intended purpose of archetypes is to empower clinicians to define the content , semantics , and data - entry interfaces of systems independently from the information systems . a feature of archetypes is the ability to separate internal model data from formal terminologies . the internal data are assigned local names which can later be bound or mapped to external terminology codes . this feature eliminatesthe need to make changes to the model whenever the terminology changes . in archetype models , snomed - ct aims to be a comprehensive terminology that provides clinical content and expressivity for clinical documentation and reporting . snomed has been developed using the description logic ontylog to allow formal representation of the meanings of concepts and their inter - relationship . the snomed hierarchy is easy to compute , which was the primary reason for selecting the terminology for the research . snomed - ct has approximately 370,000 concepts and 1.5 million triples i.e. relationships of one concept with another in the terminology ( figure 2 ) . besides medical records , various medical data measured from sensors and devices must be interchangeable in ubiquitous healthcare . the most common data exchange standards used in healthcare it are hl7 for general health information , digital imaging and communications in medicine ( dicom ) for medical images , and iso / ieee 11073 for medical devices . hl7 version 3 has evolved from a pure data interchangeable format to include a reference information model and a suite of other standards for capturing the conceptual structure of health information systems . both hl7 and dicom are built on the " free open source " philosophy therefore , most of the enabling and editing tools are " free open source " software as well . a list of the major " free open source " tools available for data interchange implementation is shown in table 1 . although not stated in the above sections , national and international standards developments , connectivity using hl7 , and document exchange using hl7 cda are also contributing to the implementation of content - based ubiquitous healthcare systems . in the health informatics community , a considerable amount of progress has been made in the area of for semantic interoperable electronic health records for ubiquitous use of patient information and health knowledge management . these methodological developments , particularly the ontological approach and openehr archetype , have changed ubiquitous healthcare allowing it to become more semantically interoperable and individual patient - based , these advances will allow healthcare professionals to manage complete electronic healthcare records of the patients regardless of which institution generates each clinical session .
there appears to be evidence that aspects of trunk control in the sitting position can be used as predictors of comprehensive activities of daily living function in patients with stroke1 . the following characteristics of the sitting position in post - stroke hemiparetic patients have been reported : the stability of the sitting position is lower in patients with stroke than in age - matched healthy subjects2,3,4 ; a delay of activation of the trunk muscles to control the sitting position is observed in patients with stroke5 , 6 ; and synchronization between activation of pertinent trunk muscle pairs is reduced in patients with stroke7 . the sacral sitting posture with a great degree of spinal flexion and neck extension is frequently observed in patients with stroke . this sitting posture is adopted to prevent falling over backwards due to poor abdominal muscle activation and excessive hip extension8 . numerous studies investigating trunk movement have considered the supine position as one segment ignoring the complexity of intervertebral movement9 . . reported on pelvic movement during lateral reach movement in the sitting position10 , and messier et al . described the movements of the upper trunk and pelvis when subjects touched a target placed in front of them with the forehead11 . riley et al . suggested that the sit - to - stand activity is the most mechanically demanding task undertaken during daily activities12 . to smoothly execute the sit - to - stand activity , the pelvis has to be leaned forward to flex the hip joint , and the trunk has to be flexed to use the hip extension moment , reduce the knee extension moment , and project the center of gravity into the base of support13,14,15,16,17,18 . therefore , the sacral sitting posture that is characteristic of patients with stroke is not the ideal posture for smoothly executing the sit - to - stand activity . maintaining the sitting position with the pelvis retroverted may be necessary to increase the sitting stability of patients with stroke . however , the ability to antevert the pelvis is required to execute the sit - to - stand activity . the purpose of this study was to investigate the relationships between the pelvic anteversion and retroversion angles and the ability to perform the sit - to - stand movement . we hypothesized that patients with stroke who are able to stand from sitting in a chair have a larger maximum pelvic anteversion angle than patients who are unable to stand from sitting in a chair . thirty - two hemiparetic subjects ( 15 females , 17 males ; mean age standard deviation ( sd ) , 66.7 7.6 years ) and 50 age - matched healthy control subjects ( 40 females , 10 males ; 64.2 8.2 years ) gave their informed consent to participate in the present study . inclusion criteria were predetermined as more than 3 months post - stroke , and an ablity to maintain the sitting position without using aids . the hemiparetic subjects were classified into two groups according to the sit - to - stand movement test described later : the group that was able to stand up ( the stand - able group ) ( 18 persons ) and the group that was unable to stand up ( the stand - unable group ) ( 14 persons ) . patients with a history of low back pain or surgery , hemispatial neglect , bilateral stroke , visual deficit , comprehension impairment , cognitive and/or communication deficits that precluded cooperation , as well as neurological or musculoskeletal disorders unrelated to the current stroke , were excluded . the exclusion criteria for the healthy subjects were known vestibular dysfunction , previous history of neurological disease , or orthopedic conditions that could have interfered with the experiment . this study was approved by the institutional ethics committee of kanazawa university and conformed to the ethical principles of the declaration of helsinki . the chair s seating face was square , 50 cm on each side , and 3 cm thick . the participants sat down on the chair 66% of their thigh length from the greater trochanter on the seat . keeping both arms crossed on the chest , the subjects sat with their feet parallel , with no support for the trunk or upper extremities . the chair seat height was adjusted to 100% of the subject s lower leg length , determined as the distance from the lateral femoral condyle to the ground , and the knee flexion angle was 90. in this study , pelvic angles were evaluated using a simple method for measuring the sacral inclination angle19 . a manual goniometer attached to an inclinometer with a resolution of one degree was used to measure pelvic angles ( fig . ( a ) anteversion angle , ( b ) retroversion angle)19 . the basic axis and the moving axis of this goniometer were defined as the vertical line and the longitudinal axis through the midline of the dorsal surface of the sacrum , respectively . therefore , pelvic anteversion was reported as a positive angle and pelvic retroversion was reported as a negative angle . an experimenter operated the goniometer that was attached gently to the midline of the dorsal surface of the sacrum , and another experimenter measured the angle with 1-degree resolution . ( a ) anteversion angle , ( b ) retroversion angle after maintaining a quiet sitting position for 20 seconds , the participants performed maximum pelvic anteversion and retroversion five times alternately . the subjects were instructed to keep the initial acromion anteroposterior position during the movements to avoid trunk anteroposterior movement . the maximum value and the minimum value of the five measurements were discarded and the mean value of the three remaining measurements were recorded as each participant s representative value . the range of pelvic motion was defined as the angle difference between the maximum pelvic anteversion and retroversion angles . next , the stroke patients were asked to stand up barefoot at a self - selected speed keeping their arms folded across the chest . three trials were performed with no restrictions on the position of the feet . before each trial , a brief rest interval was allowed . patients with stroke who could independently perform all three trials were classified as the stand - able group . the normality of the maximum pelvic anteversion angle , the maximum pelvic retroversion angle , and the range of pelvic motion in each group was evaluated by the shapiro - wilk test . normality was not observed for the maximum pelvic anteversion angle of the stand - unable group . therefore , the kruskal - wallis test was used to assess the effect of group on the maximum pelvic anteversion angle . when a significant main effect of group was found , multiple - comparison analysis was performed using the mann - whitney u test with the bonferroni correction . one - way anova was performed for the maximum pelvic retroversion angle and the range of pelvic motion . the differences indicated by one - way anova were examined by post hoc multiple - comparison analyses with the newman - keuls test . all statistical analyses were performed using spss 14.0 j ( spss japan , tokyo , japan ) . the maximum pelvic anteversion angle was distributed between 5 and 4 in the stand - able group , between 5 and 22 in the stand - unable group , and between 10 and 13 in the control group ( table 1table 1.mean , standard deviation and range of the pelvic angles in each groupthe stand - ablegroup ( n = 18 ) the stand - unablegroup ( n = 14)control group(n = 50)the maximum pelvic anteversion angle ( )mean sd1.2 2.812.4 6.11.6 5.0range ( max min)5 to 45 to 2210 to 13the maximum pelvic retroversion angle ( )mean sd18.5 5.619.6 4.627.6 8.1range ( max min)30 to 1027 to 1046 to 10the range of pelvic motion ( )mean sd19.7 5.17.2 5.125.9 7.6range ( max min)28 to 1015 to 049 to 9significant difference from the stand - able group . the maximum pelvic anteversion angles in the stand - able group were distributed above 5 , in contrast to the stand - unable group angles that were distributed below 5 ( table 1 ) . the means and standard deviations of the maximum pelvic anteversion angles were 1.2 2.8 , 12.4 6.1 , and 1.6 5.0 in the stand - able group , the stand - unable group , and the control group , respectively . a significant main effect of group was found ( p<0.001 ) . the value of the maximum pelvic anteversion angle was significantly smaller in the stand - unable group than in the control group and the stand - able group . therefore , the pelvic anteversion movement was significantly more limited in the stand - unable group than in the control group and the stand - able group . the maximum pelvic retroversion angles were distributed between 10 and 30 in the stand - able group , between 10 and 27 in the stand - unable group , and between 10 and 46 in the control group ( table 1 ) . the means and standard deviations of the maximum pelvic retroversion angles were 18.5 5.6 , 19.6 4.6 , and 27.6 8.1 in the stand - able group , the stand - unable group , and the control group , respectively . a significant main effect of group was found ( f(2 , 87 ) = 11.9 , p<0.001 ) . significant differences were found between the control group and the stand - able group ( p<0.001 ) , and between the control group and the stand - unable group ( p<0.001 ) . the range of pelvic motion was distributed between 10 and 28 in the stand - able group , between 0 and 15 in the stand - unable group , and between 9 and 49 in the control group ( table 1 ) . the means and standard deviations of the ranges of pelvic motion in each group were 19.7 5.1 , 7.2 5.1 , and 25.9 7.6 in the stand - able group , the stand - unable group , and the control group , respectively . a significant main effect of group was found ( f(2 , 87 ) = 43.7 , p<0.001 ) . significant differences were found among the groups ( the control group and the stand - able group : p<0.001 ; the control group and the stand - unable group : p<0.001 ; the stand - able group and the stand - unable group : p<0.001 ) . the maximum pelvic anteversion angle was significantly smaller in the stand - unable group than in the stand - able group and the control group . the maximum pelvic retroversion angle was significantly larger in the control group than in both the stroke patient groups . no significant difference was observed in the maximum pelvic retroversion angle between the stroke patient groups . the range of pelvic motion was significantly smaller in the stand - unable group than in the stand - able group and the control group . therefore , the hypotheses that the maximum pelvic anteversion angle in the stand - able group and the range of pelvic motion are significantly larger than those in the stand - unable group were confirmed . it is noteworthy that there was a value of the maximum pelvic anteversion angle that discriminated between patients with stroke into the stand - able group and the stand - unable group . the data suggest that the maximum pelvic anteversion angle required to perform the sit - to - stand movement in patients with stroke needs to be greater than 5 . to smoothly execute the sit - to - stand movement , the pelvis is anteverted to flex the hip joint and the trunk to use the hip extension moment , reduce the knee extension moment , and project the center of gravity into the base of support13,14,15,16,17,18 . sitting position stability was worse in patients with stroke than in the age - matched healthy subjects2,3,4 . the patients with stroke could not sufficiently flex the hip joint when the trunk extensor muscles were required to be activated during sitting8 . patients with stroke usually sit with kyphosis and pelvic retroversion to avoid falling backward , due to insufficient abdominal muscle activity . hence , the sit - to - stand movement in patients with stroke may require larger trunk forward leaning , because of kyphosis and pelvic retroversion , to shift the center of gravity into the base of support of the feet . observed that trunk forward leaning angles are larger in patients with stroke than in healthy subjects when performing the sit - to - stand movement20 . reported that the average center of gravity projection in the base of support of patients with stroke was 3 cm posterior to that of healthy subjects during the seat - off phase in the sit - to - stand movement21 . in addition , when the trunk is flexed , the hip extension moment is reduced due to a lack of pelvic anteversion , and patients with stroke may depend primarily on knee extension moment to stand up . some studies have reported a high correlation between pelvic inclination in the sitting position and the degree of lumbar lordosis9 , and a strong relationship between the sacral angle of inclination and the lumbar lordosis22 , 23 . hence , pelvic inclination ( anteversion and retroversion ) reflects lumbar movement ( lordosis and kyphosis ) . the range of pelvic motion in the stand - unable group was extremely limited , being only 28% of the control group and 36% of the stand - able group values . accordingly , lumbar movement in the sagittal plane ( lordosis and kyphosis ) in the stand - unable group was probably limited compared to the control group and the stand - able group . the pelvic angle measurements were conducted in the sitting position while maintaining 90 of knee flexion with the feet in contact with the ground . the hip joints work as pivotal axes in pelvic anteversion and retroversion in the sitting position . one factor that should influence the hip range of motion is the extensibility of the hamstrings , which drive the hip and knee as bi - articular muscles . hamstring stretching improves the pelvic anteversion angle24 and mobility of the hips of elderly people25 . the sitting position in this study fixed the knee flexion angle at 90 , which should have increased hamstring tension during the measurement . for this reason , pelvic anteversion should have been restrained by the increased tension of the hamstrings . on the other hand , since there was no significant difference in the maximum pelvic retroversion angle between the two stroke groups , it indicates that the pelvic retroversion angle did not affect the stroke patients ability to perform the sit - to - stand movement . however , the range of pelvic motion was markedly restricted in the stroke groups compared to the control group . in patients with stroke , pelvic anteversion appears to be an important factor for patients to regain the ability to perform the sit - to - stand movement . our results are in agreement with a previous study ( otao et al . ) , which concluded that the ability to anteriorly tilt the pelvis during active exercise may be related to basic movements , such as the sit - to - stand movement , in patients with stroke26 . first , the sample sizes of the stroke groups were smaller than that of the control group . second , although the results indicate the importance of the maximum pelvic anteversion angle for the ability of patients with stroke to perform the sit - to - stand movement , other factors , such as muscle strength , equilibrium , coordination , and hip and ankle range of motions should be investigated in future studies . third , the present pelvic angles were not measured during the sit - to - stand movement . the range of pelvic motion is one of the factors involved in the sit - to - stand movement , and its contribution should be clarified in future studies . finally , this study investigated japanese patients whose culture and life style are different from those of other countries , which means that the findings of this study may not be universally applicable .
mycobacterium kansasii is a slow - growing acid - fast bacillius ( afb ) and belongs to the group of environmental mycobacteria , also known as atypical mycobacteria or nontuberculosis mycobacteria ( ntm ) . local water supplies are considered as the major reservoir for the m. kansasii , and evidence of person - to - person transmission has not been reported . the most common presentation of m. kansasii infection is a chronic bronchopulmonary disease , which manifests typically in adult patients with chronic obstructive pulmonary disease or cystic fibrosis . in addition , m. kansasii can cause skeletal infections , skin and soft tissue infection , cervical or other lymphadenitis , and disseminated infection ( 1 ) . disseminated infection by m. kansasii occurs almost exclusively in immunocompromised patients , such as solid organ transplant recipients , hiv - infected individuals , patients with hematologic malignancy , or patients receiving long - term steroid regimens ( 2 ) . in the case of disseminated m. kansasii infection , involvement of multiple organs including the lungs , liver , spleen , bone marrow , lymph node ( ln ) , bowels , central nervous system , pericardium , pleura or kidneys , has been reported ( 3 ) but disseminated m. kansasii infection associated with skin involvement is not frequent ( 4 ) . recently , we encountered a rare case of disseminated m. kansasii infection involving multiple skin areas together with lung and multiple lns . to our knowledge , this is the first case of disseminated m. kansasii infection that has involved the skin in korea . therefore , we report this unusual case with a comprehensive review of previously reported disseminated m. kansasii infections in non hiv - infected patients . a 48-yr - old man was admitted with a 1-month history of fever and a 2-week history of dyspnea on exertion at severance hospital in seoul , korea . he had a history of myelodysplastic syndrome ( mds ) diagnosed 21 months ago prior to admission and had been treated with oral glucocorticoid ( prednisolone , 10 mg daily ) with regular follow - up . a year after mds was diagnosed , multiple erythematous tender nodules developed on both lower legs , and a skin biopsy of the calf revealed sweet 's syndrome . he continuously had these skin lesions without complete resolution until admission . on admission , several papulonodular skin lesions on his arms , chest , back , abdomen , buttocks , and legs were noted ( fig . multiple lns were palpated on the medial side of the right thigh and left cervical area . initial laboratory tests showed leukopenia with a white blood cell count of 1,950/l ; severe anemia with a hb level of 6.8 g / dl ; mild thrombocytopenia with a platelet count of 113,000/l ; an elevated esr ( 73 mm / hr ) and c - reactive protein level ( 10.8 mg / dl ) . chest computer tomography ( ct ) confirmed multiple lns enlargement at the mediastium , paratracheal area , subcarina and right perihilar bronchovascular interstitial and interlobular septal thickening . meanwhile , both excisional ln biopsies , which were performed at the palpable lns of the thigh and neck , and skin and mediastinoscopic paratracheal ln biopsies revealed necrotizing granuloma with many afb . also , an afb smear of a pus - like discharge obtained from the paratracheal ln revealed a positive finding . with a presumptive diagnosis of disseminated tuberculosis , anti - tuberculosis therapy was started with herz ( isoniazid [ inh ] , rifampin [ rfp ] , ethambutol [ emb ] , and pyrazinamide [ pza ] ) regimens on hospital day ( hd ) 16 . however , as the skin lesions progressed rapidly and high spiking fever persisted despite herz treatment , we assumed he had a rapidly growing ntm such as m. abscessus or m. fortuitum , and started him on amikacin , clarithromycin , levofloxacin and cefoxitin instead of emb and pza . however , improvement of the skin lesions was not evident . three weeks after anti - tuberculosis therapy was started , a mycobacterium culture of skin and pus - like discharge obtained from the paratracheal ln revealed ntm , and repeated sputum mycobacterium culture also revealed ntm . at hd 43 , all ntms cultured in sputum , paratracheal lns , and skin were identified as m. kansasii by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) of the polymorphic region of the rpob gene . in vitro drug susceptibility testing of m. kansasii showed that the isolate was susceptible to rfp , emb , pza , streptomycin , moxifloxacin , and cycloserine but resistant to inh and para - aminosalicylic acid . at hd 43 , we altered the anti - mycobacterial treatment regimens to inh , rfp , emb , and clarithromycin . gradual improvement of the general condition and symptoms with regression of skin lesions was noted . sputum afb , which was examined at hd 51 , was converted into negative and mycobacterial culture of sputum did not identify any mycobacteria . however , during treatment for m. kansasii , the patient developed small bowel obstruction and ischemic colitis . although he underwent a small bowel resection and intensive conservative management , the patient died on hd 121 . m. kansasii is the second most frequently recognized ntm pathogen and second most frequent cause of disseminated ntm disease , after m. avium complex ( mac ) , in the unites states and japan ( 2 , 5 , 6 ) . furthermore , in southeast england , m. kansasii is more common than mac ( 7 ) . in south korea , m. kansasii is the fourth most commonly isolated ntm pathogen , after mac , m. abscessus - chelonae complex , and m. fortuitum , but its incidence has increased , especially in highly industrialized areas ( 8) . in agreement with previously established risk factors ( 2 ) , our patient 's risk factors for disseminated m. kansasii infection included a history of hematological malignancy and long - term steroid use . the patient had a disseminated m. kansasii infection with multiple skin lesions , as well as lung and multiple lns . in addition , because an abdominal ct scan revealed a splenic abscess , we speculated that splenic infection with m. kansasii was also probable . we comprehensively reviewed the literature written in english and available in abstract or full text form that reported disseminated m. kansasii infection in non hiv - infected patients ( 3 , 4 , 6 , 9 - 21 ) . among a total of 67 cases including the present case , 4 cases were excluded from analysis because of insufficient information . table 1 shows the characteristics of the 63 remaining cases of disseminated m. kansasii infection in non - hiv infected patients . however , the frequency of previously healthy persons with no underlying diseases was relatively high as 23.8% . also , the table shows that m. kansasii caused infection in diverse visceral organs ; commonly involved sites included the lungs , lns , spleen , liver , and bone marrow . the prognosis of disseminated m. kansasii infection was poor as the percentage of patients that died was 60.3% . as previously known that the presence of underlying disease and/or immunosuppression seemed to be the best predictor of outcome of disseminated m. kansasii infection ( 4 ) , the mortality of patients with underlying disease was higher than those without underlying disease ( 75% and 53.3% respectively ) . we summarized the clinical characteristics of 18 patients with disseminated m. kansasii infection in non hiv - infected patients reported since 1990 yr at table 2 . the m. kansasii isolates cultured from our patient were resistant to an inh in vitro susceptibility test . the concentrations of inh used in susceptibility testing are those chosen for their usefulness with m. tuberculosis . some m. kansasii isolates may be reported resistant to inh at 0.2 or 1.0 g / ml . however , these isolates of m. kansasii are susceptible to slightly higher inh concentrations , and are still susceptible to achievable blood levels . thus , inh should be used regardless of the in vitro susceptibility test results ( 8) . we also used anti - mycobacterial regimens containing inh and noted a gradual improvement of skin lesions and negative sputum mycobacterial culture after this treatment . cutaneous ntm disease is most often caused by rapidly growing mycobacteria such as m. abscessus - chelonae complex and m. fortuitum rather than m. kansasii . it is known that m. kansasii can rarely cause a primary cutaneous infection , which usually results from penetrating injuries or disseminated disease ( 22 ) . the patient described in this case had skin lesions for a long time before disseminated infection at the multiple lns and lung developed . we surmised that minor local trauma by initial skin lesions of sweet 's syndrome resulted in the inoculation of m. kansasii and caused disseminated infection in the immunocompromised condition brought about by long - term steroid use . the natural course of untreated , non - disseminated skin infection by m. kansasii is one of non - serious , indolent progression . however , as seen in this case , skin infection associated with systemic dissemination in a patient with underlying disease in immunosuppressive conditions is associated with poor outcome ( 22 ) . in conclusion , our case emphasizes that chronic skin lesions can lead to severe , disseminated m. kansasii infection in an immunocompromised patient . particular attention to the aggressive diagnostic work - up , such as biopsy , should be given in immunocompromised patients with chronic skin lesions to diagnose infection by an unusual pathogen , such as ntm , before the infection disseminates .
production of acute inflammatory cytokines by cells of the innate immune system , including macrophages and mast cells , plays an essential role in inflammation - driven tumor development . cytokines such as tnf , interleukin ( il)-6 , and il-1 have myriad effects in the tumor microenvironment , promoting cell growth and survival as well as angiogenesis and the recruitment of immune effector cells ( 2 ) . which cytokines are required for tumor development depends largely on the model being examined . tnf plays an essential role in several models of cancer , and is a critical inflammatory mediator in many autoimmune diseases of both mice and humans , primarily acting via induction of nf-b ( 2 ) . il-6 acts both as a mitogen and an angiogenic factor and has been implicated in many of the same processes as tnf . il-6 plays an important role in carcinogen - driven liver cancer , and has recently been identified as an important driving factor in non smoking - related lung cancer in humans ( 810 ) . il-1 can activate nf-b in a manner similar to tnf , and polymorphisms in il-1 have been linked to gastric cancer ( 11 ) . not surprisingly , tumor - promoting inflammation can be induced by the same pathways that respond to microbial infections , suggesting that tumors may be aberrant consequences of initially physiological immune responses . recent work has positioned myeloid differentiation factor 88 ( myd88 ) , which is a critical downstream signaling molecule for both the toll - like receptor ( tlr ) family of microbial pattern recognition receptors and the il-1 and -18 receptors ( il-1r and -18r ) , as a central player in inflammation - driven tumorigenesis . in the diethylnitrosamine model of liver cancer , myd88-dependent myd88-deficient mice treated with the topical carcinogen 7,12-dimethylbenz[a]anthracene ( dmba ) , followed by treatment with the proinflammatory phorbol - ester tpa , developed fewer epithelial tumors than did wild - type mice ( 13 ) . surprisingly , 3-methylcholanthrene ( mca)induced sarcomas were also reduced in myd88-deficient mice , despite the lack of an obvious role for inflammation in this model ( 13 ) . myd88-dependent signaling is also required in some genetic tumor models . in the apc model of human fap , myd88 deficiency was associated with decreased inflammatory cytokine production within the tumor microenvironment ( 14 ) . this reduction correlated with a decrease in both the number and size of spontaneously arising polyps . although these findings bring us one step closer to understanding the nature of the signals driving tumor - promoting inflammation , the factors responsible for engaging myd88-dependent pathways are still obscure . tlrs can recognize a wide range of highly conserved microbial products , potentially implicating occult infections or normal flora in tumor - associated inflammation . myd88 may also facilitate so - called sterile inflammation , either through tlr - dependent recognition of endogenous adjuvants or through il-1r signaling . il-1r signaling is important for the initiation of neutrophil infiltration in response to necrotic cells ( 15 ) , and it may also have important tumor - intrinsic effects . il-1 was recently shown to be required for mca - induced tumor formation , suggesting that , in this system , myd88 may function through the il-1r ( 16 ) . consistent with the importance of sterile inflammation in tumor promotion , the findings of gebhart et al . ( 7 ) in this issue implicate endogenous proteins released during cell necrosis in inciting carcinogenic inflammation . rage activation can occur through the recognition of at least three self - proteins released from cells during necrosis : the dna - binding protein hmgb1 and the two calcium - binding cytokines s100a8 and s100a9 . intriguingly , rage - dependent recognition of hmgb1 has been shown to act in a costimulatory capacity for tlr - mediated responses to dna , potentially providing a link between rage and myd88 ( 17 ) . failure of normal antiinflammatory mechanisms is thus an essential feature of tumor - promoting inflammation . although in many cases , such as infection or autoimmunity , the mechanisms that prevent the resolution of inflammation are still unclear , genetic defects in key regulatory proteins can enhance tumor formation . loss of tir8 , which is a negative regulator of il-1r / tlr signaling , exacerbates inflammation in the dextran sulfate sodium model of colitis , leading to a substantially increased risk of colon cancer ( 1819 ) . similarly , loss of il-1 receptor antagonist , a secreted protein that blocks il-1 function , accelerates tumor onset and increases tumor aggressiveness in the dmba / tpa model ( 16 ) . infusion of regulatory t ( t reg ) cell depleted t cells into apc mice increases polyp formation and leads to the development of spontaneous mammary tumors ( 20 ) . one of the more intriguing elements of rage - dependent inflammation is the ability of rage to up - regulate its own ligands . ( 7 ) demonstrate that rage signaling in the dmba / tpa model induces s100a8 and s100a9 synthesis in epithelial cells , likely leading to a feed - forward loop that further aggravates the inflammatory environment . regulatory circuits limiting rage - mediated inflammation undoubtedly exist , but these as yet uncharacterized pathways are clearly not sufficient to prevent tumor onset in the time frame of these experiments . interestingly , the requirement for rage can be bypassed by more frequent application of tpa , suggesting that other , more rapidly self - limiting inflammatory pathways can substitute for rage if the inflammation - inciting agent is allowed to persist . in contrast to the tumor - promoting effects of chronic inflammation , increasing evidence suggests that adaptive immunity is responsible for recognizing and rejecting malignant cells ( for review see reference 21 ) . the protective effect of adaptive immunity is most obvious in the case of tumors with viral etiologies , but immunodeficiencies in both mice and humans have also been associated with an increased incidence of many tumors without a clear viral etiology ( 21 ) . consistent with a role for adaptive immunity in regulating tumor growth , spontaneous lymphocytic infiltrates can be observed in a variety of different human cancers and , in some , these infiltrates correlate with a favorable prognosis ( 2122 ) . once a tumor is established , immunosuppression is a common feature of the microenvironment , with most tumors infiltrated by immunosuppressive myeloid and lymphoid cells ( 21 ) . the expression of antiinflammatory factors is also common in tumors , suggesting that overcoming an antitumor immune response is an important step in tumorigenesis . the mechanism by which adaptive immune cells can control tumor growth has been studied extensively using the mca tumor model . in this system , mice with defects in t cell immunity exhibit an increased incidence of tumor formation after mca application , and the tumors that arise in these mice are more easily rejected when transferred into immunocompetent animals than are similar tumors that arise in wild - type mice ( 21 ) . these findings provide the most direct evidence to date that the immune system can restrict tumor formation . recent evidence also indicates that many immunocompetent animals treated with mca harbor occult tumors that are kept in check by continuous immune surveillance ( 23 ) . although direct evidence for spontaneous , immune - mediated tumor control in humans is lacking , rare cases of tumor recurrence decades after the disappearance of the primary tumors suggest that similar mechanisms may be in play in humans as well ( 21 ) . in most models used to study tumor - promoting inflammation , cytokines produced by cells of the innate immune system play an indispensable role . protective antitumor effects , in contrast , derive largely from adaptive immune cells , particularly t cells . yet , dividing the immune response to cancer into this innate - adaptive dichotomy is too simplistic . inflammatory cytokines can restrict the growth of certain tumors , and adaptive immunity can drive tumor - promoting inflammation in chronic infections and in autoimmunity . recent work has started to explain the dual role for adaptive immune cells in tumor development . overexpression of the cytokine il-23 , for example , is highly correlated with an increased risk of tumor development ( 24 ) . il-23 expands a subset of cd4 t cells that secrete the proinflammatory cytokine il-17 , which promotes angiogenesis , as well as the production of a range of other inflammatory factors from epithelial cells ( for review see reference 25 ) . at the same time , however , il-23 expression in the tumor microenvironment reduces cytotoxic t cell infiltrates , potentially blocking antitumor immunity ( 24 ) . il-17secreting t cells are found in many chronic inflammatory diseases , and may provide an important link between chronic inflammation and cancer ( 25 ) . ultimately , the consequences of the interaction between the immune system and tumors will likely depend on the context in which the immune system is engaged . feed - forward signals such as those initiated by rage lead to uncontrolled , chronic immune activation . in this setting , any potentially protective effect from the immune system is overwhelmed by tumor - promoting inflammation ( fig . we can hope to circumvent these responses , thus opening the door to novel approaches to cancer treatment and prevention . signals through innate immune receptors such as rage maintain chronic inflammation in the tumor microenvironment . chronic inflammatory responses can be initiated by microbial or endogenous tlr ligands , or by rage ligands ( s100a8 , s100a9 , and hmgb1 ) released from necrotic cells . these signals drive the expression of nf-b activating cytokines , such as tnf , il-1 , and il-6 , which act as growth factors for the tumor . il-17 produced by cd4 t cells may also promote angiogenesis . at the same time , the tumor microenvironment disables potentially protective immune responses mediated by ifn-secreting cd4 and cd8 t cells .
population ageing is one of humanity 's greatest challenges in the 21st century , which will undoubtedly put increased health , economic and social demands on all countries . in singapore , an integrated approach is being undertaken by the various government agencies to manage the silver tsunami. from the healthcare front , a central body the agency for integrated care ( aic)has been set up to facilitate and coordinate care services so that patients can be cared for at the most appropriate settings . to discuss the concept and principles of care coordination in singapore context and its impact on aged care landscape . one of the projects undertaken by aic is the aged care transition ( action ) project . piloting at five acute hospitals in singapore , action is a 4-year , 22-million project funded by the government . the aim of aged care transition ( action ) team is to help patients make an important transition from hospital into their home or community . this is done by streamlining and coordinating care services thereby optimizing outcomes throughout and following an episode of illness . with trained care coordinators at these pilot sites , the project helps to establish a line of open communication as well as point of contact amongst acute setting , aic headquarters and the community service providers . it is also through this project that the effectiveness of care coordination ( in the form of unplanned readmissions and attendances at a&e ) as well as areas of improvement in both acute and community setting are identified and intervened . the preliminary findings of action shows that this new initiative helps to reduce average length of stay ( alos ) in acute settings , foster interagency collaboration and improve access to services for patients .
it is the most common motor neuron disease in european countries ( 4 , 5 ) . its incidence and prevalence in these countries are 1 - 2/100,000/year and 4 - 13/100,000/year , respectively ( 6 , 7 ) . its clinical features depend on several factors , including age at onset of symptoms ( one to 94 years ) ( 8 , 9 ) , site of onset of symptoms ( limbs or bulbar ) ( 10 ) , rate of progression ( 11 , 12 ) , and survival time ( few months to over 10 years ) ( 6 , 10 ) . the ultimate cause of death in als patients is usually respiratory failure . genetics is the source of at least a part of the variability associated with als . the majority of patients are sporadic ( sals ) , while 1 - 13% of cases in different epidemiological studies were reported to have more than one affected individual in their families and such cases are known as familial als ( fals ) ( 13 ) . mean age at onset of fals patients is approximately 10 years lower than sals patients , but they are clinically indistinguishable ( 9 ) . to date , at least 19 als causing genes have been identified ( http://alsod.iop.kcl.ac.uk/ ) ( 14 - 16 ) and approximately , 50 - 60% of fals patients carry mutations in these genes ( 6 ) . mutations in sod1 and c9orf72 are the most common causes of disease , although their relative contributions vary in different populations ( 11 , 17 - 24 ) . sod1 and c9orf72 are , respectively , the first and one of the most recently identified als genes ( 17 , 18 , 24 ) . recently , we showed that mutations in sod1 are more common than mutations in c9orf72 among iranian als patients ( 11 , 21 ) . here , we describe the clinical features of an iranian fals patient who harbors a mutation in sod1 that causes p.val48phe . before , the mutation was once reported in an italian patient , but detailed clinical features of the patient were not presented ( 25 ) . this project was performed in accordance with the helsinki declaration and approved by the ethics board of the university of tehran , tehran , iran . all participants or their responsible guardians consented to participate after being informed about the project . based on el escorial criteria ( 26 ) , the proband was diagnosed as definite als by a neurologist ( sn ) in neuromuscular clinic of shariati hospital , tehran , iran . he belonged to a large fals pedigree that in addition to the proband , includes six als patients from four generations ( figure 1 ) . five exons of sod1 were amplified by polymerase chain reaction ( pcr ) ( supplementary table 1 , 2 ) ( 11 ) . all pcr products were sequenced with the same primers that were used in the pcrs , using the abi big dye chemistry and an abi prism 3700 instrument ( applied biosystems , foster city , ca ) . upon identification of the c.142g > t variation that affects p.val48phe in the encoded protein , the variation was screened in 100 iranian control individuals who were over 60 years old using an allele specific pcr protocol . to assess conservation of p.val48 , amino acid sequences of sod1 proteins from 17 species were obtained from uniprot ; http://www.uniprot.org/uniprot/ and aligned using clustalw2 software ; http://www.ebi.ac.uk / tools / msa / clustalw2/. additionally , the sift ; http:// blocks.fhcrc . org / sift/ sift.html , polyphen ; http://genetics.bwh.harvard.edu/pph2 , panther ; http://www.pantherdb.org/tools/ csnpscoreform.jsp and snap ; https://rostlab.org/ services / snap / submit bioinformatics tools were used to predict the potential pathological effects of p.val48phe on the encoded protein . in the encoded sod1 protein , c.142g > t variation that causes p.val48phe was observed in the heterozygous state in the dna of the proband ( figure 2 ) . the only surviving affected individual in the pedigree ( iii-1 ) lives in europe and was not available for genetic analysis . furthermore , segregation analysis in the pedigree was not possible because none of the unaffected members of the pedigree consented to genetic analysis ; they did not want to know whether or not they carried the mutated allele . however , the c.142g > t variation was not observed in 100 unrelated healthy elderly iranian control individuals . furthermore , valine at positions corresponding to p.48 in the human sod1 protein is well conserved across species from caenorhabditis elegans to homo sapiens ( table 1 ) . the sift , polyphen , panther and snap tools predicted , respectively , that the substitution is damaging , probably damaging , deleterious , non - neutral . before , the same variation was once reported as the cause of als in an italian als family ( 25 ) . all togeths1er , our data led us to conclude that the p.val48phe causing variation in sod1 was the probable cause of als in the proband and his affected relatives . the inheritance pattern of als in the pedigree suggests an autosomal dominant mode of inheritance , consistent with observation of a single mutated allele in the proband ( figure 1 ) . the als patient who was studied here is a member of a fals pedigree ( als164 ) that includes seven als - diagnosed patients . five patients had died before the start of the study , and now the proband is also deceased . available clinical information on five affected members of the pedigree belonging to generations iii and iv is presented in table 2 . the average of age at onset of symptoms was 34.6 years ( range : 29 - 45 years ) . four patients died 2.5 to 3 years following the onset of symptoms , and the earliest presentations involved the limbs in these individuals . presentation in the fifth patient ( iii-1 ) was bulbar ; age at onset for this individual was approximately 13 years more than the average age at onset of the other patients . patient iii-1 is now , two years after the onset , in the final stages of disease . he breathes with the help of a ventilator and is completely paralyzed , unable to speak and swallow . the proband was a 31-year - old man ( iv-13 , figure 1 ) who presented with a two year history of weakness and atrophy of the limbs which had been started in the left hand and gradually progressed sequentially to involve the right leg , the right hand and the left leg . he mentioned that there was muscle twitching at the beginning , but it was disappeared after a few months . neurological examination showed normal mental state , tongue atrophy and fasciculation and wasting of left upper extremity . asymmetric quadriparesis which was more severe in left upper and right lower extremities was seen . biceps and triceps reflexes were increased on the right side , and were absent on the left side . electromyography that was performed two years after onset of symptoms , showed denervation , fasciculation and reinnervation in various muscles innervated by cranial , cervical , thoracic and lumbar segments . the findings were interpreted as definite motor neuron disease according to awaji criteria ( 27 ) . laboratory studies were normal , as were results of brain and cervical spine magnetic resonance imaging ( mri ) . he voluntarily entered a clinical trial of autologous mesenchymal stem cell transplantation with intraspinal injection ( irct201107221696n3 ) . repeated electro - myography - nerve conduction velocity ( emg - ncv ) performed prior to transplantation evidenced reduced compound muscle action potential ( cmap ) motor amplitude as compared to his first electromyography . three months after transplantation , his pulmonary function test showed a forced vital capacity of 75% . the patient remained in a stable and good condition during a four months follow - up . then , one night he developed severe dyspnea , was admitted to the hospital with a possible diagnosis of pulmonary emboli , and died a few hours later . in the encoded sod1 protein , c.142g > t variation that causes p.val48phe was observed in the heterozygous state in the dna of the proband ( figure 2 ) . the only surviving affected individual in the pedigree ( iii-1 ) lives in europe and was not available for genetic analysis . furthermore , segregation analysis in the pedigree was not possible because none of the unaffected members of the pedigree consented to genetic analysis ; they did not want to know whether or not they carried the mutated allele . however , the c.142g > t variation was not observed in 100 unrelated healthy elderly iranian control individuals . furthermore , valine at positions corresponding to p.48 in the human sod1 protein is well conserved across species from caenorhabditis elegans to homo sapiens ( table 1 ) . the sift , polyphen , panther and snap tools predicted , respectively , that the substitution is damaging , probably damaging , deleterious , non - neutral . before , the same variation was once reported as the cause of als in an italian als family ( 25 ) . all togeths1er , our data led us to conclude that the p.val48phe causing variation in sod1 was the probable cause of als in the proband and his affected relatives . the inheritance pattern of als in the pedigree suggests an autosomal dominant mode of inheritance , consistent with observation of a single mutated allele in the proband ( figure 1 ) . the als patient who was studied here is a member of a fals pedigree ( als164 ) that includes seven als - diagnosed patients . five patients had died before the start of the study , and now the proband is also deceased . available clinical information on five affected members of the pedigree belonging to generations iii and iv is presented in table 2 . the average of age at onset of symptoms was 34.6 years ( range : 29 - 45 years ) . four patients died 2.5 to 3 years following the onset of symptoms , and the earliest presentations involved the limbs in these individuals . presentation in the fifth patient ( iii-1 ) was bulbar ; age at onset for this individual was approximately 13 years more than the average age at onset of the other patients . patient iii-1 is now , two years after the onset , in the final stages of disease . he breathes with the help of a ventilator and is completely paralyzed , unable to speak and swallow . the proband was a 31-year - old man ( iv-13 , figure 1 ) who presented with a two year history of weakness and atrophy of the limbs which had been started in the left hand and gradually progressed sequentially to involve the right leg , the right hand and the left leg . he mentioned that there was muscle twitching at the beginning , but it was disappeared after a few months . neurological examination showed normal mental state , tongue atrophy and fasciculation and wasting of left upper extremity . asymmetric quadriparesis which was more severe in left upper and right lower extremities was seen . biceps and triceps reflexes were increased on the right side , and were absent on the left side . electromyography that was performed two years after onset of symptoms , showed denervation , fasciculation and reinnervation in various muscles innervated by cranial , cervical , thoracic and lumbar segments . the findings were interpreted as definite motor neuron disease according to awaji criteria ( 27 ) . laboratory studies were normal , as were results of brain and cervical spine magnetic resonance imaging ( mri ) . he voluntarily entered a clinical trial of autologous mesenchymal stem cell transplantation with intraspinal injection ( irct201107221696n3 ) . repeated electro - myography - nerve conduction velocity ( emg - ncv ) performed prior to transplantation evidenced reduced compound muscle action potential ( cmap ) motor amplitude as compared to his first electromyography . three months after transplantation , his pulmonary function test showed a forced vital capacity of 75% . the patient remained in a stable and good condition during a four months follow - up . then , one night he developed severe dyspnea , was admitted to the hospital with a possible diagnosis of pulmonary emboli , and died a few hours later . sod1 encodes copper - zinc superoxide dismutase , which is an evolutionarily highly conserved enzyme that catalyzes the conversion of toxic superoxide anion to hydrogen peroxide and molecular oxygen . in various studies , mutations in sod1 were observed in 12 - 23% of fals patients ( average : 20% ) and in 0 to 7 percent of sals patients ( average : 3% ) ( 13 , 15 , 28 ) . in iranian als patients , these mutations were found in 38.5% of the fals probands , and 4.25% of the sals cases ( 11,12 ) . over 170 different sod1 mutations have been reported so far ( 11 , 14 , 21 , 22 ) . while it has been generally difficult to establish clear genotype- phenotype correlations for specific mutations and even for different causative genes , mutations in sod1 that cause p.asp90ala and p.leu144ser are associated with long survival time ( 11 , 29 ) , while p.ala4val and p.gly85ser are associated with rapid progression of disease ( 30 ) . in the present study the proband of the pedigree harbored a mutation in sod1 that causes p.val48phe in the encoded protein . als inheritance in the pedigree was autosomal dominant , without evidence of anticipation . before , although detailed clinical findings were not presented in the earlier finding , age at onset of symptoms in the proband was reported to be about 36 years . this is close to age at onset in patients of als164 pedigree ( average : 34.6 years ) . the father of the italian proband had died from als at the age of 39 . age at onset , survival time and limb onset presentation were notably uniform among four of five patients in the als164 pedigree . the fifth patient ( iii-1 ) differed from the others and had a much higher age at onset and a bulbar presentation . these observations provide evidence that the p.val48phe mutation can result in different clinical features even within a single pedigree . the efficacy of the autologous mesenchymal stem cell transplantation in the proband could not be assessed . as stated above , a clear genotype - phenotype correlation exists for only a few als causing sod1 mutations . based on clinical data on the iranian family which was described here and the available data on a previously reported italian als patient who harbored the same mutation , it appears that p.val48phe causing mutation in sod1 mutation causes als with an early onset . this having been said , there was some variability in just how early symptoms manifested . age at onset of four out of five patients ranged between 29 and 36 years while it was notably higher ( 45 years ) for one of them . limb onset presentation was a common feature among four out of five iranian patients , but onset was bulbar in the patient who had showed the latest onset . it can be concluded that while the window of clinical presentation for the p.val48phe mutation is relatively narrow , particularly with respect to age at onset , it is not strictly uniform .
giant vascular eccrine spiradenoma ( gves ) is a rare highly vascular variant of eccrine spiradenoma ( es ) , as only four cases are reported in the literature ( 1 - 3 ) . clinically , it might be mistaken for angiomatous lesions in view of its florid vascularity and hemorrhagic features . it is well known that tumor lobules are composed of two types of epithelial cells , namely small , dark staining basaloid cells located at the periphery and larger cells with a pale nucleus situated mostly in the center . however , cellular differentiation of gves remains under discussion and the cells of which is composed are not clearly described . this tumor is considered to differentiate toward both the dermal duct and the secretory segment of the eccrine sweat gland like es ( 4 ) . this has been substantiated by the identification of occasional myoepithelial cells at the periphery of tubular structures ( 5 ) . however , other authors have not found myoepithelial cells by ultrastuctural and histochemical studies . furthermore , immunohistochemistry has failed to demonstrate myoepithelial differentiation ( 6 ) . to clarify the nature of the cells of which is composed , we performed immunohistochemical stainings using various monoclonal antibodies for cytokeratins ( cks : ck , ck7 , ck20 , cam5.2 ) , epithelial membrane antigen ( ema ) , and carcinoembryonic antigen ( cea ) . double - marker analysis was performed using p63 and smooth muscle actin ( sma ) and triple - marker analysis was also performed using p63 , sma , and ck7 . a 56-yr - old healthy woman had a solitary violaceous protruding mass on her lower back . the tumor had begun as a small soft nodule approximately 3 yr before , and had grown slowly . physical examination showed a 2 cm sized , erythematous to violaceous hemispheric firm nodule on the left side of the lower back ( fig . an excisional biopsy was performed with the clinical impression of a painful tumor of the skin such as angiolipoma or neuroma . formalin - fixed paraffin embedded tissue sections ( 4 to 5 m thick ) were used . the antibody panel for epithelial cells included ck ( immunotech , marseille , france ) , ck7 ( scytec , logan , ut , u.s.a . ) , ck20 ( biomeda , foster city , ca , u.s.a . ) , cam5.2 ( becton - dickinson , san jose , ca , u.s.a . ) , ema ( signet laboratories , dedham , ma , u.s.a . ) , and cea ( immunotech ) . the antibody panel for nerve fibers included neurofilament ( signet ) , and s-100 protein ( signet ) . for characterize myoepithelial cells , we used monoclonal antibodies for sma ( immunotech ) and p63 ( clone 4a4 , oncogene research products , boston , ma , u.s.a . ) . recently , p63 has been proposed as a specific marker of precursor / stem cells ( 7 ) and n - p63 is the predominant p63 isoform preferrentially expressed in the epithelial basal cells of organs such as the skin , breast , prostate and uterine cervix . it is also expressed in ductal structures of skin appendages and is also used as a marker of myoepithelial cells ( 8) . clone 4a4 is a mouse monoclonal antibody obtained from mice hyperimmunized with an aminoterminal fragment of the n - p63 isoform expressed in e. coli as a gst fusion protein . double marker analysis was performed using monoclonal antibody for p63 and sma to differentiate basal cells and myoepithelial cells ( 8) . triple marker analysis using monoclonal antibody for p63 , sma , and ck7 was also performed to clarify three type of the cells es is composed of , namely pale epithelial cells , small basal cells , and myoepithelial cells ( 9 ) . microscopic examination revealed one large well - circumscribed encapsulated lobule and a small satellite lobule involving the dermis and subcutis . unlike usual es , a lobule consisted of peculiarly abundant stroma and compressed cellular cords or sheets that were composed of two types of cells : cells with large pale nuclei in the center and basaloid cells with small , dark nuclei at the periphery ( fig . 2 , 3 ) . the stroma showed greatly dilated vascular spaces containing pale pinkish lymph fluid and red blood cells ( fig . 2 ) . marked edematous or hyalinized perivascular stroma and a sprinkling of lymphocytes among tumor cells were characteristic histologic findings ( fig . 3 ) . the luminal large , pale epithelial cells were strongly positive for ck , ck7 , cam5.2 , and ema ( fig . the outer layer of small basaloid cells were strongly positive for p63 and negative for sma , ck , ck7 , cam5.2 , and ema . in addition , many p63+/sma+ myoepithelial cells were present among tubules and sometimes around tubules ( fig . 5 ) . compressed cords of tumor cells were mostly composed of myoepithelial cells although there were scattered abortive tubules which were clearly recognized on ck , ck7 , cam5.2 , and ema immunostaining . based on the results of immunohistochemical findings , we concluded that the tumor was composed of pale epithelial cells , small basal cells , and myoepithelial cells . the tubules were composed of pale epithelial cells and small basal cells which were surrounded by the basement membrane with or without myoepithelial cells . these were clearly recognized on double- and triple - marker analysis ( fig . 5 , 6 ) . s-100+/neurofilament+ compressed nerve fibers were present in the vicinity of the tumor lobules but not in the tumor lobules . formalin - fixed paraffin embedded tissue sections ( 4 to 5 m thick ) were used . the antibody panel for epithelial cells included ck ( immunotech , marseille , france ) , ck7 ( scytec , logan , ut , u.s.a . ) , ck20 ( biomeda , foster city , ca , u.s.a . ) , cam5.2 ( becton - dickinson , san jose , ca , u.s.a . ) , ema ( signet laboratories , dedham , ma , u.s.a . ) , and cea ( immunotech ) . the antibody panel for nerve fibers included neurofilament ( signet ) , and s-100 protein ( signet ) . for characterize myoepithelial cells , we used monoclonal antibodies for sma ( immunotech ) and p63 ( clone 4a4 , oncogene research products , boston , ma , u.s.a . ) . recently , p63 has been proposed as a specific marker of precursor / stem cells ( 7 ) and n - p63 is the predominant p63 isoform preferrentially expressed in the epithelial basal cells of organs such as the skin , breast , prostate and uterine cervix . it is also expressed in ductal structures of skin appendages and is also used as a marker of myoepithelial cells ( 8) . clone 4a4 is a mouse monoclonal antibody obtained from mice hyperimmunized with an aminoterminal fragment of the n - p63 isoform expressed in e. coli as a gst fusion protein . double marker analysis was performed using monoclonal antibody for p63 and sma to differentiate basal cells and myoepithelial cells ( 8) . triple marker analysis using monoclonal antibody for p63 , sma , and ck7 was also performed to clarify three type of the cells es is composed of , namely pale epithelial cells , small basal cells , and myoepithelial cells ( 9 ) . microscopic examination revealed one large well - circumscribed encapsulated lobule and a small satellite lobule involving the dermis and subcutis . unlike usual es , a lobule consisted of peculiarly abundant stroma and compressed cellular cords or sheets that were composed of two types of cells : cells with large pale nuclei in the center and basaloid cells with small , dark nuclei at the periphery ( fig . 2 , 3 ) . the stroma showed greatly dilated vascular spaces containing pale pinkish lymph fluid and red blood cells ( fig . 2 ) . marked edematous or hyalinized perivascular stroma and a sprinkling of lymphocytes among tumor cells were characteristic histologic findings ( fig . the luminal large , pale epithelial cells were strongly positive for ck , ck7 , cam5.2 , and ema ( fig . the outer layer of small basaloid cells were strongly positive for p63 and negative for sma , ck , ck7 , cam5.2 , and ema . in addition , many p63+/sma+ myoepithelial cells were present among tubules and sometimes around tubules ( fig . 5 ) . compressed cords of tumor cells were mostly composed of myoepithelial cells although there were scattered abortive tubules which were clearly recognized on ck , ck7 , cam5.2 , and ema immunostaining . based on the results of immunohistochemical findings , we concluded that the tumor was composed of pale epithelial cells , small basal cells , and myoepithelial cells . the tubules were composed of pale epithelial cells and small basal cells which were surrounded by the basement membrane with or without myoepithelial cells . these were clearly recognized on double- and triple - marker analysis ( fig . 5 , 6 ) . s-100+/neurofilament+ compressed nerve fibers were present in the vicinity of the tumor lobules but not in the tumor lobules . gves , first described by cotton et al . ( 1 ) in 1986 , is a rare variant of es . they report two cases of unusually large es with marked degree of vascularity . both were above 2 cm in size and histologically showed prominent blood - filled vascular spaces . this marked vascularity is an uncommon feature in sweat gland tumors and might suggest that this type of es arise from a highly vascular region of the normal sweat gland ( 1 ) . as far as we know , this is the fifth case of gves in the literature . the clinical features of the previously reported cases of gves are summarized in table 1 ( 1 - 3 ) . all cases of gves , including our case , had made a faulty clinical diagnosis of angiolipoma , angiosarcoma , malignant melanoma , neuroma , sebaceous cyst , or venous thrombosis . it is emphasized that this rare type of es may result in the erroneous diagnosis of angiomatous lesions by both clinicians and pathologists because of the florid vascularity and hemorrhagic features . to clarify the histogenesis of gves , we performed double- and triple - marker analysis . we found numerous p63+/sma+ myoepithelial cells that were mostly arranged as compressing cellular cords or strands around edematous or hyalinized perivascular spaces or among tubules or ducts . tubular structures were mostly composed of two types of epithelial cells , namely inner luminal ( ck+/ck7+/cam5.2 + ) and outer basal cells ( p63+/sma- ) and had basement membrane . normal intradermal and intraepidermal eccrine duct and ductal portion of eccrine secretory coil are lined by two types of epithelial cells , inner luminal cells ( ck+/ck7-/cam5.2- ) and basal cells ( p63+/sma- ) without myoepithelial cells and basement membrane . but secretory portion of eccrine secretory coil is composed of inner luminal cells ( ck+/ck7+/cam5.2 + ) and outer myoepithelial cells ( p63+/sma+ ) with basement membrane . therefore , tubule or ducts seen in gves did not strictly correspond to intradermal eccrine duct and ductal portion of secretory coil . furthermore , findings of numerous myoepithelial cells and cea+/ema+ intercellular canaliculi support that gves differentiates toward secretory portion of eccrine secretory coil ( table 2 , 3 ) . therefore , we conclude that gves is a rare benign tumor originated from eccrine gland and mainly differentiates toward secretory portion of secretory coil .
phosphodiesterases ( pdes ) are isoenzymes that control the level of intracellular cyclic guanosine monophosphate ( cgmp ) and cyclic adenosine monophosphate ( camp ) by hydrolyzing them . pde isoenzyme 5 ( pde-5 ) selectively breaks down the cgmp , a critical smooth muscle tone regulator . nitric oxide ( no ) , produced by nitric oxide synthase , signals the conversion of gmp into cgmp which accumulates inside the cell . inhibition of the pde-5 enzyme increases the available intracellular cgmp which leads to vasodilatation . aside from corpus cavernosum , pde-5 is found on a variety of tissues , including platelets , lungs , muscle , brain , retina , thymus , heart , liver , esophagus , stomach , pancreas , small intestine , arterial and venous vasculature , and endothelial cells . sildenafil , vardenafil , and tadalafil are the three commercially available pde-5 inhibitors ( pde-5is ) . all three pde-5is are available in oral formulation , are rapidly absorbed from the gastrointestinal tract , and are metabolized by hepatic enzymes via cytochrome p450 . sildenafil and vardenafil have similar molecular structures , while the tadalafil molecule is different , the difference being reflected in the pharmacokinetic properties ( figure 1 ) . tadalafil is not affected by food ingestion and has a terminal half - life of 17.5 hours as opposed to sildenafil and vardenafil which are affected by fatty food intake and both have a half - life of approximately 4 hours . the primary food and drug administration- ( fda- ) approved indication for the pde-5is is erectile dysfunction . in recent years , sildenafil ( 2005 ) and tadalafil ( 2009 ) have also been approved for use in pulmonary arterial hypertension . vardenafil was recently shown to improve hemodynamic parameters in patients with pulmonary arterial hypertension in a randomized trial of 66 patients . raynaud 's phenomenon ( rp ) is an exaggerated vasoconstrictive response to cold and stress and is the presenting symptom in the majority of patients with systemic sclerosis ( ssc ) . an important clinical manifestation of the scleroderma - related vasculopathy is the ischemic digital ulcer ( du ) which is associated with significant morbidity . use of pde-5is in ssc - related rp and du makes pathophysiologic sense and has been explored in randomized fashion . as with penicillin or tnf- blockers , the pde-5is history is interesting . the initial intent was to develop pde-5is as a new anti - ischemic therapy , but the early cardiac trials failed to excite any interest . another pde-5i , vardenafil ( levitra ) , came to market in september of 2003 , followed shortly by the weekend each of these individual drugs ' use in ssc - rp and du will be reviewed below . the popular blue pill for erectile dysfunction has been used off - label by rheumatologists for symptomatic improvement of secondary rp and ssc - dus . a retrospective chart review of 10 ssc patients at a single center briefly described the response to sildenafil dosed from 12.5 mg to 100 mg daily . as the letter to the editor reports in 2005 , eight of the ten patients [ ] had a response within few weeks , with significant reduction in the frequency and severity of rp . of the eight patients who had digital ulcers [ ] six experienced complete healing of the ulcers . no other details were provided regarding the specific measures used to quantify the rp improvement . the physiological benefit of sildenafil citrate in patients with ssc - rp was assessed in a group of 5 patients and published as a letter to the editor in 2006 . in this small study , the objective measure of the skin temperature response to mild cold challenge after a single dose of 50 mg of sildenafil citrate was conducted by thermography ( thermal images of the hands were collected every minute for 15 minutes after the cold challenge to enable an area under the curve ) and by percentage recovery to mean baseline temperature postcold challenge . although this letter reported that 3 out of the 5 patients had clear and significant improvement in digital temperature responses to mild cold challenge , no other details were provided . based on this limited information , sildenafil citrate seems to be well tolerated in patients with ssc - rp . a randomized double - blind cross - over trial of single dose of 50 mg sildenafil or -tocopherol ( 100 mg ) was reported in abstract form in 15 patients with rp . the outcome measures reported were 75% improvement in forearm blood flow ( as measured by near - infrared time - resolved spectroscopy ) and 32% increase in serum cgmp concentration ( p < 0.01 ) in the sildenafil group . the study reported no adverse events , aside from a mild decrease in the systolic and diastolic blood pressure ( p < 0.05 ) in the patients exposed to sildenafil . the only published trial that evaluated efficacy of sildenafil in rp is a double - blinded , placebo - controlled , fixed - dose , cross - over study of 50 mg sildenafil twice daily for 4 weeks versus placebo . most subjects had secondary rp ( 16/18 ) , and 6 of the patients with secondary rp had dus . primary outcome variables included rp frequency and duration as assessed by diary cards , raynaud 's condition score ( rcs ) , capillary flow velocity by laser doppler anemometry , and healing of the dus . at the end of the study , there was significant improvement in the frequency ( 35 versus 52 , p = 0.0064 ) and duration ( 581 versus 1046 minutes , p = 0.0038 ) of rp attacks and in the rcs ( 2.2 versus 3.0 , p = 0.0386 ) . after 4 weeks of active therapy with sildenafil , 2 of the 6 subjects with dus completely healed their ulcers , while the rest of the subjects noted visible healing . the mean capillary blood flow velocity increased by more than 400% ( from 0.13 to 0.53 , p = 0.0004 ) during the sildenafil treatment . despite its small size , this trial confidently shows improvement in the peripheral circulation after exposure to sildenafil and improvement of traditional measures of rp . aside from case reports , the effect of sildenafil on ssc - dus was not systematically studied . in 2010 , brueckner et al . reported the results of a pilot open - label study of the effects of maximum tolerated sildenafil in ssc - dus . sixteen patients were treated with a mean sildenafil dose of 114 mg / day for a mean duration of 5.2 months . there was significant improvement ( p < 0.001 ) in the number of dus from baseline ( mean of 3.1/patient ) to the end of sildenafil therapy ( mean of 1.1/patient ) . nine subjects developed a total of 12 new dus while taking sildenafil , of which one of the subjects was diagnosed with calcinosis . based on this small , open label trial , it seems that sildenafil is well tolerated , and it could be a viable option for ssc - du therapy . an important caveat of this study is the lack of uniformity in defining ssc - du ( dus due to calcinosis tend to respond less to vasoactive therapies ) and the absence of a control group . vardenafil is similar to sildenafil in terms of pharmacokinetic properties , with rapid onset of action , maximal benefit at 1 hour , and a half - life of about 4 hours . it is the second pde-5i to make its debut in the erectile dysfunction arena at a dose of 10 mg as needed . published the results of an open - label pilot study of vardenafil ( 10 mg twice daily for 2 weeks ) in patients with rp in 2006 . all the vasoactive medications were discontinued at least one week prior to recruitment , and the outcome measures included rcs , frequency , and duration of rp attacks , and measures of peripheral blood flow by laser doppler 's flowmetry at room temperature and in the cold exposure test room . laser doppler 's flowmetry revealed that 70% ( 28 ) of the subjects had improved digital flow , and , in those individuals , the digital blood flow measured at room temperature increased by a mean of 21% and 30% at 1 hour and 2 weeks compared to baseline at ( p < 0.01 ) . the rcs improved significantly from baseline to the second week of vardenafil therapy ( 5.05 versus 3.54 , p < 0.001 ) . based on this information vardenafil seems to be well tolerated and shows rp improvement . the longer half - life of this pde-5i made tadalafil an attractive option as a daily dose for ssc - related peripheral vasculopathy . a small open - label study of tadalafil ( between 5 and 20 mg every other day as needed ) was published in abstract format in 2005 . of the 15 patients studied , 11 had ssc spectrum of diseases and the rcs was reported to be 3.8 while on tadalafil versus 6.9 without . due to the limited data available in the abstract , no meaningful conclusions can be derived from this study . a physiological study of tadalafil in rp ( mostly primary rp , 18/20 ) was reported in 2007 : this was a double - blind , placebo - controlled cross - over study of 20 patients with rp that received a single dose of 10 mg of tadalafil versus placebo . the hypothesis that tadalafil improves cold - induced vasoconstriction was tested by measuring the digital blood flow with laser doppler flowmetry at rest and during two graduated local heat and cold exposure cycles ; skin blood flow ( flux ) and skin temperature were recorded at baseline and 90 minutes after receiving the drug . tadalafil did not affect the baseline flux ( p = 0.57 ) or skin temperature ( p = 0.69 ) . tadalafil neither increased the maximal flux flow during heating nor decreased the vasoconstriction during cooling , which might mean that tadalafil improves rp through a different mechanism . there is evidence that tadalafil improves peripheral circulation in ssc - rp . a randomized controlled trial of tadalafil ( 20 mg 2 - 3 times / week ) versus pentoxifylline for 4 weeks in men with severe rp associated with autoimmune diseases was reported in abstract form in 2006 . the frequency ( decline of 59% with tadalafil versus 36% with pentoxifylline ) and duration of rp attacks and the rcs were improved in the patients receiving tadalafil . also , physician and patient assessments of rp improved at 4 weeks ( p < 0.05 compared to 2 weeks , and p < 0.05 versus controls ) . our interpretation of this study is limited as no specific information is provided about the number of ssc patients included in this study or about the statistical significance of some of the results . a more recent open - label study of 20 male patients with ssc - rp receiving 10 mg of daily tadalafil for 12 weeks was published in 2009 . the primary endpoint showed improvement in the rcs , the number of rp attacks and a decrease in the plasma adrenomedullin and endothelin-1 levels compared to baseline . in 2009 , schiopu et al . reported a randomized , double - blinded , placebo - controlled , crossover trial of tadalafil at a fixed dose of 20 mg daily versus placebo in 39 women with ssc - rp . the trial design prohibited use of any other vasodilator therapies for rp , required a run - in period to document presence of a minimum of 6 rp attacks per week and excluded smokers . there were not sufficient dus to permit an adequate statistical analysis of the effects on dus . although all measures showed overall improvement , there was no significant difference between the change from baseline rcs , duration , and frequency of attacks among the tadalafil and the placebo groups ( rcs 2.43 versus 2.53 , frequency 2.08 versus 2.1 , duration 40.61 versus 47.0 ) . a year later , shenoy et al . described the results of a single - center , randomized , double - blind cross - over trial of tadalafil at 20 mg on alternate days versus placebo in subjects with ssc - rp which was conducted in india . the trial design , although similar to the previous cross - over trial of tadalafil , included a longer treatment block ( 6 weeks ) and a shorter washout ( 1 week ) , and allowed subjects to continue all the previous rp - specific therapies . when compared to the schiopu et al . trial , the patients recruited needed to have 4 rp attacks / week , as opposed to 6 , and the mean age of the 24 participants was younger ( 36.87 versus 52.9 ) . the primary outcomes were similar : mean change in rcs , duration , and frequency of rp attacks from baseline . the secondary outcomes included assessment of dus , quality of life ( qol ) measures , endothelial function , and flow - mediated dilatation of brachial artery . in this study , the primary outcomes were significantly better in the tadalafil group : frequency ( 2.29 versus 3.37 , p < 0.001 ) , duration ( 33.81 versus 54.89 , p = 0.023 ) , and rcs ( 3.86 versus 5.20 , p < 0.0005 ) . all the 24 digital lesions healed during the tadalafil treatment versus only 3 of the 13 dus during the placebo treatment . the brachial artery reactivity was measured using b - mode ultrasound imaging and improved significantly while subjects received active drug ( p < 0.05 ) . the levels of e - selectin and endothelin 1 were not significantly different between placebo and the active treatment groups ( p = 0.5 and 0.81 , resp . ) . following this above - mentioned single - center trial by shenoy et al , a multicenter randomized , double - blinded , placebo - controlled study of tadalafil at 20 mg every other day versus placebo was conducted in 4 centers from north and northeastern india and reported in an abstract form . similar to shenoy et al . , the mean age of the recruited subjects was lower than the mean age of the subjects in the us tadalafil trial ( 36.8 years in both indian studies versus 52.9 years in the us study ) . this trial recruited 53 subjects , and , after a 2 week run - in period , they were randomized to tadalafil 20 mg every other day or placebo for the 8-week treatment period . trial , improvement in rcs , duration , and frequency of rp attacks was demonstrated ( p < 0.05 , p < 0.001 , p < 0.001 , resp . ) , along with significant ssc - du healing ( 14 out of 18 dus healed in the tadalafil group compared to 5 out of 13 in the placebo group ) . it is likely that the differences in the trial design , specifically the concomitant rp therapies , along with the lower average age ( less disease severity and more reversal changes , consistent with younger age ) could explain the differences in the results of these controlled trials . a significant aspect on which all three studies seem to agree was the excellent tolerability of the oral tadalafil as a daily or every other day therapy for ssc - rp . a phase iia , randomized double - blinded , placebo - controlled , cross - over trial to assess the efficacy of a novel , once daily pde-5i ( pf-00489791 ) for the treatment of primary and ssc - related secondary rp has just finished recruiting . another phase ii / iii clinical trial is comparing daily use of amlodipine ( 10 mg ) versus udenafil daily ( 100 mg ) in secondary rp in a double - blind , randomized , cross - over design ; aside from the rp common outcome measures , the investigators are also assessing dus and arterial flow velocity ( http://www.clinicaltrials.gov/ identifier : nct01280266 ) . the effects of sildenafil in ssc - rp as measured by the microcirculator blood flow , endothelial progenitor cells , and serum levels of vascular endothelium growth factor is ongoing ( http://www.clinicaltrials.gov/ identifier : nct01347008 ) . a multicenter double - blinded , placebo - controlled trial is also currently randomizing participants to either sildenafil ( 20 mg three times a day ) or placebo for 90 days to assess healing of ssc - dus ( http://www.clinicaltrials.gov/ identifier : nct01295736 ) . the ssc - related vasculopathy ( including rp and dus ) lacks fda - approved therapies . the class of pde-5is is a well - tolerated vasoactive therapy for patients with ssc - related vasculopathy . after a surge of clinical case reports , case series and open - label studies published in the mid 2000s , large , multicenter , controlled trials of pde-5is in ssc - related vasculopathy remain underdeveloped . most of the published clinical trials in ssc - rp focus on sildenafil and tadalafil although the one study reported with vardenafil has shown it to be well tolerated with a beneficial impact on rp . currently , the available evidence for the efficacy of pde-5i 's for ssc rp / dus is thin . there are efforts to address the questions of placebo response in rp by designing trials focused on objective vascular markers , qol , and physicians ' and patients ' global assessments . robust clinical evidence that pde-5is , alone or in conjunction with traditional vasoactive therapy , improve ssc - rp and heal ssc - dus is still lacking . the great clinical need for a tolerable , affordable therapeutic option for ssc - rp and ssc - du remains . the clinical research community recognizes pde-5is as an excellent potential option for ssc - related vasculopathy .
determination of mrna half - life is important to our understanding of gene expression and mechanisms involved in the regulation of the level of transcripts in response to environmental changes or developmental cues . in addition , the stability of mrna may determine how rapidly the synthesis of the encoded protein can be shut down after transcription ceases . mrna half - life can be determined by densitometric analysis of in situ hybridization histochemistry or by northern blot analysis of rna samples removed from cells treated with transcriptional inhibitors such as actinomycin d ( actd ) , -amanitin , or 5,6-dichloro-1--d - ribofuranosylbenzimidazole ( drb ) . although reliable , these multi - step methods are laborious and time - consuming . the advent of new technologies such as the real - time pcr allows rapid and exact measurement of copy number of molecules present in the sample . real - time reverse transcriptase pcr ( rt - pcr ) allows precise and reproducible quantitative determination of the number of mrna transcripts synthesized . we have developed a rapid and reliable real - time quantitative rt - pcr approach to determine mrna half - life based on the sybr green i fluorogenic dye ( molecular probes , inc . , eugene , or , usa ) and relative to the amount of total rna per cell samples . to evaluate that approach , actin proteins are components of the microfilament which play a crucial role in maintaining cell shape and motility . expression of -actin has been shown to be relatively constant as cells progress through the cell cycle and has been used as a standard for an unchanging protein and mrna in studies of gene regulation . in this study , we used the human leukemia cell lines ccrf - cem ( t - cell lineage , acute lymphoblastic leukemia ( all ) ) and nalm-6 ( b - cell precursor , all ) that respond differently to antifolate drugs , such as methotrexate ( mtx ) . nalm-6 cells were shown to be more sensitive to mtx when compared to ccrf - cem . the -actin mrna half - life was determined from ccrf - cem and nalm-6 cell lines treated with 0.5 m , 1 m , and 5 m actd ( sigma - genosys , woodlands , tx , usa ) . under these conditions , naml-6 cells were more sensitive than ccrf - cem cells as measured by viable cell counts . it has been shown that actd inhibits cell proliferation by forming a stable complex with single - stranded dna and blocking the movement of rna polymerase that interferes with dna - dependent rna synthesis . based on their respective ic50 for actd , -actin mrna half - life was evaluated from nalm-6 cells treated with 0.5 m and 1 m actd , and from ccrf - cem cells treated with 1 m and 5 m actd . twenty - four h before treatment , the cells were transferred to a tissue culture flask at a concentration of 6 10 cells / ml , and aliquots of 3 10 cells were collected every 2 or 4 h for a period of 8 to 24 h. total rna was isolated and its concentration determined as described in materials and methods . for each sample , the amount of -actin mrna was quantified relative to 1 g of total rna by real - time rt - pcr . first - strand cdna was synthesized using 1 g of total rna ( dnase - treated ) and a region of the -actin mrna was amplified using primers ba67 and ba68 . serial ten - fold dilutions ( 10 to 10 molecules ) of pbactin-231 were used as a reference molecule for the standard curve calculation ( figure 2 ) . all real - time pcr quantitations were performed using the bio - rad icycler iq system ( biorad , hercules , ca , usa ) . a representative example of rt - pcr amplification plots is shown in figure 1 . a fluorescence threshold value ( ct ) ranged from 0.988 to 0.995 indicating a high degree of confidence for the measurement of the copy number of molecules in the samples . as shown in figure 2 , the number of -actin mrna molecules ranged between 10 to 10 molecules/l of cdna for most of the cdna preparations . amounts of -actin mrna molecules were calculated for each time - point and plotted as a function of time ( figure 3 ) . in nalm-6 cells , the -actin mrna exhibited a half - life of 6.6 h. this result is comparable to the previously reported published value of approximately 5.5 h using the traditional northern blot procedure . a significant higher half - life value of ~13.5 h was obtained with the ccrf - cem cells . it is interesting to observe a two - fold difference for -actin mrna half - life between these two cell lines . this difference can not be explained by the concentrations of the actd used to block transcription because similar half - life values were obtained with different actd concentrations ( see figure 3 ) . however , since the expression of the -actin gene is regulated as a function of the cell cycle with transcription in the g1 phase and mrna decay in g2 phase , it is possible that this difference in -actin mrna half - life could be explained by their respective cellular growth rate . indeed , ccrf - cem and nalm-6 cells exhibited different cellular growth rate with generation times of 24 and 36 h , respectively . taken together , this suggests that cells growing at a slower rate such as nalm-6 would process the -actin mrna at a faster rate in order to respond to its cytoskeleton requirement . representative rt - pcr plot resulting from the amplification of -actin cdna templates ( 1/20 volume ) synthesized from 1 g of total rna ( see materials and methods for details ) . for each time point , the cdna was generated and quantified from three independent experiments and run in triplicates . the calculated cycle threshold ( ct = 99 ) provides an arbitrary cut off point at which a ct value is assigned for each sample . standard curve showing amplification efficiencies of ten - fold serial dilutions of the pbactin-231 template . the calculated ct values were plotted versus the log of the initial amount of pbactin-231 molecules ( 10 to 10 ) to generate the standard curve . the ccrf - cem cells were treated with 5 m ( squares ) or 1 m ( circles ) act - d for various times to block mrna synthesis . similarly , the nalm6 cells were treated with 1 m ( lozenges ) or 0.5 m ( triangles ) act - d . total rna , cdna , and real - time pcr amplification were performed as described in the text . the values represent the mean ( ) the standard deviation ( sd ) of the -actin rna copy number per g of total rna from three independent experiments . the inset shows the calculated half - life for the -actin mrna in each cell line for each treatment . in summary , we described an alternative method using real - time rt - pcr to determine the rate of mrna degradation by accurately measuring the number of mrna molecules relative to total rna . using this approach , we obtained a values for the -actin mrna half - life in nalm-6 cells that are comparable to those estimated from northern blot studies using normal human leukocytes . therefore , real - time rt - pcr is a reliable method for measuring mrna half - life . because of its sensitivity , half - life of mrnas expressed at very low level can be determined in cases in which northern blots may not be sensitive enough . the length of the amplified fragment is important to determine the mrna half - life because incomplete or degraded mrna can interfere with the measurement of the actual mrna half - life . ideally , full - length cdna molecules should be amplified to ensure integrity and identity of the mrna species . the use of the fluorogenic sybr green i dye limits the length of the amplified product ( cdna recommended to be less than 200 bp ) . however , the use of taqman ( applied biosystems , foster city , ca , usa ) , molecular beacons ( molecular probes , inc . , eugene , or , usa ) , or fluorescence resonance energy transfer ( fret ) probes ( roche molecular biochemicals , indianapolis , in , usa ) could overcome this limitation and allow amplification of longer pcr products . in addition , since multiplex real - time rt - pcr can be achieved in the same reaction tube using different fluorogenic dyes , this method could be modified to simultaneously estimate mrna half - life of several genes . thus , our approach represents a rapid and sensitive assay to determine mrna half - life . the human leukemia cell lines ccrf - cem ( t - cell , all ) and nalm-6 ( b - cell precursor , all ) were obtained from the american type culture collection ( atcc , rockville , md , usa ) and dsmz ( braunschweig , germany ) , respectively . both cell lines were grown in rpmi 1640 ( sigma - genosys , woodlands , tx , usa ) supplemented with 10% fetal bovine serum at 37c under a 5% co2 atmosphere . valencia , ca , usa ) and its concentration determined using the ribogreen fluorescent dye ( molecular probes , inc . , eugene , or , usa ) with the versafluor fluorometer system ( biorad , hercules , ca , usa ) . quality and integrity of total rna was assessed on 1% formaldehyde - agarose gels . first - strand cdna was synthesized using 1 g of total rna ( dnase - treated ) in a 20 l reverse transcriptase reaction mixture as described by leclerc and barredo . a region of the -actin mrna was amplified using primers ba67 ( 5'-gcgggaaatcgtgcgtgacatt ) and ba68 ( 5'-gatggagttgaa ggtagtttcgtg ) , as described by lenz et al . . the cdna amplified fragment ( 231 bp ) was cloned into the pcr2.1-topo vector ( invitrogen , carlsbad , ca , usa ) to generate the plasmid pbactin-231 ( 4139 bp ) . serial ten - fold dilutions ( 10 to 10 molecules ) of pbactin-231 were used as a reference molecule for the standard curve calculation ( figure 2 ) . all real - time pcr reactions were performed in a 25 l mixture containing 1/20 volume of cdna preparation ( 1 l ) , 1x sybr green buffer ( pe applied biosystems , foster city , ca , usa ) , 4 mm mgcl2 , 0.2 m of each primers ( ba67 and ba68 ) , 0.2 mm dntps mix and 0.025 unit of amplitaq gold thermostable dna polymerase ( applied biosystems , foster city , ca , usa ) . real - time quantitations were performed using the bio - rad icycler iq system ( biorad , hercules , ca , usa ) . the human leukemia cell lines ccrf - cem ( t - cell , all ) and nalm-6 ( b - cell precursor , all ) were obtained from the american type culture collection ( atcc , rockville , md , usa ) and dsmz ( braunschweig , germany ) , respectively . both cell lines were grown in rpmi 1640 ( sigma - genosys , woodlands , tx , usa ) supplemented with 10% fetal bovine serum at 37c under a 5% co2 atmosphere . valencia , ca , usa ) and its concentration determined using the ribogreen fluorescent dye ( molecular probes , inc . , eugene , or , usa ) with the versafluor fluorometer system ( biorad , hercules , ca , usa ) . quality and integrity of total rna was assessed on 1% formaldehyde - agarose gels . first - strand cdna was synthesized using 1 g of total rna ( dnase - treated ) in a 20 l reverse transcriptase reaction mixture as described by leclerc and barredo . a region of the -actin mrna was amplified using primers ba67 ( 5'-gcgggaaatcgtgcgtgacatt ) and ba68 ( 5'-gatggagttgaa ggtagtttcgtg ) , as described by lenz et al . . the cdna amplified fragment ( 231 bp ) was cloned into the pcr2.1-topo vector ( invitrogen , carlsbad , ca , usa ) to generate the plasmid pbactin-231 ( 4139 bp ) . serial ten - fold dilutions ( 10 to 10 molecules ) of pbactin-231 were used as a reference molecule for the standard curve calculation ( figure 2 ) . all real - time pcr reactions were performed in a 25 l mixture containing 1/20 volume of cdna preparation ( 1 l ) , 1x sybr green buffer ( pe applied biosystems , foster city , ca , usa ) , 4 mm mgcl2 , 0.2 m of each primers ( ba67 and ba68 ) , 0.2 mm dntps mix and 0.025 unit of amplitaq gold thermostable dna polymerase ( applied biosystems , foster city , ca , usa ) . real - time quantitations were performed using the bio - rad icycler iq system ( biorad , hercules , ca , usa ) . the authors would like to thank florence b. leclerc and sanja altman - hamamdzic for helpful discussion , and the medical university of south carolina , biotechnology resource laboratory , charleston , sc , for their assistance with dna sequence analysis . this work was supported in part by research grant nih / nci ca72734 ( to j. c. b. ) and the jolly foundation award ( to g. j. l. and j. c. b. ) and by the monica kreber golf classic , charleston , sc .
every anesthesiologist worth their salt is guilty of administering a wrong drug at least once in their career . most of the time the consequences have been harmless ( albeit not without feeling of guilt or remorse ) , but in some cases they have caused an undesired iatrogenic morbidity and/or mortality . the high duress milieu of an operation theatre ( ot ) , intensive care unit ( icu ) or emergency room ( er ) predisposes flawed actions . pediatric population in ot , icu , or er is at considerable hazard for medication blunders . once injected into the blood stream , oxford english dictionary defines error as something incorrectly done through ignorance or inadvertence ; a mistake , e.g. , in calculation , judgment , speech , writing , action , etc . kohn described it as a failure to complete a planned action as intended , or the use of an incorrect plan of action to achieve a given aim . a medication error can be defined as a failure in the treatment process that leads to , or has the potential to lead to , harm to the patient . failure here implies that the method used was beneath the usual realistic standards being practiced . while includes management of symptoms or their causes or investigation or prevention of disease or physiological changes . the aviation industry has adopted a definitive safety culture , whereas anesthesia professionals exhibit an attitudinal barrier to safety . both accidents and incidents in the aviation industry are reviewed and the risks are assessed , providing an opportunity for safety improvement and risk mitigation . accidents during periods of anesthesia are often not reported due to the fear of being blamed for carelessness , forgetfulness and sometimes character weakness . the institute for safe medication practices uses an index for categorizing medication errors written by the national coordinating council for medication error reporting and prevention . stelfox et al . have reported that medication errors are the seventh most common cause of death in the health care system . a canadian survey from 687 anesthesiologists ( 30% response rate ) revealed that 85% of the participants had experienced at least one drug error or near miss . although most errors ( 1,038 ) were of minor consequence ( 98% ) , four deaths were reported . the most common incident involved the administration of muscle relaxants instead of a reversal agent . syringe swaps ( 70.4% ) and the misidentification of the label ( 46.8% ) were common contributing factors . most of the time , although the label color was an important secondary cue . about 84% agreed that improved standards for drug labels would reduce the incidence of error . in norway , drug error was recorded in 63 cases ( 0.11% ) in anesthesia - related information from all anesthetic cases for 36 months totaling 55,426 procedures . there were 28 syringe swaps , nine ampoule swaps , 15 instances where muscle relaxants were erroneously given , eight wrong drug cases and 18 instances where a wrong dose of the correct drug was administered . another study done in australia identified 144 incidents in which the wrong drug was nearly or actually administered to a patient . of these errors , the most common was actually giving the wrong drug from a correctly labeled syringe . interestingly , they found that communication failure was a significant factor in syringe incidents when two or more staff was involved . in a critical care related study by rothschild et al . , 120 adverse events were identified in 79 patients , including 66 ( 55% ) non - preventable and 54 ( 45% ) preventable adverse events . most serious medical errors occurred during the ordering or execution of treatments , especially in administering medications ( 61% ) . of 5744 observations in 851 patients , calabrese et al . found 187 ( 3.3% ) medication administration errors . therapeutic classes most commonly associated with those errors were vasoactive drugs 61 ( 32.6% ) and sedative / analgesics 48 ( 25.7% ) . we believe the key instigator of erroneous drug administration ( eda ) is the adaptation of the universal leur locking mechanism to intravenous drug delivery systems . all syringe ports on the fluid delivery system are able to interlock with any syringe nozzle by nature of the leur design . this inherently provides the opportunity for an adverse event to occur especially in a situation of high duress . the development of a fluid intake manifold used for multiple intravenous drug delivery featuring specially designed syringe ports , which can only interlock with a predispositioned syringe . an acronym which stands for vassopressors , emergency drugs , induction agents , reversal agents , opioids , and miscellaneous drugs . these seven categories encompass most of the intravenous drugs that are used frequently in anesthesia . the envisioned fluid delivery system , which we named veinrom , shall harbor one syringe port for each of the seven drug class categories that are most commonly used drugs in anesthesiology and critical care . veinrom is a unique drug delivery manifold and syringe assembly which has incorporated mechanical and electronic mechanisms that will make it very difficult to administer wrong drugs intravenously . these defense mechanisms are : veinrom manifold : the fluid intake manifold shall have seven differently designed syringe ports which feature a lock - and - key interaction between the port and designated syringe . through the improvised veinrom lock - and - key mechanism , it is impossible to incorrectly administer one category of drug into any one of the other six ports . incompatible syringes will not be able to enter the manifold ports , and thus the drug administrator will not be able to inject the drug [ figures 1 and 2 ] . figure 1ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) figure 2ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines veinrom syringes : preloaded syringes will further decrease the potential for human error when administering drugs instead of loading - labeling them perioperatively . the veinrom syringes shall have following features specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3].color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . this feature promotes visual memory and obliterates the need to manually label the syringes.texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3].scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3].inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . not only does this improve patient data logging practices , it implements practitioner accountability . veinrom manifold : the fluid intake manifold shall have seven differently designed syringe ports which feature a lock - and - key interaction between the port and designated syringe . through the improvised veinrom lock - and - key mechanism , it is impossible to incorrectly administer one category of drug into any one of the other six ports . incompatible syringes will not be able to enter the manifold ports , and thus the drug administrator will not be able to inject the drug [ figures 1 and 2 ] . figure 1ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) figure 2ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines veinrom syringes : preloaded syringes will further decrease the potential for human error when administering drugs instead of loading - labeling them perioperatively . the veinrom syringes shall have following features specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3].color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . this feature promotes visual memory and obliterates the need to manually label the syringes.texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3].scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3].inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . not only does this improve patient data logging practices , it implements practitioner accountability . specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 503 , 603 are distinctive locking mechanisms for specified ports on the manifold . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 503 , 603 are distinctive locking mechanisms for specified ports on the manifold . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3 ] . color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3 ] . scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3 ] . inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . we believe the key instigator of erroneous drug administration ( eda ) is the adaptation of the universal leur locking mechanism to intravenous drug delivery systems . all syringe ports on the fluid delivery system are able to interlock with any syringe nozzle by nature of the leur design . this inherently provides the opportunity for an adverse event to occur especially in a situation of high duress . the development of a fluid intake manifold used for multiple intravenous drug delivery featuring specially designed syringe ports , which can only interlock with a predispositioned syringe . an acronym which stands for vassopressors , emergency drugs , induction agents , reversal agents , opioids , and miscellaneous drugs . these seven categories encompass most of the intravenous drugs that are used frequently in anesthesia . the envisioned fluid delivery system , which we named veinrom , shall harbor one syringe port for each of the seven drug class categories that are most commonly used drugs in anesthesiology and critical care . veinrom is a unique drug delivery manifold and syringe assembly which has incorporated mechanical and electronic mechanisms that will make it very difficult to administer wrong drugs intravenously . these defense mechanisms are : veinrom manifold : the fluid intake manifold shall have seven differently designed syringe ports which feature a lock - and - key interaction between the port and designated syringe . through the improvised veinrom lock - and - key mechanism , it is impossible to incorrectly administer one category of drug into any one of the other six ports . incompatible syringes will not be able to enter the manifold ports , and thus the drug administrator will not be able to inject the drug [ figures 1 and 2 ] . figure 1ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) figure 2ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines veinrom syringes : preloaded syringes will further decrease the potential for human error when administering drugs instead of loading - labeling them perioperatively . the veinrom syringes shall have following features specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 503 , 603 are distinctive locking mechanisms for specified ports on the manifold . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3].color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . this feature promotes visual memory and obliterates the need to manually label the syringes.texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3].scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3].inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . not only does this improve patient data logging practices , it implements practitioner accountability . veinrom manifold : the fluid intake manifold shall have seven differently designed syringe ports which feature a lock - and - key interaction between the port and designated syringe . through the improvised veinrom lock - and - key mechanism , it is impossible to incorrectly administer one category of drug into any one of the other six ports . incompatible syringes will not be able to enter the manifold ports , and thus the drug administrator will not be able to inject the drug [ figures 1 and 2 ] . figure 1ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) figure 2ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines ariel and lateral view of the dome shaped vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 210 a , b , and c are the port for miscellaneous drugs ( this will retain their universal port characteristics ) ariel and side view of the liner vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous manifold . 111 - 112 is for connection to the intravenous lines veinrom syringes : preloaded syringes will further decrease the potential for human error when administering drugs instead of loading - labeling them perioperatively . the veinrom syringes shall have following features specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 503 , 603 are distinctive locking mechanisms for specified ports on the manifold . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3].color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . this feature promotes visual memory and obliterates the need to manually label the syringes.texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3].scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3].inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . not only does this improve patient data logging practices , it implements practitioner accountability . specific male ports : each syringe shall have uniquely designed tips that can only mate with their destined manifold ports [ figure 3 ] . figure 3vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors vassopressors , emergency drugs , induction agents , reversal agents , opioids and miscellaneous syringes prototypes with its unique physiognomies . 504 , 604 are the special texture , 506 and 606 are scan - able bar codes . each syringe will have american society for testing and materials specified colors engraved labels : acting as a visual reinforcement to the user , each syringe shall display what category it belongs to by being boldly engraved on to the syringe body , obviating the need for colored sticker labeling [ figure 3 ] . color coded : syringe pistons and bodies for each drug class shall be color coded per american society for testing and materials standards . texture coded : each syringe class shall have a specific external texture embedded within the syringe body , producing tactile stimuli which generates neurogenic memory rendering identification of syringes easier [ figure 3 ] . scan able bar codes : each syringe shall have a barcode at its distal end that identifies drug class and lot number . before delivery , the drug administrator swipes the syringe in front of a barcode scanner which in turn enters the drug information and delivery time into patient specific electronic medical records [ figure 3 ] . inherent electronic data collection : veinrom shall incorporate a medical electrical system designed to identify and register the connection of a syringe to any port , thereby logging the port i d and time of delivery into the patient 's medical records . as indicated previously , medical errors are prevalent within this field and current safety protocol has not been changed in over 60 years . not only will the implementation of a device like veinrom increase practitioner 's accountability , update patient records in real time and improve the overall health care system , it will most importantly save lives . it is an obligation for standards committee members and medical device manufacturers to implement safeguards that prevent human error . the institute of medicine estimates that at least 1.5 million americans are injured each year as a result of eda , costing the us healthcare field more than 3.5 billion usd annually . the global health care system is in the process of implementing improved standards and regulations that require syringes to be pre - filled by outside pharmacies rather than medical practitioners during the pre - operation period . to support this claim , transparency market research estimates that the global pre - filled syringe market will grow by a 13.3% compound annual rate , reaching a market value of 4.98 billion usd by the year 2019 . these trends point to an estimated 3 billion usd in profit opportunity within the next 7 years . it is our moral and hippocratic duty to continue risk management processes that decrease the probability of iatrogenic morbidities . for a device such as veinrom , veinrom is the next step in making the art of anesthesia safer for all involved .
gene therapy is defined as the transfer of nucleic acid molecules ( usually dna ) to a patients somatic cells in order to prevent , treat , or alleviate a specific condition . different gene therapy strategies have been designed to suit different types of diseases , the most classical of which involves gene delivery to target cells in order to obtain optimal expression of the gene introduced . this therapeutic approach is particularly well suited for inherited diseases that are caused by recessive mutations , since these are typically associated with the absence of a functional gene product or the drastic decrease in the expression of a gene . in these cases , the therapeutic gene must be inserted within a dna molecule ( usually a bacterial plasmid ) along with all its essential regulatory sequences in order to ensure the correct expression of the gene in the target cells . to facilitate the adequate cellular uptake of dna molecules viral vectors have become the preferred gene delivery vehicles in the field of gene therapy due to their extremely high efficiency of gene transfer in somatic cells . however , viral vectors fail to fulfil all the requirements for an optimal gene therapy vector . ideally , the best gene therapy protocol would involve a single administration of the therapeutic gene to the organism , such that it could replicate and segregate alongside the endogenous chromosomes , if necessary , thereby permitting long - term maintenance in any dividing cell . however , most viral vectors are either nonintegrating episomes that are unable to replicate , segregate , and persist in dividing cells , or they are integrating vectors . these latter vectors insert into the chromosomes of the host cell , and although they can therefore persist in proliferating cells , they carry the risk of producing insertional mutagenesis . these viruses can be used as nonintegrating vectors since their genome is maintained as an episome once it is released into the nucleus of the host cell . however , these episomes are lost after several cell divisions . moreover , the first - generation adenoviral vectors elicited a strong immune response that led to elimination of the transduced cells , and the humoral response excluded them after future administrations [ 1 , 2 ] . to diminish or overcome this reaction , multiple deletions have been introduced into the subsequent generations of high - capacity gutless adenoviral vectors [ 4 , 5 ] . nevertheless , while these vectors can now infect a wide range of cells , their lack of persistence in dividing cells represents an important weakness . retroviral - based vectors are capable of transducing a wide variety of dividing cells and they are efficiently integrated in the genome of the host cells . however , this latter characteristic represents their major disadvantage since random integration may cause insertional mutagenesis , and the ensuing activation and/or silencing of undesirable genes might even trigger oncogenic transformation in the host cells [ 7 , 8 ] . indeed , some patients treated with retroviral vectors in a clinical trial for severe combined immunodeficiency ( scid ) developed leukemia as a consequence of vector integration . while lentiviruses belong to the retrovirus family , they are able to transduce both proliferating and non - proliferating cells , augmenting the range of target cells [ 10 , 11 ] . these viruses are currently the most widely used gene therapy vectors , as they sometimes permit prolonged gene expression , although gradually silencing of gene expression has also been reported on occasion . finally , most viral vectors have a limited packaging capacity and they can not accommodate large genes . for this reason , most constructs used for gene therapy are minigenes , consisting of cdna sequences ( instead of entire genes ) under the control of heterologous promoters ( usually small compacted promoter sequences of viral origin ) . minigene constructs often exhibit variable expression or lack tissue specificity , and they may not be properly regulated in somatic cells , being completely silenced in many cases , which obviously restricts their usefulness in gene therapy . indeed , studies performed in transgenic mice have demonstrated that these minigene constructs are very susceptible to being switched off by neighbouring chromatin . on the other hand , there are cases where the cdna is too big to be contained in one of these vector systems and some nonessential regions have to be eliminated . by contrast , studies performed in transgenic mice have highlighted the advantages of using large fragments of genomic dna that permit the delivery of intact mammalian genes with all their introns , promoters , enhancers , and long - range controlling elements . by using gene 's normal promoter and controlling elements , the level and control of expression is comparable to the endogenous expression of the gene . the experience gained from using yeast artificial chromosomes ( yacs ) and bacterial artificial chromosomes ( bacs ) has shown that large genomic fragments drive tissue - specific expression at endogenous levels [ 1520 ] . for example , transgenic mice carrying the intact human cystic fibrosis transmembrane regulator gene ( cftr ) , which spans 200 kb of a 300 kb yac , express cftr in an appropriate tissue specific manner and complement the cftr defect in null mice . similar physiological expression has also been achieved with the huntingtin gene , which has been difficult to express from minigene constructs . likewise , the use of a yac or bac carrying the entire frataxin gene has rescued frataxin knock - out mice from embryonic lethality [ 16 , 19 ] . in view of these data , large dna molecules seem to be better candidates as vectors for gene therapy than the typical viral vectors , with their limited capacity . as a result of the human genome project , bacs are available that cover the whole genome . given that the average size of a bac is approximately 150 kb , most genes , together with all their regulatory elements , can be included in such a construction . a system for linking overlapping bacs by homologous recombination in e. coli has been described for when a bac containing a whole locus is not available , or if new regions need to be added upstream or downstream of a given gene within a bac [ 2325 ] . also recombination has been used to modify bacs with different tags , which is very useful in studies of protein - protein or dna - protein interactions , and of protein localization . we will focus on recent advances on the use of bacs containing genomic loci as a platform for the design of artificial minichromosome - like vectors for gene therapy applications , and on the delivery vehicles that can accommodate such large dna molecules . we will also analyse two examples of applications of these artificial minichromosome - like vectors for rare genetic diseases ( haemophilia a and friedreich 's ataxia ) . an obvious vector to stably carry large genomic dna fragments would be an artificial minichromosome that must have a replication origin and a centromere , enabling the dna molecule to replicate during s phase and to correctly segregate during mitosis . in addition , if the dna molecule is lineal telomeres must be present at both ends to guarantee its stability . the centromere is the most important chromosomal element since it is crucial for the attachment of chromosomes to the mitotic spindle and their correct segregation in mitosis . centromeric dna binds the cenp proteins that form the scaffold for the kinetochore , the structure connecting the centromere to the spindle microtubules . artificial chromosomes have been built in yeast without any difficulty , since yeast chromosomes contain small centromeres . however , mammalian centromeres are very large and extraordinarily difficult to engineer , which has complicated the construction of mammalian artificial chromosomes ( macs ) or human artificial chromosomes ( hacs ) . all normal human centromeres contain large arrays of alphoid satellite dna ( -dna ) , a family of repetitive dna sequences first discovered in the african green monkey . satellite dna corresponds to only a small fraction of the human genome ( less than 2% ) whereas in other species like the rat , it is much more abundant . human -dna consists of tandem repeats of a 171 bp monomer arranged in highly ordered repeats [ 30 , 31 ] . the divergent intermonomeric sequences lying between the independent monomers vary from 20% to 40% whereas the sequence divergence is around 5% in the higher order repeats . despite this monomeric variation , each human centromere is thought to be formed by arrays of a unique monomer spanning from several kilobases up to megabases [ 33 , 34 ] . several attempts to generate artificial chromosomes have used alphoid dna arrays from different chromosomes ( x , y , 17 , and 21 ) , although not all have succeeded . alphoid dna from chromosome y has failed to form active centromeres in some cases [ 35 , 36 ] , probably because of the lack of cenp - b boxes . masumoto and collaborators have also showed how two different alphoid dna loci from the same chromosome behave in a completely opposite manner . they found that alphoid - derived yac unable to form hacs do not have cenp - b boxes in addition to a more diverged alphoid repeats . in terms of specific dna sequences , alphoid dna may not be the only element needed for centromeres to fulfil their function , implicating additional mechanisms . this view is supported by the lack of a consensus sequence for -dna , the existence of dicentric chromosomes with two identical regions of -dna but only one active centromere [ 4042 ] , or the fact that functional neocentromeres can be formed from noncentromeric dna . however , it is clear that no dna other than alphoid dna can efficiently form a centromere after transfection into human cells . it has been already mention that not all alphoid dnas can form active centromeres with the same efficiency and it appears that functional centromeres required a minimum size , estimated to be around 100 kb . lo and collaborators analysed all chromosomes in order to determine the minimum length of alphoid dna tolerated by chromosomes and still able to seed de novo centromere formation . in general , chromosomes can not bear an important reduction in the length of alphoid dna array , with the exception of chromosome 21 whose alphoid dna could oscillate between 50 and 100 kb , with an average size of 78 kb . despite all this evidence , there are several reports where introducing different arrays of alphoid dna does indeed lead to centromere formation . several studies have shown how when large ( > 50 kb ) arrays of alphoid dna are transfected into a human cell line ( ht1080 ) in culture , de novo chromosomes may form with a functional centromere [ 35 , 46 , 47 ] . nevertheless , much work is still needed in order to elucidate the mechanisms underlying de novo centromere formation . the bottom - up approach consists of assembling the elements needed for hac formation in a vector and with this aim , a universal system to introduce alphoid dna into any bac using in vivo recombineering has been design , containing a 70 kb array of this alphoid dna proven to be capable of forming de novo macs [ 38 , 46 ] . with the second strategy or top - down approach an existing chromosome is manipulated in order to reduce its size , eliminating unneeded elements except for the centromere region . in the first studies carried out on the formation of centromeres and de novo human artificial chromosomes ( hacs ) , synthetic alpha satellite arrays were cotransfected with telomeric dna and other genomic sequences to obtain linear minichromosomes of 6 to 10 mb in size . due to the already mention sequence divergence between alphoid dna , arrays from 2 different chromosomes were used to obtain 4 minichromosomes - containing cell lines , two with alphoid dna from the y chromosome and the other two with alphoid dna from chromosome 17 . however , three of these minichromosomes had acquired dna from other centromeres or neo - centromeres , and only one of them contained alphoid dna exclusively from the transfected -dna . the hacs were cytogenetically stable , in terms of the presence and absence of selection , and their copy number or size did not change over time . only one year later , yac - based mammalian artificial chromosomes ( macs ) were generated , yeast artificial chromosomes having been widely used to introduce mammalian genes into mice as they can harbour large dna fragments [ 15 , 50 , 51 ] . in these studies , a recombination deficient yeast host was used in which alphoid arrays from chromosome 21 ( 21-i and 21-ii ) , telomere sequences and selectable markers were introduced , and the modified yac was transfected into ht1080 cells . the results obtained showed some variability regarding the proportion of cell lines containing minichromosomes ( from 18% to 68% ) , although the number of macs per cell line was most frequently one . the macs obtained segregated accurately , they bound centromeric proteins , and they were about 15 mb in size . in addition , they did not gain host dna and presented a loss rate of less than 1% per generation in the absence of selection . a further advance came when it was demonstrated that no telomere sequences are needed when the input dna is circular . accordingly , it was established that macs can be formed in ht1080 cells from either linear or circular input dna , with circular alphoid constructs being very effective at forming macs , whether or not human telomere sequences are added . by contrast , it was ascertained how linear dna molecules require telomere sequences to maintain and protect minichromosome ends . when considering the de novo formation of hacs , an important inconvenience is the fact that these structures are not stable in murine cell lines , making it difficult to perform studies in mice as a model system . when a previously established hac was transferred into several murine cell lines ( rag , sto and la-9 ) , the hac dna rearranged and was different to the original hac . however , murine dna was not acquired and no integration events were observed . when selection was withdrawn , the artificial chromosomes were rapidly lost at a rate between 2%5% per day , depending on the cell line . these results show how centromere activity is retained to some extent , as they were still able to bind a marker of active centromere , yet as pointed out above , they also suggested that something else is needed to provide correct segregation . the next advance in the development of human artificial chromosomes was probably the demonstration that any gene cloned into those constructs could be expressed normally , as addressed in several reports . there are two studies where the entire human hprt gene was expressed from such mammalian artificial chromosomes ( macs ) [ 53 , 54 ] , using slightly different strategies but with the same results . a pac containing about 140 kb of genomic dna , including the human hprt gene , and a second pac including a 70 kb array of -dna derived from chromosome 21 ( 21-i ) were transfected into hprt - deficient ht1080 cells . the results obtained showed how two of the three mac - containing cell lines generated were mitotically stable and that hprt expression was maintained over long periods of time , even in the absence of selection . in a second study , a 404 kb hac made by in vivo recombination and introduced into hprt - deficient ht1080 cells successfully ensured expression of the gene after two months without any selection . more recently , a 21-derived minichromosome made in dt-40 chicken cells using the top - down approach was introduced into human ht1080 cells by microcell - mediated chromosome transfer to check its stability . subsequently , the erythropoietin gene was added by cre - loxp recombination and transgene expression and vector stability was finally tested in hfl-1 cells . accordingly , the vector was mainly maintained as a single copy per cell with no translocation and/or insertion events , and epo expression was sustained over 12 weeks without any loss in the absence of selection . using the same strategy , the dystrophin gene ( about 2.4 mb in length ) has been cloned into a hac , a construction that enabled all the tissue - specific isoforms of human dystrophin gene to be detected , overcoming the problem shared by other vector systems that only produce one of the isoforms . one year later , the same group used the dystrophin hac to correct patient - derived fibroblasts and then transform the corrected cells into human ips ( induced pluripotent stem ) cells . this is a great achievement as probably autologous - corrected cells would be the best candidate cells to be used in gene therapy protocols . it seems that the size and/or structure of the hac matters since artificial chromosomes have a higher rate of missegregation than normal chromosomes [ 60 , 61 ] . it is also notable that different hacs behave in a different way in different cell lines , suggesting that centromere activity could probably be maintained by trans - acting factors with different specificities between different cell lines , even within the same species . a better understanding of centromere function seems crucial in order to be able to generate centromeres , which should improve the development of macs . thus , despite some of the advances and successes , the engineering of hacs / macs to carry genes of interest is evidently quite difficult , which has restricted their use as vectors for gene therapy . in view of the difficulties associated with macs , other alternative vectors have been developed to carry large genomic constructs . among them , stable non - integrating and replicating episomes have emerged as very promising gene therapy vectors for dividing cells . interestingly , these stable episomes do not become integrated within host cell chromosomes and are therefore not associated with any risk of insertional mutagenesis . perhaps the best characterized artificial minichromosome - like episomes of this type are the so called orip / ebna1 vectors . these vectors are bacterial plasmids or bacterial artificial chromosomes in which episomal replication and segregation are achieved through dna sequences derived from the epstein - barr virus ( ebv ) . this herpes virus has a genome consisting of dsdna of approximately 172 kb and it is characterized by a latent phase during which it is maintained for life asymptomatically in lymphocytes of about 90% of the human population . in the latent stage , the viral genome is maintained as a circular extrachromosomal replicating episome within the nucleus of infected cells . the only viral elements needed to confer replication and segregation on this episome are the latent origin of replication , orip , and the viral gene encoding the ebna1 protein [ 6466 ] . the origin of plasmid replication ( orip ) from epstein - barr virus is a cis - acting element which has been proven to confer replication autonomy and maintenance to recombinant plasmids in cells harbouring latent ebv . orip is formed by two elements about 1 kb apart : the family of repeats ( fr ) , a 20 member family of 30 bp direct repeats , and the dyad symmetry ( ds ) element , a 65 bp ds containing four copies of the repeat [ 67 , 68 ] . orip resides within a 1.8 kb segment in the short unique region of the ebv genome , and it appears to be the only element needed in cis [ 65 , 68 ] . the trans - acting gene allowing orip function lies in a 2.6 kb region encoding the epstein - barr nuclear antigen 1 ( ebna-1 ) . the ebna-1 protein can bind to both the fr and ds sequences , whereby the ds element is the initiation site of episomal dna replication whereas fr acts as a replication fork barrier and termination site . ebna-1 also activates the 30 bp repeats of orip in trans , the latter acting as a transcriptional enhancer for genes linked to the repeat . plasmids containing these two elements ( orip and ebna-1 ) are replicated once per cell cycle and they segregate passively by attachment to the mitotic chromosomes [ 72 , 73 ] . such plasmids are maintained in human cell lines in tissue culture [ 63 , 66 ] , as well as in mouse cell lines if the plasmid contains large fragments of genomic dna that permit replication . although standard ebv - derived vectors usually carry both orip and ebna-1 , it is well established that vectors containing only the orip fr and ebna-1 as well as inserts of genomic dna greater than 100 kb can be replicated and maintained as episomes , and can thus be used for studies in human and rodent cells . as the segregation of episomes is passive , by binding to endogeneous chromosomes instead of by direct attachment to the microtubules of the mitotic spindle , selection is sometimes required to maintain a population of cells that carry the episomes ( especially in cases of a low copy number of episomes per cell ) . the typical structure of orip / ebna1 vectors and their localization in the interphase and mitotic cells is shown in figure 1 . the replication and segregation of orip / ebna1 vectors depends only on the presence of ebna-1 protein . as mentioned above , the binding of ebna-1 to the ds element of orip destabilizes nucleosomes and aids the recruitment of cellular replication factors to orip . the segregation function of ebna-1 depends on its ability to bind to both the fr element of orip and to the periphery of chromosomes . it is thought that ebna-1 interacts with chromosomes in two different ways . during interphase ( and probably also in mitosis ) , ebna-1 associates with chromatin through its binding to either cellular dna or chromatin - associated proteins . during metaphase / anaphase , there is additional binding of ebna-1 to a cellular protein referred to as ebp2 ( ebna1-binding protein 2 ) , which strengthens the association of ebna-1 with mitotic chromosomes . one of the strategies for cloning a eukaryotic gene relies on the ability to select for the functional expression of the gene of interest in mammalian cells . a subcloning vector has been designed containing orip / ebna-1 that enables the expression of very rare clones to be selected directly . these clones were stably maintained as episomes , 2 to 10 copies per cell , as long as selection was applied . other studies using orip / ebna-1 vectors have expressed cdna from heterologous promoters , all producing high levels of expression for several weeks or months , and all showing stable retention of the episomes in the cells for long periods of time [ 7881 ] . there are also several studies showing how these two elements are sufficient for large plasmids to be maintained indefinitely in cells , expressing the gene they contain in a physiological fashion . for example , 2 yacs of 90 and 660 kb were created that contained orip and when they were introduced into human cells expressing ebna-1 , these constructs were maintained as stable episomes for more than 8 months in the presence of selection , and for up to 5.5 months without selection . three cell lines were produced with the 90 kb yac , all of which contained unrearranged episomes . however , of the 3 cell lines containing the 660 kb yac only two presented the intact form of the 660 kb molecule . fish analysis also demonstrated how episomes , some visible as pairs , associated with the host cell chromosomes . this association may explain how they are so efficiently maintained , even in the absence of selection , indicating that stability is achieved by attachment to host chromosomes as proposed for the ebv genome . an intact cftr gene has also been expressed from an orip / ebna1 episome in mouse cells . by introducing circularized yacs into cmt-93 and la-9 cells by fusion with yeast spheroplasts , nonrearranged yacs were obtained and maintained as episomes of either 320 or 640 kb . the copy number varied between 2 to 56 in the different cell lines obtained and the rate of loss varied from 0.4% to 5% in the absence of selection , very similar to the data from the 293-cell lines expressing orip - yacs . the level of expression of the exogenous cftr gene was dependent on its copy number and each copy of the yac produced about 20% of the level of each endogenous gene . a further advance was made by changing the constructs based on yacs to others based on bacs , as the latter are easier to use and manipulate , allowing purification of much larger quantities of dna . in order to modify bacs for their use in gene therapy protocols , an efficient system for retrofitting bacs with orip / ebna1 and reporter genes has been described using loxp / cre recombination [ 86 , 87 ] . these vectors not only contain the elements needed for episomal maintenance , they also include suitable marker genes for selection and monitoring of the mammalian cells into which the constructs are introduced . as mentioned above , the type of dna molecules used for gene therapy is crucial but the dna delivery vehicles indeed , the transfer of large dna molecules to mammalian cells is not an easy task . medium size bacs can be delivered into mammalian cells using lipofectamine 2000 , and more than half of the transfected cells contain intact delivered bac [ 87 , 88 ] . however , when working with bacs , the purification of supercoiled dna may be quite cumbersome and it becomes increasingly difficult with larger bacs . different methods involving less manipulation and/or higher efficiency of delivery should be tested in order to optimize the whole system . to date , two methods appear to be quite promising to transfer bacs - based vectors to mammalian cells : bactofection and packaging within herpes simplex virus 1 ( hsv-1 ) particles . bactofection refers to the use of bacteria for the delivery of dna molecules to mammalian cells . one advantage of this method is that it minimizes the need to manipulate the dna molecules , which is especially useful in the case of very large molecules such as bacs . indeed , different studies have proven how dna transfer can occur from bacteria to mammalian cells [ 8991 ] . hence , bacterial strains that are naturally or artificially engineered to be invasive , but that are attenuated to prevent pathogenesis , are particularly useful for bactofection . accordingly , attenuated intracellular bacteria engineered to lyse after cell invasion have been shown to transfer functional genes to a very broad range of mammalian cells [ 92 , 93 ] . the main bacterial species used as delivery vectors are facultative intracellular pathogens such as salmonella sp , shigella sp , listeria sp , or yersinia sp . however , more recently , it was demonstrated that almost any species can be used provided they are appropriately engineered . anaerobic or facultative anaerobic bacteria are also being applied as anticancer agents to target solid tumours taking advantage of their ability to grow in the hypoxic region of tumours [ 96 , 98103 ] . indeed , bacterial delivery has been assessed as a way to administer dna vaccines using different intracellular pathogen strains [ 104 , 105 ] . for certain purposes , a different approach for bacterial delivery relies on the use of a genetically modified e. coli strains that express two additional genes ; inv from yersinia pseudotuberculosis , and hly from lysteria monocytogenes [ 107 , 108 ] . inv permits binding to the integrins expressed on the surface of mammalian cells and hly , the escape from lysosomes once in the cytosol of the mammalian cells . e. coli is also auxotrophic for aminopimelic acid ( dap ) , which means that it is not able to form a new cellular membrane once in the mammalian cytosol . for this reason it lyses and freely liberates the dna it contains . the y. pseudotuberculosis invasin protein ( inv ) is encoded by a 3.8 kb gene expressing a 103 kda protein localized to the outer membrane , part of a family of adhesins encoded by enteropathogenic bacteria . all the members of this family have a region of similarity in the amino terminal 500 amino acids of the y. pseudotuberculosis invasin ( 36% identity ) . this conserved domain is required for protein localization in the outer membrane and for export of the carboxyl termini of these proteins . indeed , the terminal 192 amino acids of invasins represents the most divergent region and it is that which binds to integrin receptors [ 110 , 111 ] . invasin is the only protein needed to invade mammalian cells and it can be cloned into non - invasive strains of bacteria making them invasive [ 89 , 112 ] . the second gene introduced in the modified e. coli strain is the hly locus from l. monocytogenes , encoding listeriolysin o. this protein is a member of the cytolysin family that are produced by various gram positive species . this 58 kda cholesterol - dependent pore - forming toxin allows the bacteria to escape from phagolysosomes into the cytosol of the infected cell . in order to promote lysis of the internalized bacteria , the strain used is unable to synthesise a new cell wall when it divides since it is a diaminopimelic acid ( dap ) auxotrophic strain , a substrate not present in the mammalian cells . this ensures that the bacteria will not survive in the cells , and that the plasmid dna will be liberated into the cytosol and eventually reach the nucleus . using this bacterial vehicle , functional dna can be transferred into a variety of mammalian cell lines by simple coincubation with the engineered e. coli strain . different studies have showed that this strain is also able to transfer large dna fragments [ 116118 ] , establishing the proof - of - principle for the use of this kind of delivery method as a true alternative to efficiently deliver intact bacs into recipient mammalian cells . currently , the use of bactofection seems to be limited to ex vivo gene transfer to cultured mammalian cells , although some examples of in vivo applications to cells of the gastrointestinal tract are envisaged . however , there are very serious concerns about the biosafety of bacteria as gene delivery vehicles due to the risks of important adverse immunological and inflammatory reactions , which will probably restrict their wider application in gene therapy . vectors based on herpes simplex virus 1 ( hsv-1 ) represent one of the most promising alternatives for the delivery of large dna molecules , including many artificial minichromosome - like episomes . hsv-1 has a genome of 152 kb , so viral vectors based on hsv-1 have a notably larger capacity to accommodate dna than other conventional viral vectors used for gene therapy , such as retroviruses and adenoviruses . hsv-1 derived amplicon vectors are plasmids ( or bacs ) bearing only two sequences of viral origin : an oris to permit replication in packaging cells ; and a pac sequence to permit packaging into hsv-1 viral particles . hence , the remaining genomic capacity is available for exogenous dna ( up to approximately 150 kb ) , which may include entire genomic loci with all their regulatory elements to ensure persistent and physiologically regulated levels of expression [ 120123 ] . this feature is probably the most promising characteristic of this type of vector as , to date , they are the only viral vectors with such a characteristic high efficiency of gene transduction that are capable of carrying such large dna fragments that they may include a whole genomic locus . other relevant aspects of hsv-1 amplicons as gene delivery vehicles include their capability to transduce both dividing and non - dividing cells , and their ability to persist as non - integrating episomes , eliminating the risk of insertional mutagenesis . as amplicon vectors only contain the oris and the pac packaging signal as viral dna sequences , generating them requires the use of a helper virus encoding all the viral genes necessary for the assembly and packaging of dna into viral particles . this helper virus , although replication - defective , quite often produces amplicons contaminated with helper viruses that can trigger immune and inflammatory responses . recently , a new system has been developed where all the genes needed for packaging are encoded in a large bac ( which is unable to be packed within virions ) , eliminating all the genes unnecessary to generate the amplicon - containing particles [ 123 , 125 , 126 ] . hsv-1 amplicons can play an important role in gene therapy protocols for neurological disorders due to their notable ability to deliver genes into neurons , both in vitro and in vivo [ 127131 ] . although they can express foreign genes driven by viral promoters only for a period of several days , genes under the control of a neuronal promoter can be stably expressed . in this context , it is not surprising that expression from a genomic locus where the gene is under the control of its own promoter renders physiological levels of expression that are maintained stably . as neurons are postmitotic cells , there is no need for reinoculation of the therapeutic gene should a gene therapy protocol use this kind of vector . however , for dividing cells some modifications would have to be made to the amplicon vectors in order for them to persist during cell divisions . one approach is to use hybrid amplicons containing elements from hsv-1 and elements from the epstein - barr virus that confer episomal retention [ 121 , 122 ] . figure 2 shows the structure of an hsv-1-derived amplicon vector encompassing an ebv - based minichromosome - like episome , and the procedure to pack it into hsv-1 virions . given the high capacity of these vectors , even artificial minichromosomes may be delivered to mammalian cells with an efficiency higher than that obtained by other methods . thus , hsv-1 hac amplicons containing alphoid dna for episomal maintenance and a hprt minigene have efficiently transduced cultured cells , producing stable hprt expression for over three months . curiously , not all hacs behave in the same way in the different cell lines tested , which was probably due to the level of expression of some proteins implicated in cell cycle control . whereas hsv-1 virions are unable to pack dna molecules larger than 160 kb , viral vectors based on other herpes viruses may have a greater capacity to accommodate exogenous dna . thus , cytomegalovirus , which has a 250 kb genome , may permit larger dna fragments to be used . in this way , herpes virus - based vectors appear to be highly promising delivery vehicles of artificial minichromosome - like episomes for gene therapy applications . haemophilia a is an x - linked recessive bleeding disorder caused by mutations in the factor viii gene ( fviii ) that encodes for a clotting factor . it affects about 1 in 5,000 male births in all populations and it is currently treated by infusion of plasma - derived or recombinant factor viii protein . the frequency of the disorder combined with the high cost of recombinant factor viii make it a major burden on the healthcare systems . there are different degrees of severity for this disease depending on the levels of the circulating - factors ( severe , less than 1% ; moderate , between 2%5% ; and mild , between 6%30% ) , thus , low levels of expression may have a large positive effect on the health of the individual . interestingly , fviii is normally expressed in the liver , an organ that is particularly easy to access for gene delivery . much work has been carried out towards preparing gene therapies for both fviii ( haemophilia a ) and factor ix ( fix ) deficiencies ( hemophilia b ) . fix is a much smaller protein encoded by a 1385 bp open reading frame that can be easily packaged as a minigene in retroviral , adenoviral and adeno - associated ( aav ) vectors driven by either a viral or a mammalian promoter [ 136139 ] . long - term delivery and physiological expression have been achieved for fix in mouse models , although many problems remain and gene therapy suitable for human patients is still not available . by contrast , the open reading frame of fviii is 7055 bp long and it has been much harder to express in viral vectors . different strategies have been applied to make the fviii cdna a bit shorter , and hence easier to introduce into different vectors , including using a b - domain deleted version of fviii cdna [ 141 , 142 ] as this domain does not appear to be essential for coagulation [ 14 , 143145 ] . in view of these data , it seems that the election of the vector into which the fviii gene has to be introduced is not a minor issue for haemophilia a gene therapy . first generation adenovirus - based vectors still contained viral gene sequences that produced immunogenic reactions and as a consequence , transgene expression was only short - lived as a result of vector clearance . nevertheless , even with these vectors it might be possible to achieve curative levels of fviii for several months in haemophilic mice [ 147 , 148 ] . however , the results from larger animals were not so promising [ 149 , 150 ] and although some improvements in adenoviral vectors have been made , particularly regarding transgene expression , some toxicity still persists when these vectors are used [ 151 , 152 ] . there is evidence of the expression of the fviii cdna with the b - domain deleted from retroviral vectors in different cells lines , from human skin fibroblasts [ 153 , 154 ] to bone marrow stromal cells . in each case , a functional fviii factor is expressed but at low levels , due to the repressive sequences found in the cdna of fviii gene [ 156159 ] . however , this difficulty has been overcome by using slightly different retroviral vectors based on the mfg retroviral vector system . yet , in these systems , the therapeutic levels of fviii in circulation were obtained over just one week . an important drawback of retroviral vectors is their inability to transduce nondividing cells , particularly important if gene therapy is directed to the liver . one way to bypass this limitation has been to use fviii - expressing retroviral vectors to transduce neonatal mice where hepatocytes are undergoing rapid cell division , thereby obtaining complete correction of the disease . a different approach is to employ lentivirus - based vectors which can transduce both dividing and non - dividing cells [ 163 , 164 ] . due to the small genome of adeno - associated virus ( aav ) vectors , they have been used little with the fviii gene . nonetheless , there are reports of therapeutic levels of fviii expression for a limited period of time using this viral system [ 165 , 166 ] . perhaps due to the disappointing results obtained with other viral vectors , delivery systems based on aav vectors , three phase i trials have been launched to test the safety of the procedure and to check the levels of expression from these vectors . in an ex vivo gene therapy protocol , a b - domain deleted fviii cdna expressed from a non - viral plasmid was electroporated into dermal fibroblasts and , after selection and expansion , the fibroblasts were reintroduced into the patients . fviii levels increased in four of the six subjects studied without producing adverse events . in the patient with the highest levels of fviii expression , the therapeutic effects lasted for 10 months , although fviii expression finally disappeared . in a second trial , a b - domain - deleted cdna was expressed from a moloney murine leukaemia virus - based ( retroviral ) vector , which only produced levels of fviii expression above 1% sporadically , concluding that efficient retroviral transduction would probably require higher doses and some degree of mitotic induction . in 2004 , a third trial using an adenovirus vector encoding the complete fviii cdna was carried out in just a single patient . despite obtaining sustained expression of fviii above 1% of the normal levels for several months , adverse effects were observed associated with the appearance of thrombocytopenia and an elevation of transaminases . there is only one study where factor viii has been expressed from a bac containing the entire genomic locus . as no single fviii - containing bac was available , a new one was constructed by homologous recombination and then retrofitted with different elements in order to provide episomal maintenance [ 48 , 88 ] . the different vectors obtained were introduced into hepatic and nonhepatic human cell lines to check whether the construct could drive expression from the transgene , achieving detectable fviii levels with all of them and in some cases , even stronger expression than the endogenous gene . although a demonstration of functional fviii expression from the bac awaits the use of cell lines or mice with null endogenous expression , this is the first characterized genomic clone carrying the intact locus which could potentially be useful for therapeutic applications . friedreich 's ataxia ( fa ) is the most common form of autosomal recessive ataxia and it is caused by a decrease in the levels of frataxin , a mitochondrial protein encoded by the nuclear frda ( fxn ) gene . fa is a predominantly neurodegenerative disease with an estimated prevalence of 1 - 2 in 50,000 individuals , and it is characterized by the progressive loss of large sensory neurons and spinocerebellar tracts the most common cause of the disease is an expansion of the gaa triplet within the first intron of the frda gene , which has a dramatic effect in reducing mrna levels and consequently frataxin protein levels . although most patients have both chromosomes affected by the gaa expansion , it is possible to find some cases with one expanded allele and a point mutation in the other . frataxin is an 18 kda protein implicated in functions such as mitochondrial iron homeostasis [ 176 , 177 ] , iron - sulfur cluster biosynthesis and oxidative phosphorylation . frataxin is expressed in all cell types , although cells from the nervous system , heart and muscle present the highest levels of the protein . homozygous frda knockout mice are embryonic lethal a few days after implantation , demonstrating the essential role for frataxin during early mammalian embryo development . thus , it appears that the milder phenotype associated with the human disease is due to the residual frataxin expression observed in patients with the expansion mutations . these results may also explain why no patients with homozygous point mutations have been encountered . it has been difficult to generate mouse models for fa because of the embryonic lethality associated with the null mutations . neuron - specific and conditional knock - out models generally exhibit a wider and more prominent neurodegenerative phenotype than the human disease [ 182 , 183 ] . some knock - in mice bearing the human gene with an expansion mutation surprisingly failed to develop any clinical phenotype , despite the significant reduction in frataxin levels to 25%36% the wild - type levels . more recently , representative fa mouse models have been generated by crossbreeding lines of a human fxn yac transgenic mice that contain unstable gaa repeat expansions with heterozygous frda knock - out mice . the resultant transgenic - knockout mice express comparatively little human - derived frataxin , and they exhibit a neurodegenerative and cardiac pathological phenotype similar to human disease sufferers , although significantly milder . with their limitations , these mice currently constitute the most useful model to study the physiopathology of fa and to test for possible therapeutic approaches . there is no effective cure for fa , although antioxidants may have a mildly positive effect on some clinical signs . there has been great interest in the possibility of boosting frataxin gene expression as a therapeutic approach and indeed , erythropoietin has been shown to increase frataxin protein levels in cultured cells from fa patients . in a pilot clinical trial , 12 patients were treated with human recombinant erythropoietin ( rheepo ) to establish whether it could produce an increment in frataxin levels . after 8 weeks of treatment , only two patients experienced a net increment in frataxin levels in peripheral blood lymphocytes , patients who also showed a reduction in markers of oxidative stress . however , it is not yet clear whether or not erythropoietin raises frataxin levels within affected neurons . since chromatin condensation around the gaa repeat is thought to be responsible , to some extent , for the low levels of frataxin mrna expression in fa , histone deacetylase ( hdac ) inhibitors have also been investigated with the same encouraging results [ 189 , 190 ] . fa is a good candidate for treatment with gene therapy as it is a monogenic disease and an increases in frataxin level could substantially improved the symptoms given that healthy carriers may have around 40%50% of normal frataxin levels . a human frataxin cdna has been delivered into fa patient fibroblasts using lentiviral or adeno - associated viral ( aav ) vectors , which has resulted in a partial correction of their sensitivity to oxidant stress . however , the nonphysiological overexpression of frataxin driven by these vectors has been shown to be cytopathological . interestingly , the expression of frataxin cdna to physiological levels driven by a hsv-1 amplicon vector can rescue the neurodegeneration triggered by frataxin deficiency both in cultured neurons and in vivo . these results constitute the first proof of principle that neurological function can be recovered through a gene therapy approach aimed at correcting frataxin deficiency . as mentioned above , it seems that optimal frataxin levels are required for correct cell function , since both deficiency and massive overexpression are associated with cell pathology . thus , physiologically regulated expression of frataxin gene seems to be important for the gene therapy to succeed in fa . an interesting possibility is the use of the entire genomic locus of frataxin , since its large size may ensure the inclusion of all the regulatory elements required for proper gene expression . previous reports of transgenic mice indeed support the use of the frataxin genomic locus to correct frataxin deficiency in vivo . thus , the entire fxn gene within a human yac clone of 370 kb was reported to rescue the embryonic lethality of frataxin knock - out mice . likewise , a human bac clone of 188 kb containing the entire genomic fxn locus has also been demonstrated to rescue the lethal phenotype of frataxin deficiency . this indicates that the frda - bac contains all the regulatory elements needed for the frda gene to be expressed in a physiological fashion . a slightly smaller frda - bac of 135 kb containing the entire 80 kb fxn genomic locus has been used to generate an hsv-1 amplicon ( ibac - frda ) in order to test its ability to correct the fa phenotype . this vector is capable of restoring physiological levels of frataxin in fibroblasts from fa patients and hence , of almost completely rescuing the cell phenotype of susceptibility to oxidative stress . another key issue when using this kind of vector is the ability to infect cells that are hard to transfect with other techniques . once frda expression from the ibac has been demonstrated , the ibac might be used for in vivo delivery of the frda gene to fa - affected regions since delivery of hsv-1 amplicons to determined brain regions has already been shown . indeed , we have results indicating that hsv-1 amplicons containing the entire frataxin genomic locus produce persistent gene expression in the nervous system in vivo ( corona et al . , all these results support the promise of these vectors for gene therapy of fa . in this regard , it is particularly interesting that herpes viral vectors are now in clinical trials for gene therapy of chronic pain . the results of this trial will provide the first clues about the safety and efficacy of herpes viral vectors as gene - delivery vehicles in the human nervous system . artificial chromosomes and minichromosome - like episomes based on bacs containing entire genomic loci are very promising tools for gene therapy of inherited diseases caused by recessive mutations , such as haemophilia or friedreich 's ataxia . the development of vehicles capable of accommodate whole genes emerges like a priority due to the importance of correct and accurate expression of most genes . even if this is the key question , we should not forget that the way these molecules are delivered into target cells is as important as the former . so , improved development of delivery methods should be as well crucial for the success of gene therapy using these large dna molecules . in this respect , amplicon vectors based on herpes viruses are currently a very useful tool regarding average bacs but still much work is needed in order to increase their dna capacity or finding alternative methods for delivery .
a 38-year - old man with cystic fibrosis had undergone bilateral lung transplantation in 1998 and had been well . in september 2000 , he visited a physician with a 3-day history of fever as high as 38.3c , myalgias , and headache . a resident of columbia , missouri , the patient had spent much time outdoors but did not recall tick infestation or recent tick bite . his medications included cyclosporine , mycophenolate , prednisone , diltiazem , trimethoprim - sulfamethoxazole , and valacyclovir . on physical examination , the patient appeared acutely ill with temperature 38.3 c , blood pressure 140/64 , heart rate 110 per minute , and respiratory rate 20 per minute . serum creatinine was 4.6 mg / dl , aspartate aminotransferase 420 u / l , alanine aminotransferase 96 u / l , and bilirubin 3.2 mg / dl . four days after admission , the bone marrow was examined because of worsening pancytopenia ; intracytoplasmic morulae were seen in monocytoid cells , characteristic of monocytic ehrlichiosis ( figure ) . whole - blood polymerase chain reaction ( pcr ) ( viromed , minneapolis , mn ) in the first week of illness was subsequently reported positive for e. chaffeensis dna . serology by immunofluorescence antibody testing for both e. equi and e. chaffeensis performed 2 weeks after onset of illness was negative , with titers < 1:40 . intraleukocytic morulae of ehrlichia can be seen ( arrow ) within monocytoid cells . despite treatment with doxycycline , the patient s confusion , thrombocytopenia , and microangiopathic anemia did not improve , and on the fifth hospital day he was transferred to the university of wisconsin hospital and clinics . physical examination showed blood pressure 144/94 mmhg , heart rate 77/minute , temperature 36.5c , and respiratory rate 24/minute . serum creatinine was 6.2 mg / dl ( 548 mol / l ) , total bilirubin 2.0 mg / dl , aspartate aminotransferase 105 u / l , and alanine aminotransferase 55 u / l . the patient s fever resolved 2 days after doxcycline was started ; however , oliguric renal failure necessitated hemodialysis . hematologic studies showed progressive microangiopathic anemia and thrombocytopenia with a normal inr , suggestive of ttp , presumably secondary to ehrlichia infection . daily plasmaphereses were begun and continued for 8 weeks . gradually the hematologic abnormalities and renal function improved , and the patient s mental status returned to normal . cyclosporine was not resumed , and he was maintained on sirolimus and prednisone to prevent transplant rejection . no rejection occurred , despite a reduction in immunosuppressive therapy during the treatment of ehrlichia infection . ehrlichiosis is a zoonotic illness caused by ehrlichia species , which are pleomorphic , intracellular , rickettsia - like organisms ( 24 ) . the clinical spectrum of ehrlichiois varies from a mild , influenzalike illness to a fulminant sepsis syndrome , but in most patients is self - limiting and not fatal . death rates of documented ehrlichial infection in large , unselected series have been 1% to 8% ( 3,68 ) . this low rate contrasts sharply with the high death rate of ehrliochiosis in immunocompromised patients ( table ) . hme = human monocytic ehrlichiosis ; hge = human granulocytic ehrlichiosis ; ns = not specified cellular immunity represents the most important host defense against rickettsial infection ( 23 ) . acute - phase sera of patients with hge contain elevated levels of interferon gamma , which is associated with the clearance of ehrlichia from peripheral blood ( 24 ) . in a mouse model of ehrlichiosis , impairment of cellular immunity , whether from immunosuppresssive therapies or underlying disease , retards recovery , leading to more severe disease and higher death rates . the population of immunocompromised patients is large and growing ; many have asplenia or solid organ or bone marrow transplants ( 26 ) . an analysis of the published reports of ehrlichial infection shows that the disease in immunocompromised patients is far more severe and prolonged and more likely to be fatal ( table ) ( 5,722 ) . virtually all these patients had signs of organ dysfunction , including pancytopenia ( 40% ) , renal failure ( 24% ) , respiratory distress ( 14% ) , shock ( 28% ) , and neurologic dysfunction ( 18% ) . six ( 25% ) of 23 patients died ; 4 of the 6 deaths were in hiv - infected patients . two patients died within 24 hours after coming to medical attention , despite initiation of appropriate antimicrobial therapy ; in the third , the diagnosis was not considered until late in the hospital course ; and in the fourth , the diagnosis was made postmortem . two deaths occurred in asplenic patients ; in both , ehrlichia infection was not suspected until 1 week after onset of illness . in a recent series of ehrlichial infection in 21 hiv - infected patients , 6 of which reported a high frequency ( 71% ) of moderate to severe disease in hiv - infected patients , particularly with e. chaffeensis ( 27 ) . to our knowledge , this is the first reported case of acute ehrlichiosis in a lung transplant recipient . our patient had laboratory features typical of ehrlichia infection ( thrombocytopenia , leukopenia , and transaminase elevation ) . however , he also had microangiopathic anemia , renal failure , and neurologic dysfunction characteristic of ttp . ehrlichiosis with features of ttp has been described in two reports ( 28,29 ) , one case each of hme and hge . both cases were in immunocompetent persons : one was treated with doxycycline and plasmapheresis ; in the other , the diagnosis was made postmortem . our patient 's multiorgan failure and hematologic aberrations persisted , despite doxycycline therapy , until he underwent plasmapheresis . he was receiving cyclosporine , which is a well - known cause of a rare hemolytic uremic syndrome - ttp - like condition that does not respond to plasmapheresis and nearly always proves fatal ( 30 ) . that our patient s ttp - like illness coincided with ehrlichia infection and responded to doxycycline and plasmapheresis makes it most likely that it was a consequence of acute ehrlichiosis , not cyclosporine . neurologic manifestations , ranging from confusion to frank meningitis , have been reported in up to 20% of patients with erhlichiosis ( 31 ) . our patient had obtundation and delirium that persisted after doxycycline therapy was initiated and his fever had resolved . the presence of headache and confusion in conjunction with pancytopenia and transaminase elevation should raise suspicion of ehrlichia infection , especially if the patient has had potential tick exposures . the diagnosis of ehrlichiosis is often delayed because of its nonspecific clinical and laboratory manifestations . in the immunocompromised person , the empiric antimicrobial regimens used in immunocompromised patients for suspected cryptogenic bacterial and fungal sepsis rarely include a drug or drugs effective against ehrlichia . pcr to detect ehrlichia dna is invaluable for the diagnosis and has > 90% sensitivity and even better specificity ( 32 ) . this technique is particularly useful in the immunocompromised host in whom rapid diagnosis is of utmost importance . peripheral blood and bone marrow examinations show intracellular morulae in hme in only 1% to 5% of cases and can not be relied on diagnostically , unless positive . serologic testing does not allow rapid diagnosis and may be negative in the immunocompromised patient ( 21 ) , as was the case with our patient . the diagnosis of ehrlichiosis should be considered in any patient with fever , transaminase elevations , and new - onset thrombocytopenia or leukopenia who has had potential tick exposures in an endemic area . in the immunocompromised host , clinical manifestations are more severe and can include neurologic deterioration , multiorgan failure , and even a ttp - like illness . response to appropriate therapy with doxycycline may be delayed . a high index of suspicion , the use of pcr for confirmatory diagnosis and early empiric therapy can be life - saving .
the enormous impact of cardiovascular disease on global health and economy demands that low - cost interventions , such as altered lifestyle ( e.g. diet and physical activity ) , be rigorously implemented for prevention and treatment . dietary intake of n-3 pufas , particularly fish oil , is a propitious and therapeutically achievable intervention that has been clearly shown to have a beneficial effect on the cardiovascular system . however , it is disappointing that despite significant clinical and experimental research on n-3 pufas and cardiovascular disease over the past 25 years , there is still no clear molecular mechanism or appropriate dosing strategy in place , making reliable and consistent therapeutic use of n-3 pufas extremely difficult . thus , a coordinated effort is needed to establish a mechanism and develop proper therapeutic paradigms to make n-3 pufas more amenable for use in prevention and treatment of cardiovascular disease . here , we provide a brief overview of the debate regarding the mechanism of n-3 pufa therapy among biomedical researchers , and present a novel hypothesis that may help reconcile this controversy and unite existing , well - characterized n-3 pufa effects with as - yet unresolved questions . compounds that exert vasodilating , anti - inflammatory , anti - thrombotic , anti - arrhythmic and heart rate - lowering effects are all effective therapies for cardiovascular disease . while n-3 pufas have shown promise in virtually all of these areas , results from clinical trials have been mixed , with some showing clear benefits and others showing no change compared with placebo . excellent comprehensive reviews of these topics are found in the recent literature . to date , the mechanisms proposed to explain these beneficial cardiovascular effects have largely focused on the ability of the two predominant n-3 pufas in fish oil , eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , to compete with arachidonic acid in the orthodox pathways of eicosanoid synthesis ( figure 1 ) . further support for eicosanoid - mediated n-3 pufa effects has come from the discovery of an exciting new family of epa / dha - derived eicosanoids by serhan and colleagues . this family of compounds , called the specialized pro - resolving mediators , exemplified by the resolvin series , were found to have potent properties that enhance the resolution phase of inflammation , in addition to other potential therapeutic effects ( e.g. analgesia ) . however , while the cardiovascular benefits of epa / dha - derived eicosanoids must be recognized , burgeoning experimental evidence suggests that attributing the mechanism of n-3 pufa therapy in cardiovascular disease solely to eicosanoid - mediated effects is grossly over - simplistic . the pathway shown in green represents the orthodox enzymatic oxidation cascade that begins with the liberation of epa or dha from phospholipids in cellular membranes by phospholipase a2 ( pla2 ) . the free fatty acid is then acted on by members of the cyclo - oxygenase ( cox ) , 5-lipoxygenase ( 5-lox ) and cytochrome p450 monooxygenase ( cyp ) family of enzymes to form eicosanoids , all of which have various roles in vascular function and innate immunity . the pathway shown in blue depicts spontaneous oxidation of epa or dha by reactive oxygen species , such as superoxide ( o2 ) , h2o2 and hydroxyl radical ( oh ) . further oxidation yields lipid peroxides of epa or dha which , if not neutralized by endogenous antioxidant systems , undergo destabilization and fragmentation to yield reactive lipids such as isoprostanes , isofuranes , alkenes , alkanes and aldehydes . pg , prostaglandin ; txa , thromboxane ; lt , leukotriene one of the more intriguing and promising therapeutic potentials for n-3 pufas is in the treatment and prevention of heart failure . indeed , fish oil ( dha in particular ) has been shown in several heart failure models to improve cardiac function and efficiency [ 6 - 9 ] . these findings have been supported recently by placebo - controlled clinical trials showing that daily intake of dha / epa for one year improved left ventricular function and exercise capacity in patients with established heart failure . furthermore , clinically significant changes in left ventricular function have been reported as early as three months after initiating n-3 pufa treatment . given that heart failure is known to result from altered cardiac energetic and structural parameters , the effects of n-3 pufas in enhancing these parameters has come under vigorous scrutiny by researchers . this is particularly important in highly oxidative , excitable tissues such as the heart , where phospholipid composition is critical for proper membrane structure , thereby ensuring that ion channel activity , charge separation and energy conservation are maintained . several studies have proposed that epa and dha directly modify cardiomyocyte plasma membrane ion channel activity , which may partially explain their anti - arrhythmic properties [ 13 - 16 ] . mitochondria are highly reliant on cardiolipin , a phospholipid unique to this organelle , to maintain membrane electrochemical gradient and capacity for oxidative phosphorylation . recent findings in animal models of heart failure have demonstrated that altered cardiolipin structure parallels the increase in left ventricular function seen with epa / dha treatment [ 17 - 19 ] , suggesting that n-3 pufa incorporation into cardiolipin may partially explain the improvements in cardiac function . however , from a biochemical / biophysical perspective , it is not clear how altering cardiolipin structure with epa / dha incorporation would enhance mitochondrial function and improve cardiac energetics . consequently , alternative metabolic pathways and roles for n-3 pufas in the heart continue to be explored . in particular , there is compelling evidence that n-3 pufas may play a significant role in gene regulation [ 20 - 22 ] , which may contribute to the many beneficial effects that have been observed . aside from the well - characterized enzymatic pathways , pufas are prone to oxidation from non - enzymatic ( i.e. spontaneous ) reactions because of their highly unsaturated structure . these reactions , initiated by reactive oxygen and nitrogen species , ultimately form lipoxidative products such as lipid peroxides , reactive aldehydes and other electrophilic lipids ( figure 1 ) . of all pufas , dha is the most susceptible to lipid peroxidation due to its chemical structure . to date , non - enzymatic oxidation pathways and lipoxidative products of n-3 pufas have been largely ignored by investigators , with a few exceptions . the reasons for this paucity of investigation are not completely clear , but could be due to either the strongly supported belief that the rate of non - enzymatic n-3 pufa oxidation in vivo is negligible , or that the previously held idea that any form of lipid peroxidation is undesirable as it is unconditionally toxic . the latter idea has recently been challenged on the basis of studies described below . lipoxidative stress can be beneficial in many contexts , particularly in cells and tissues that are highly plastic and adaptable , such as the heart . indeed , it has long been known that 4-hydroxynonenal , an aldehyde formed from n-6 pufa oxidation , exerts bi - directional effects on the heart , characterized by a beneficial hormetic effect at sub - toxic concentrations ( 10 m ) , but toxic effects at higher concentrations . concentrations of 4-hydroxynonenal are only beginning to be elucidated , but recent studies have pointed to the involvement of the eukaryotic translation initiating factor 2/activating transcription factor-4 ( eif2/atf4 ) , nf e2-related factor-2 ( nrf2 ) , and peroxisome proliferator - activated receptor ( ppar ) family of transcription factors in up - regulating amino acid biosynthesis , antioxidant / anti - inflammatory genes , and mitochondrial biogenesis , respectively . consistent with this , gao and co - workers demonstrated that oxidized derivatives of n-3 pufas up - regulate nrf2 activity in several stable cell lines , in vitro . whether similar pathways of adaptation occur in heart as a result of lipoxidative products formed from n-3 pufas remains to be determined ( figure 2 ) , but a recent study in our laboratory showed that dietary intake of n-3 pufas resulted in a time - dependent increase in 4-hydroxyhexenal adducts in the heart and up - regulation of nrf2-mediated enzymes in mice that were paralleled by decreased mitochondrial reactive oxygen species production and enhanced mitochondrial tolerance to insults such as ca overload . several clinical studies have reported marked increases in antioxidant enzymes such as superoxide dismutase , catalase and glutathione peroxidase in blood of patients taking n-3 pufas [ 32 - 37 ] and in some cases , lipid peroxidation levels increased in parallel with elevation in these enzymes . furthermore , a number of studies in animals have reported increased expression of antioxidant / anti - inflammatory enzymes in heart following n-3 pufa diet [ 40 - 43 ] , and these were supported by findings from a small clinical trial showing that pre - surgical intake of fish oil suppressed nfb activity and augmented antioxidant activity in heart tissue of patients undergoing cardiac surgery . upon entering cells , dha and epa are either used for oxidative metabolism ( not shown ) or esterified and used for phospholipids within membranes ( shown here in both plasma and mitochondrial membranes ) . liberation of dha and epa from the phospholipids is catalyzed by phospholipase a2 ( pla2 ) . in their free fatty acid form , the n-3 pufas are oxidized directly by reactive oxygen species coming from sources such as nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase or mitochondrial electron transport system . the lipid peroxides formed in this manner are then more reactive and their potency as transcriptional activators is increased , as depicted by the hydroperoxy - dha reacting with ppars and nrf2 . sustained increases of these lipid peroxides can lead to a build - up of their reactive aldehyde derivatives such as 4-hydroxyhexenal and 4-oxo-2-hexenal ( ohe ) , and these can also cause transcriptional activation or react with functional proteins directly . activation of ppars would be expected to cause increased mitochondrial gene expression and lipid oxidation capacity , particularly in the heart . possible outcomes of the direct protein modifications include changes in ion channel conductance ( shown in plasma membrane above ) or in altered activity of mitochondrial respiratory enzymes . keap1 , kelch - like ech - associated protein 1 ; pgc1 , ppar coactivator-1 ; ant , adenine nucleotide translocase ; gst , glutathione s - transferase ; gclc , -glutamylcystein ligase catalytic subunit ; gr , glutathione reductase ; trx , thioredoxin ; trx - r ; thioredoxin reductase . the context and background provided by the findings outlined above allude to a provocative question : is it possible that , to some ( or even a large ) extent , the cardiovascular benefits derived from n-3 pufas result from the lipoxidative stress that they cause ? if so , it would follow that any physiological state resulting in sustained increases in cellular and tissue reactive oxygen species ( e.g. cardiovascular and metabolic diseases ) would drive increased lipoxidative product formation when presented with epa and dha . this is undoubtedly a controversial hypothesis because it contradicts existing paradigms regarding reactive oxygen species and disease , but we offer the following evidence in support of this . first , recent reports demonstrated that oxidized dha has the highest affinity and ppar - activating effect of any ppar ligand tested , including all members of the fibrate and glitazone drug classes . this finding could have broad clinical implications because it indicates that dha peroxidation in vivo would greatly enhance its potency as a ppar activator . secondly , an interesting study by jud and colleagues showed that the electrophysiological effects of dha on the transient outward current in cardiomyocytes were only present when the dha was oxidized . this finding led the authors to speculate that perhaps much of the electrophysiological effects that investigators have attributed to dha were actually coming from lipoxidative products derived from it , since a large amount of dha oxidation occurs spontaneously upon exposure to air ( ~30% ) . as mentioned above , it is now clear that 4-hydroxynonenal can exert a beneficial effect on the heart . zhang et al . recently showed that treating cardiomyocytes with small , sub - toxic doses ( 5 m ) of 4-hydroxynonenal elicits protection from subsequent exposure to toxic doses ( 20 m ) . this group further showed the physiological relevance of this effect by pre - treating mice with 4-hydroxynonenal prior to ischemia / reperfusion , and showed that 4-hydroxynonenal - treated mice exhibited reduced infarct size . in addition , this 4-hydroxynonenal - induced cardioprotection was lost in nrf2 -/- mice , suggesting a fundamental requirement for nrf2 in the 4-hydroxynonenal effect . the role of nrf2 in cardioprotection is only beginning to be understood , and this was underscored by a recent study where overexpression of nrf2 protected against pressure overload - induced cardiac hypertrophy . 4-hydroxyhexenal is the n-3 pufa alkenal equivalent to 4-hydroxynonenal , which is similar in structure but displays different reactivity . as an electrophile , it is equipotent in activating nrf2 , but studies on heart mitochondria in our laboratory have shown that at physiological concentrations , 4-hydroxynonenal inhibits oxidative phosphorylation and also causes increased susceptibility to ca overload , whereas 4-hydroxyhexenal does not ( unpublished data ) . from a translational perspective , this observation is important because it implies that n-3 pufa - derived lipoxidative products may cause similar adaptations to those of n-6 pufa - derived lipoxidative products , without as much of the corresponding cellular and mitochondrial toxicity . many of the cardiovascular effects of n-3 pufas have been linked to their anti - thrombotic , antioxidant / anti - inflammatory , anti - arrhythmic , and vasorelaxing activities . we propose that many of these effects are mediated by n-3 pufa - derived peroxides and reactive aldehydes . moreover , to this list of well - known clinical effects of n-3 pufas we add enhanced mitochondrial biogenesis ( i.e. gene expression ) and antioxidant capacity in the myocardium , which would be expected to augment fatty acid oxidation and protect mitochondria against insults such as ca overload . the latter effects would be expected to have particular importance in clinical scenarios such as pathological cardiac hypertrophy and heart failure , conditions known to result in , or be a result of , mitochondrial dysfunction . compounds that exert vasodilating , anti - inflammatory , anti - thrombotic , anti - arrhythmic and heart rate - lowering effects are all effective therapies for cardiovascular disease . while n-3 pufas have shown promise in virtually all of these areas , results from clinical trials have been mixed , with some showing clear benefits and others showing no change compared with placebo . excellent comprehensive reviews of these topics are found in the recent literature . to date , the mechanisms proposed to explain these beneficial cardiovascular effects have largely focused on the ability of the two predominant n-3 pufas in fish oil , eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , to compete with arachidonic acid in the orthodox pathways of eicosanoid synthesis ( figure 1 ) . further support for eicosanoid - mediated n-3 pufa effects has come from the discovery of an exciting new family of epa / dha - derived eicosanoids by serhan and colleagues . this family of compounds , called the specialized pro - resolving mediators , exemplified by the resolvin series , were found to have potent properties that enhance the resolution phase of inflammation , in addition to other potential therapeutic effects ( e.g. analgesia ) . however , while the cardiovascular benefits of epa / dha - derived eicosanoids must be recognized , burgeoning experimental evidence suggests that attributing the mechanism of n-3 pufa therapy in cardiovascular disease solely to eicosanoid - mediated effects is grossly over - simplistic . the pathway shown in green represents the orthodox enzymatic oxidation cascade that begins with the liberation of epa or dha from phospholipids in cellular membranes by phospholipase a2 ( pla2 ) . the free fatty acid is then acted on by members of the cyclo - oxygenase ( cox ) , 5-lipoxygenase ( 5-lox ) and cytochrome p450 monooxygenase ( cyp ) family of enzymes to form eicosanoids , all of which have various roles in vascular function and innate immunity . the pathway shown in blue depicts spontaneous oxidation of epa or dha by reactive oxygen species , such as superoxide ( o2 ) , h2o2 and hydroxyl radical ( oh ) . further oxidation yields lipid peroxides of epa or dha which , if not neutralized by endogenous antioxidant systems , undergo destabilization and fragmentation to yield reactive lipids such as isoprostanes , isofuranes , alkenes , alkanes and aldehydes . pg , prostaglandin ; txa , thromboxane ; lt , leukotriene one of the more intriguing and promising therapeutic potentials for n-3 pufas is in the treatment and prevention of heart failure . indeed , fish oil ( dha in particular ) has been shown in several heart failure models to improve cardiac function and efficiency [ 6 - 9 ] . these findings have been supported recently by placebo - controlled clinical trials showing that daily intake of dha / epa for one year improved left ventricular function and exercise capacity in patients with established heart failure . furthermore , clinically significant changes in left ventricular function have been reported as early as three months after initiating n-3 pufa treatment . given that heart failure is known to result from altered cardiac energetic and structural parameters , the effects of n-3 pufas in enhancing these parameters has come under vigorous scrutiny by researchers . this is particularly important in highly oxidative , excitable tissues such as the heart , where phospholipid composition is critical for proper membrane structure , thereby ensuring that ion channel activity , charge separation and energy conservation are maintained . several studies have proposed that epa and dha directly modify cardiomyocyte plasma membrane ion channel activity , which may partially explain their anti - arrhythmic properties [ 13 - 16 ] . mitochondria are highly reliant on cardiolipin , a phospholipid unique to this organelle , to maintain membrane electrochemical gradient and capacity for oxidative phosphorylation . recent findings in animal models of heart failure have demonstrated that altered cardiolipin structure parallels the increase in left ventricular function seen with epa / dha treatment [ 17 - 19 ] , suggesting that n-3 pufa incorporation into cardiolipin may partially explain the improvements in cardiac function . however , from a biochemical / biophysical perspective , it is not clear how altering cardiolipin structure with epa / dha incorporation would enhance mitochondrial function and improve cardiac energetics . consequently , alternative metabolic pathways and roles for n-3 pufas in the heart continue to be explored . in particular , there is compelling evidence that n-3 pufas may play a significant role in gene regulation [ 20 - 22 ] , which may contribute to the many beneficial effects that have been observed . aside from the well - characterized enzymatic pathways , pufas are prone to oxidation from non - enzymatic ( i.e. spontaneous ) reactions because of their highly unsaturated structure . these reactions , initiated by reactive oxygen and nitrogen species , ultimately form lipoxidative products such as lipid peroxides , reactive aldehydes and other electrophilic lipids ( figure 1 ) . of all pufas , dha is the most susceptible to lipid peroxidation due to its chemical structure . to date , non - enzymatic oxidation pathways and lipoxidative products of n-3 pufas have been largely ignored by investigators , with a few exceptions . the reasons for this paucity of investigation are not completely clear , but could be due to either the strongly supported belief that the rate of non - enzymatic n-3 pufa oxidation in vivo is negligible , or that the previously held idea that any form of lipid peroxidation is undesirable as it is unconditionally toxic . lipoxidative stress can be beneficial in many contexts , particularly in cells and tissues that are highly plastic and adaptable , such as the heart . indeed , it has long been known that 4-hydroxynonenal , an aldehyde formed from n-6 pufa oxidation , exerts bi - directional effects on the heart , characterized by a beneficial effect at sub - toxic concentrations ( 10 m ) , but toxic effects at higher concentrations . concentrations of 4-hydroxynonenal are only beginning to be elucidated , but recent studies have pointed to the involvement of the eukaryotic translation initiating factor 2/activating transcription factor-4 ( eif2/atf4 ) , nf e2-related factor-2 ( nrf2 ) , and peroxisome proliferator - activated receptor ( ppar ) family of transcription factors in up - regulating amino acid biosynthesis , antioxidant / anti - inflammatory genes , and mitochondrial biogenesis , respectively . consistent with this , gao and co - workers demonstrated that oxidized derivatives of n-3 pufas up - regulate nrf2 activity in several stable cell lines , in vitro . whether similar pathways of adaptation occur in heart as a result of lipoxidative products formed from n-3 pufas remains to be determined ( figure 2 ) , but a recent study in our laboratory showed that dietary intake of n-3 pufas resulted in a time - dependent increase in 4-hydroxyhexenal adducts in the heart and up - regulation of nrf2-mediated enzymes in mice that were paralleled by decreased mitochondrial reactive oxygen species production and enhanced mitochondrial tolerance to insults such as ca overload . several clinical studies have reported marked increases in antioxidant enzymes such as superoxide dismutase , catalase and glutathione peroxidase in blood of patients taking n-3 pufas [ 32 - 37 ] and in some cases , lipid peroxidation levels increased in parallel with elevation in these enzymes . furthermore , a number of studies in animals have reported increased expression of antioxidant / anti - inflammatory enzymes in heart following n-3 pufa diet [ 40 - 43 ] , and these were supported by findings from a small clinical trial showing that pre - surgical intake of fish oil suppressed nfb activity and augmented antioxidant activity in heart tissue of patients undergoing cardiac surgery . upon entering cells , dha and epa are either used for oxidative metabolism ( not shown ) or esterified and used for phospholipids within membranes ( shown here in both plasma and mitochondrial membranes ) . liberation of dha and epa from the phospholipids is catalyzed by phospholipase a2 ( pla2 ) . in their free fatty acid form , the n-3 pufas are oxidized directly by reactive oxygen species coming from sources such as nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase or mitochondrial electron transport system . the lipid peroxides formed in this manner are then more reactive and their potency as transcriptional activators is increased , as depicted by the hydroperoxy - dha reacting with ppars and nrf2 . sustained increases of these lipid peroxides can lead to a build - up of their reactive aldehyde derivatives such as 4-hydroxyhexenal and 4-oxo-2-hexenal ( ohe ) , and these can also cause transcriptional activation or react with functional proteins directly . activation of ppars would be expected to cause increased mitochondrial gene expression and lipid oxidation capacity , particularly in the heart . possible outcomes of the direct protein modifications include changes in ion channel conductance ( shown in plasma membrane above ) or in altered activity of mitochondrial respiratory enzymes . keap1 , kelch - like ech - associated protein 1 ; pgc1 , ppar coactivator-1 ; ant , adenine nucleotide translocase ; gst , glutathione s - transferase ; gclc , -glutamylcystein ligase catalytic subunit ; gr , glutathione reductase ; trx , thioredoxin ; trx - r ; thioredoxin reductase . the context and background provided by the findings outlined above allude to a provocative question : is it possible that , to some ( or even a large ) extent , the cardiovascular benefits derived from n-3 pufas result from the lipoxidative stress that they cause ? if so , it would follow that any physiological state resulting in sustained increases in cellular and tissue reactive oxygen species ( e.g. cardiovascular and metabolic diseases ) would drive increased lipoxidative product formation when presented with epa and dha . this is undoubtedly a controversial hypothesis because it contradicts existing paradigms regarding reactive oxygen species and disease , but we offer the following evidence in support of this . first , recent reports demonstrated that oxidized dha has the highest affinity and ppar - activating effect of any ppar ligand tested , including all members of the fibrate and glitazone drug classes . this finding could have broad clinical implications because it indicates that dha peroxidation in vivo would greatly enhance its potency as a ppar activator . secondly , an interesting study by jud and colleagues showed that the electrophysiological effects of dha on the transient outward current in cardiomyocytes were only present when the dha was oxidized . this finding led the authors to speculate that perhaps much of the electrophysiological effects that investigators have attributed to dha were actually coming from lipoxidative products derived from it , since a large amount of dha oxidation occurs spontaneously upon exposure to air ( ~30% ) . as mentioned above , it is now clear that 4-hydroxynonenal can exert a beneficial effect on the heart . zhang et al . recently showed that treating cardiomyocytes with small , sub - toxic doses ( 5 m ) of 4-hydroxynonenal elicits protection from subsequent exposure to toxic doses ( 20 m ) . this group further showed the physiological relevance of this effect by pre - treating mice with 4-hydroxynonenal prior to ischemia / reperfusion , and showed that 4-hydroxynonenal - treated mice exhibited reduced infarct size . in addition , this 4-hydroxynonenal - induced cardioprotection was lost in nrf2 -/- mice , suggesting a fundamental requirement for nrf2 in the 4-hydroxynonenal effect . the role of nrf2 in cardioprotection is only beginning to be understood , and this was underscored by a recent study where overexpression of nrf2 protected against pressure overload - induced cardiac hypertrophy . 4-hydroxyhexenal is the n-3 pufa alkenal equivalent to 4-hydroxynonenal , which is similar in structure but displays different reactivity . as an electrophile , it is equipotent in activating nrf2 , but studies on heart mitochondria in our laboratory have shown that at physiological concentrations , 4-hydroxynonenal inhibits oxidative phosphorylation and also causes increased susceptibility to ca overload , whereas 4-hydroxyhexenal does not ( unpublished data ) . from a translational perspective , this observation is important because it implies that n-3 pufa - derived lipoxidative products may cause similar adaptations to those of n-6 pufa - derived lipoxidative products , without as much of the corresponding cellular and mitochondrial toxicity . many of the cardiovascular effects of n-3 pufas have been linked to their anti - thrombotic , antioxidant / anti - inflammatory , anti - arrhythmic , and vasorelaxing activities . we propose that many of these effects are mediated by n-3 pufa - derived peroxides and reactive aldehydes . moreover , to this list of well - known clinical effects of n-3 pufas we add enhanced mitochondrial biogenesis ( i.e. gene expression ) and antioxidant capacity in the myocardium , which would be expected to augment fatty acid oxidation and protect mitochondria against insults such as ca overload . the latter effects would be expected to have particular importance in clinical scenarios such as pathological cardiac hypertrophy and heart failure , conditions known to result in , or be a result of , mitochondrial dysfunction . it must be emphasized that if indeed n-3 pufa - derived lipoxidative products are partially responsible for the therapeutic effects observed , this would be manifested to the greatest extent in highly plastic and adaptable organs ( i.e. organs with large antioxidant capacities , like the heart ) . the reason for this is because 4-hydroxyhexenal , and indeed all n-3 pufa - derived lipoxidative products , will affect tissues and organ systems differently depending on their ability to positively adapt to mild lipoxidative stress. moreover , it is expected that this adaptation would require several days or weeks to become optimal . despite these considerations , our contention is that when taken together , the experimental evidence outlined here and elsewhere supports the notion that many of the broad effects of n-3 pufas in cardiovascular disease ( figure 3 ) can be explained by lipoxidative products derived from them , particularly reactive aldehydes such as 4-hydroxyhexenal . if further investigation confirms this observation , it is deserving of rigorous evaluation as it may allow for future development of novel n-3 pufa therapies in cardiovascular disease and other diseases , and assist in developing relevant and proper dosing strategies for n-3 pufa use in clinics .
spontaneous regression ( sr ) of malignant tumor was first defined by cole and everson as complete or partial clearance of malignant cells in the absence of any specific treatment , particularly antineoplastic chemotherapy or surgical resection . it is an extremely rare phenomenon and its incidence is estimated to be one per 60,000100,000 cancer patients . sr of malignant tumor has been reported mainly in neuroblastoma , renal cell carcinoma , malignant melanoma , malignant lymphoma , and leukemia . we report an unusual case of sr of hcc , possibly due to disruption of the feeding artery . a 77-year - old man with alcoholic liver cirrhosis was referred to our hospital on august , 2010 for workup of hepatic masses incidentally found by abdominal computed tomography ( ct ) . he had been a regular drinker , consuming approximately 120 g / day of alcohol every day for 50 years . examination of the head , neck , chest , heart , and extremities was unremarkable . laboratory examinations showed hb of 9.5 g / dl , platelets of 139,000/mm , and wbc count of 6,600/mm . blood chemistry tests revealed aspartate aminotransferase of 172 iu / l , alanine aminotransferase of 59 iu / l , -gtp of 461 iu / l , alkaline phosphatase of 461 iu / l , albumin of 2.9 g / dl , globulin of 3.9 g / dl , total bilirubin of 2.6 mg / dl , prothrombin time of 51.7% , and glucose of 102 mg / dl . serum alpha - fetoprotein ( afp ) was 1,825.0 ng / ml ( normal < 10 ng / ml ) and protein induced by vitamin k absence ii ( pivka ii ) was 3,043 mau / ml ( normal < 40 mau / ml ) . abdominal ultrasonography demonstrated a 50-mm hypoechoic mass with a mosaic pattern in the right anterior superior segment of the liver ( couinaud 's segment 8 , s8 ) and a 15-mm hypoechoic mass in the s8 - 7 . dynamic contrast - enhanced ct scan showed a partially hypervascular tumor with necrotic area in the s8 and a hypervascular tumor in the s7 - 8 ( fig . 1 ) and 10-mm hypervascular tumors in the other segments ( s6 and s4 ) on august , 2010 . the findings of the above high level of tumor markers and imaging examinations were consistent with hcc . he did not receive any further treatment for hcc including herbal medicine and stopped drinking alcohol for one month after the diagnosis of hcc . one month after the diagnosis , afp and pivka ii spontaneously decreased to 51.1 ng / ml and 411 mau / ml , respectively . the 50-mm mass in the s8 decreased to 30 mm and became completely necrotic on ct arteriography ( cta ) ( fig . the 15-mm mass in the s8 - 7 diminished to 10 mm and was described as an enhanced lesion on cta ( fig . 2 ) . hepatic angiography disclosed no tumor stain and occlusion in the right anterior superior arteries ( a8 ) ( fig . tumor stains were noted in the a7 ( the above mass in the s8 - 7 ) ( fig . the hypervascular tumors were treated by transcatheter arterial chemoembolization ( tace ) using an emulsion of 3 ml lipiodol and 30 mg epirubicin with gelfoam . serum afp and pivka ii decreased to the normal range ( 5.7 ng / ml and 37 mau / ml , respectively ) on october , 2010 . after that , the tumor with sr in the s8 completely became scar without local recurrence . six and 16 months after the first treatment , several new recurrent tumors that measured 10 mm were detected in the other segments of the liver . we report herein a rare case of sr of hcc . for diagnosis of sr , it is important to prove the presence of malignant cells morphologically . the limitation of the present case study is that this case was not histologically proven before sr . 2 ) and the marked elevation of serum afp and pivka ii level were useful for the diagnosis of hcc . in the previous reports of sr of hcc , there has been no common tendency about the number and size of tumor , the level of tumor markers , extrahepatic metastases , and causative liver disease . the causative factors and mechanisms leading to sr of hcc are unclear , although two possible causes have been considered in previous literature . in the first place , ischemia has been thought to be the major cause of subsequent necrosis of the oxygen - sensitive malignancy . because of the hypervascular nature of hcc , an important factor might be an insufficient blood supply to the tumor , possibly due to disruption of the feeding artery [ 5 , 6 ] , portal vein tumor thrombus [ 7 , 8 ] , deprivation of oxygen due to rapid natural tumor growth , massive bleeding , and poor arterial supply in cirrhotic liver . in the second place abstinence from alcohol or smoking [ 8 , 11 ] , blood transfusion , the use of some herbal medicine , withdrawal of anabolic steroids , and fever stimulate the production of cytokines such as tumor necrosis factor ( tnf ) and tnf may play an important role in antitumor activities . tumor regression has been observed not only in the primary site but also in metastatic regions by systemic effects such as immune responses . generally , sr of hcc develops after the above possible events ; however , none of these events could be identified in the present patient . at the first diagnosis , the 50-mm tumor in the s8 was described as a high- , iso- , and low - density lesion at the early phase on dynamic - enhanced ct . at this time , the tumor in the s8 might have already begun to lose arterial blood ( a8 ) and to become necrotic . one month later , the tumor in the s8 spontaneously became small and completely necrotic and the 15-mm tumor in the s8 - 7 decreased to 10 mm ( fig . 1 , fig . in addition , the tumors in the other segments ( s6 and s4 ) remained the same in size and character . namely , the tumor in the s7 - 8 might originally have received blood supply from the a7 and a8 and decreased in size , possibly due to disruption of the feeding a8 . two mechanisms of local ischemia due to loss of arterial blood supply are speculated : disruption of the feeding artery associated with angiography and arterial thrombosis . the former is due to a case in which , during the procedure of superselective catheterization , severe subintimal injury occurred in the proper hepatic artery . the latter is deduced from a case in which the resected specimen showed arterial thrombus in non - tumor liver tissue . the present case is unique as regards the fact that ct suggested the concern of blood supply ( a8 ) loss by the complete tumor regression in the s8 and the partial regression in the s7 - 8 . however , it is difficult to prove the local effect of deficiency of blood supply ( a8 ) in the present patient . however , it is difficult to explain the phenomenon by systemic effect of the immunological mechanisms , because the other tumors in the s6 and s4 remained . we assume that a local effect was involved in the mechanism of sr in the present patient . after all , although the precise mechanism of sr can not be fully explained in the present case , disruption of the feeding artery might have played a role in this outcome . we consider that the accumulation of clinical data obtained from patients with sr of hcc will contribute to the understanding of this phenomenon .
an estimated 30%70% of patients with chronic obstructive pulmonary disease ( copd ) have pulmonary hypertension ( ph).1 ph associated with copd is classified as group 3 ph by the world health organization ( who ) , which is associated with lung disease and/or hypoxemia.2 the relevance of a vascular pathology in copd is still somewhat unresolved,3,4 but there is clear evidence that ph plays an important role in the morbidity and mortality associated with copd . the prognosis of copd with severe ph and a resting pulmonary artery pressure ( pap ) > 3540 mmhg5,6 is very poor . systemic treatment of ph causes a lot of side effects : these drugs cause systemic hypotension as they lower the pulmonary and systemic vascular resistance and worsen the mismatch of ventilation perfusion in the lung.7 survival improves in copd patients with secondary ph by long - term oxygen therapy ( ltot).8 there is evidence that in hypoxic lung diseases , there is impaired endothelial cell release of nitric oxide ( no).9 inhaled no is currently under development for other potential clinical indications because it is a powerful endothelium - derived relaxing factor.1013 inhaled no therefore seems to be both a selective and an effective pulmonary vasodilator.12 by administering no through inhalation , it is directly delivered to the target organ , which allows for a lower dose than is necessary with systemic delivery , thereby resulting in fewer and less severe adverse effects . pulsed inhalation of no with oxygen is a safe and effective treatment for patients with copd and ph.14 inhaled no , a prescription pharmaceutical drug under the brand name inomax ( no ; ino therapeutics , madison , wi , usa ) for inhalation , is available commercially as a gaseous mixture of no and nitrogen ( n2 ) . inomax is approved in the us ( december 1999 ) , european union ( august 2001 ) , and other national entities for neonates ( > 34 weeks old ) with hypoxic respiratory failure associated with clinical or echocardiographic evidence of ph when used in conjunction with ventilatory support and other appropriate agents.1518 to our knowledge , there are no studies looking at the vessel caliber of the lung during no treatment . the aim of our study is to investigate the feasibility , safety , and acute therapeutic effect of pulsed no using functional respiratory imaging to calculate the changes in blood vessel caliber in patients with copd with ph . patients with a confirmed diagnosis of copd ( according to the global initiative for chronic obstructive lung disease [ gold ] criteria with a postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) < 0.7 and an fev1 value < 60% predicted ) and using ltot for > 10 hours a day , were included in this study . ph was determined within the previous 12 months by a right heart catheterization with a mean pap ( mpap ) 25 mmhg , or an echocardiogram with a tricuspid regurgitant velocity 2.9 m / s or systolic pap ( spap ) 38 mmhg . patients were excluded from the study if they had any of the following : an exacerbation during the past month requiring the start of or increase in systemic oral corticosteroid therapy and/or hospitalization , left ventricular dysfunction with echocardiographic evidence of a left ventricular ejection fraction ( lvef ) < 40% . also excluded were patients with moderate lv diastolic dysfunction ( ie , > grade 2 ) or any history of increased pulmonary capillary wedge pressure , left atrial pressure , or left ventricular end diastolic pressure > 18 mmhg as measured during cardiac catheterization within the past 6 months . in addition , patients with renal impairment ( ie , an estimated glomerular filtration rate ( gfr ) modification of diet in renal disease ( mdrd ) study value < 60 ml / min/1.73 m ) or history of renal failure , those with clinically significant valvular heart disease that may contribute to ph , and patients using an approved ph medication , such as sildenafil or bosentan , currently or within 30 days of screening were excluded . for the indication of copd - associated ph , inhaled nitrogen oxide ( ino ) is being developed as a drug / device combination product to be used with the investigational inopulse ds - c delivery device ( bellerophon therapeutics , warren , nj , usa ) . the investigational inopulse ds - c delivery system is made up of a device that uses 0.16 l mini cylinders containing 3.0 mg / l ( 2,440 ppm ) or 6.0 mg / l ( 4,880 ppm ) of no gas ( figure 1 ) . the device is lightweight , portable , and can be used in an ambulatory setting . the main advantage of the inopulse ds - c device for spontaneously breathing patients lies in its ability to deliver precise , preset ino doses over time , independent of the patients respiration rate and tidal volume . prescribed doses of ino are delivered through inopulse ds - c device according to the ideal body weight ( ibw ) per hour ( micrograms / kilogram ibw / hour ) . the ino dose is pulsed during the beginning of the subject s inspiration rather than throughout the entire inspiratory period , and the hourly set dose is accurately delivered each hour . because the amount of drug delivered by the inopulse ds - c device is independent of the subject s respiratory frequency and tidal volume , the dose of ino is tightly controlled . a direct comparison between pulsatile delivery and constant concentration delivery is difficult as the delivered dose varies with the breath rate for constant concentration delivery , while it is constant for the pulsatile delivery of the inopulse device . the level of exposure between pulsatile and constant concentration deliveries can be compared by making assumptions regarding respiration rate and tidal volume , as well as the ibw , for the inopulse device . one advantage of pulsed delivery of ino early in inspiration is that it presumably delivers the drug selectively to the healthiest well - ventilated lung segments by using a short pulse width . the objective of this exploratory study was to assess the effect of pulsed ino on vascular geometry in subjects with copd - related ph who are on ltot . the primary end point was the change in lobar blood volume at total lung capacity ( tlc ) , measured by functional respiratory imaging ( fri ) , after dosing with pulsed ino . additional end points were internal airflow distribution ( to link regional vasodilation with regional ventilation ) and patient feeling of dyspnea and exercise tolerance . the study protocol ( figure 2 ) was approved by the ethical committe of the university hospital of antwerp , ( 14/35/361 ) and informed consent was given by each patient at the time of entry to the study . six patients ( three males and three females ) with adequate renal function ( estimated gfrmdrd ) 60 ml / min/1.73 m and meeting all inclusion and none of the exclusion criteria were enrolled ( table 1 ) . at screening , treatment visit , and posttreatment , the following were measured : vital signs [ heart rate ( hr ) , respiratory rate ( rr ) , noninvasive blood pressure ( nibp ) and oxygen partial pressure ( spo2 ) ] and methemoglobin ( methb ) ( via pulse oximeter ) . after no treatment also four low - dose high - resolution computed tomography ( hrct ) scans were obtained while the patient was on ltot and room air for at least 20 minutes ( baseline phase ) . two baseline tlc scans were taken quickly after each other with contrast , given before the first tlc scan . these two baseline hrct scans were performed at tlc to demonstrate baseline variability and/or repeatability . next , one baseline functional residual capacity ( frc ) scan and one upper airway ct scan was performed . after the patient received 30 g / kg ibw / hour , the patient remained lying down , not removed from the motorized bed of the ct scanner , starting at least 20 minutes before the baseline ct scans were taken . the baseline tlc and frc scans ( preceding the ino dose ) were used to compute the baseline values for the primary end point ( ie , blood vessel density ) and the secondary end points ( ie , internal airflow distribution ) . in the fri workflow , ct images are converted into three - dimensional images of the lung lobes , the airway tree , and vessels.1921 by segmenting the lung lobes at frc and tlc ( video s1 ) , the internal airflow distribution can be derived from the relative volume change between these two lung volumes . , the airway structure can be segmented down to bronchi with diameter of about 12 mm . beyond this point , a typical airway model includes five to ten generations , depending mainly on the disease state of the individual patient . distal airway volumes ( ivaw ) can be assessed at individual airways and in different regions . anatomical structures that were analyzed include lung volumes , lobar volumes , and local airway volumes , mainly lung blood vessels volumes that were obtained before and after ino administration . the contrast ct scans make it possible to extract the blood vessels by performing hounsfield thresholding , combined with region growing , starting from the central vessels . the hrct parameters are extracted using a semiautomated tool ( mimics 15.0 , materialise nv , leuven , belgium ; food and drug administration , k073468 ; conformit europenne certificate , be 05/1191 . ce.01 ) , which identified the fissures separating the lung lobes . for the calculation of vasodilation , all statistical analyses were conducted using r version 3.0.2 ( the r foundation for statistical computing , vienna , austria ) . the global differences before and after the treatment phase were assessed using a paired t - test . the data were also analyzed on a lobar level using linear mixed - effects models , whereby different subjects are combined in a random factor . in this mixed - effect approach , goodness of fit patients with a confirmed diagnosis of copd ( according to the global initiative for chronic obstructive lung disease [ gold ] criteria with a postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) < 0.7 and an fev1 value < 60% predicted ) and using ltot for > 10 hours a day , were included in this study . ph was determined within the previous 12 months by a right heart catheterization with a mean pap ( mpap ) 25 mmhg , or an echocardiogram with a tricuspid regurgitant velocity 2.9 m / s or systolic pap ( spap ) 38 mmhg . patients were excluded from the study if they had any of the following : an exacerbation during the past month requiring the start of or increase in systemic oral corticosteroid therapy and/or hospitalization , left ventricular dysfunction with echocardiographic evidence of a left ventricular ejection fraction ( lvef ) < 40% . also excluded were patients with moderate lv diastolic dysfunction ( ie , > grade 2 ) or any history of increased pulmonary capillary wedge pressure , left atrial pressure , or left ventricular end diastolic pressure > 18 mmhg as measured during cardiac catheterization within the past 6 months . in addition , patients with renal impairment ( ie , an estimated glomerular filtration rate ( gfr ) modification of diet in renal disease ( mdrd ) study value < 60 ml / min/1.73 m ) or history of renal failure , those with clinically significant valvular heart disease that may contribute to ph , and patients using an approved ph medication , such as sildenafil or bosentan , currently or within 30 days of screening were excluded . for the indication of copd - associated ph , inhaled nitrogen oxide ( ino ) is being developed as a drug / device combination product to be used with the investigational inopulse ds - c delivery device ( bellerophon therapeutics , warren , nj , usa ) . the investigational inopulse ds - c delivery system is made up of a device that uses 0.16 l mini cylinders containing 3.0 mg / l ( 2,440 ppm ) or 6.0 mg / l ( 4,880 ppm ) of no gas ( figure 1 ) . the device is lightweight , portable , and can be used in an ambulatory setting . the main advantage of the inopulse ds - c device for spontaneously breathing patients lies in its ability to deliver precise , preset ino doses over time , independent of the patients respiration rate and tidal volume . prescribed doses of ino are delivered through inopulse ds - c device according to the ideal body weight ( ibw ) per hour ( micrograms / kilogram ibw / hour ) . the ino dose is pulsed during the beginning of the subject s inspiration rather than throughout the entire inspiratory period , and the hourly set dose is accurately delivered each hour . because the amount of drug delivered by the inopulse ds - c device is independent of the subject s respiratory frequency and tidal volume , the dose of ino is tightly controlled . a direct comparison between pulsatile delivery and constant concentration delivery is difficult as the delivered dose varies with the breath rate for constant concentration delivery , while it is constant for the pulsatile delivery of the inopulse device . the level of exposure between pulsatile and constant concentration deliveries can be compared by making assumptions regarding respiration rate and tidal volume , as well as the ibw , for the inopulse device . patients received 30 g / kg ibw / hour doses ( inopulse ds - c setting of 0.030 mg / kg ibw / hour ) . one advantage of pulsed delivery of ino early in inspiration is that it presumably delivers the drug selectively to the healthiest well - ventilated lung segments by using a short pulse width . the objective of this exploratory study was to assess the effect of pulsed ino on vascular geometry in subjects with copd - related ph who are on ltot . the primary end point was the change in lobar blood volume at total lung capacity ( tlc ) , measured by functional respiratory imaging ( fri ) , after dosing with pulsed ino . additional end points were internal airflow distribution ( to link regional vasodilation with regional ventilation ) and patient feeling of dyspnea and exercise tolerance . the study protocol ( figure 2 ) was approved by the ethical committe of the university hospital of antwerp , ( 14/35/361 ) and informed consent was given by each patient at the time of entry to the study . six patients ( three males and three females ) with adequate renal function ( estimated gfrmdrd ) 60 ml / min/1.73 m and meeting all inclusion and none of the exclusion criteria were enrolled ( table 1 ) . at screening , treatment visit , and posttreatment , the following were measured : vital signs [ heart rate ( hr ) , respiratory rate ( rr ) , noninvasive blood pressure ( nibp ) and oxygen partial pressure ( spo2 ) ] and methemoglobin ( methb ) ( via pulse oximeter ) . after no treatment also four low - dose high - resolution computed tomography ( hrct ) scans were obtained while the patient was on ltot and room air for at least 20 minutes ( baseline phase ) . two baseline tlc scans were taken quickly after each other with contrast , given before the first tlc scan . these two baseline hrct scans were performed at tlc to demonstrate baseline variability and/or repeatability . next , one baseline functional residual capacity ( frc ) scan and one upper airway ct scan was performed . after the patient received 30 g / kg ibw / hour , the patient remained lying down , not removed from the motorized bed of the ct scanner , starting at least 20 minutes before the baseline ct scans were taken . the baseline tlc and frc scans ( preceding the ino dose ) were used to compute the baseline values for the primary end point ( ie , blood vessel density ) and the secondary end points ( ie , internal airflow distribution ) . in the fri workflow , ct images are converted into three - dimensional images of the lung lobes , the airway tree , and vessels.1921 by segmenting the lung lobes at frc and tlc ( video s1 ) , the internal airflow distribution can be derived from the relative volume change between these two lung volumes . , the airway structure can be segmented down to bronchi with diameter of about 12 mm . beyond this point , a typical airway model includes five to ten generations , depending mainly on the disease state of the individual patient . distal airway volumes ( ivaw ) can be assessed at individual airways and in different regions . anatomical structures that were analyzed include lung volumes , lobar volumes , and local airway volumes , mainly lung blood vessels volumes that were obtained before and after ino administration . the contrast ct scans make it possible to extract the blood vessels by performing hounsfield thresholding , combined with region growing , starting from the central vessels . the hrct parameters are extracted using a semiautomated tool ( mimics 15.0 , materialise nv , leuven , belgium ; food and drug administration , k073468 ; conformit europenne certificate , be 05/1191 . ce.01 ) , which identified the fissures separating the lung lobes . for the calculation of vasodilation , all statistical analyses were conducted using r version 3.0.2 ( the r foundation for statistical computing , vienna , austria ) . the significance level was set at 0.05 . the global differences before and after the treatment phase were assessed using a paired t - test . the data were also analyzed on a lobar level using linear mixed - effects models , whereby different subjects are combined in a random factor . in this mixed - effect approach , goodness of fit changes in blood vessel caliber could be observed in all patients , as shown in figure 3 . the vasodilation is heterogeneous ( figure 4 ) , but the overall effect is that there is big improvement in the volume of the vessels . in some areas , figure 5 shows the vascular changes for the total lung and for the individual lobes . in addition , the figure depicts the variability of the fri measurement ( 1.56%3.57% ) relative to the treatment effect ( 7.06%5.88% ) . it can be observed that the variability of the measurement , as determined using the test the changes in the volume of the blood vessels correlate well with regional ventilation , also derived from the functional ct based on lobe expansion . , we can anticipate that the ventilation perfusion ratio is preserved ( figure 6 ) . there was no decrease in the oxygen saturation ( figure 7 ) , which confirms the fact that the vasodilation is in line with the ventilation . the patients improved in their subjective feelings of dyspnea and exercise tolerance 24 hours after treatment ( figure 8) . combined inhalation of no and oxygen with a pulsed device for 20 minutes in patients with chronic ph due to copd caused a significant vasodilation in a majority of the vessels visualized by fri . the changes , mostly increases , in blood vessel caliber seem to occur in well - ventilated areas of the lung . there was no decrease in the oxygen saturation , suggesting that ventilation / perfusion ratio was preserved . compared with oxygen alone , the combined inhalation caused a significant decrease in mpap and pulmonary arterial resistance index , without decreasing arterial oxygenation . this was the first controlled randomized trial showing that inhalation of no with oxygen can safely be used for treating ph in patients with copd . vonbank et al14 nevertheless demonstrate that it is possible to give a small concentration of no at the beginning of the inspiration . as a consequence , the exposure of the lung to no is reduced to a very small volume and systemic overall toxicity of no is avoided . delivery of no in pulsed doses early in inspiration , using a short pulse width , has the potential to follow the flow and to be delivered to the well - ventilated zones . in our study no - mediated vasodilation occurs via the activation of soluble guanylate cyclase , the production of cyclic guanosine monophosphate ( cgmp ) , and subsequent relaxation of vascular smooth muscle . ino produces pulmonary vasodilation with minimal effect on systemic vascular beds due to its high affinity for hemoglobin and rapid inactivation . patients with end - stage copd have impaired no - mediated endothelium - dependent pulmonary artery relaxation912 because of a reduced expression of endothelial no synthase.23,24 several groups14,25,26 could show that short - term use of no can improve the hemodynamics in copd patients . the effect on the partial pressure of arterial oxygen ( pao2 ) is however controversial . in some studies , no lowers pao2,27 whereas in others , it improves the same,26 and in some other studies , there seems to be no effect.14 therefore , there are probably several phenotypes for no response in copd . it is possible that the controversial results are linked to the dosage of ino used . finding the right dose to reduce the mpap and pulmonary vascular resistance ( pvr ) while maintaining pao2 is a challenge.14,2730 in a number of animal species and under several vasoconstrictive stimuli,31,32 ino produced rapid and effective pulmonary vasodilation at concentrations between 5 ppm and 80 ppm . evaluations in the new - born lamb demonstrated that ino selectively reverses hypoxic pulmonary vasoconstriction , with maximal pulmonary vasodilation produced by no concentrations of 80 ppm . in an animal model of persistent ph of the newborn , neonatal lambs displayed a marked and rapid pulmonary vasodilation that improved oxygenation , at ino concentrations of 100 ppm . too high doses of no,28 however , may decrease the pao2 due to worsening of the ventilation perfusion distributions , caused by a greater increase in blood flow compared to the increase in airflow reaching the vasodilated areas . taking into account these earlier studies , our patients received 30 g / kg ibw / hour doses , which obviously prevented excess vasodilation and lowering of ventilation / perfusion ratio ( v / q ) ratio . this dose was chosen because it was the highest dose safely tested so far and the objective of this study was to observe with fri the vascular effect induced by the strongest safe dose . one can also hypothesize that the severity of the ph may play an essential role in the response to no treatment . our patients had relatively high severity of ph , the mean spap was 49 mmhg . this was also confirmed by katayama.29 they looked at the effect of no in nine patients with chronic obstructive disease , eleven patients with severe ph , and 14 healthy volunteers . the patients were randomized for inhalation of 40 ppm no or air for 20 minutes . there was a decrease in transcutaneous arterial oxygen tension in normal subjects and in patients with copd overall . there was , however , no change in arterial oxygen tension in patients with severe ph . the responding phenotype of copd patients needs to have a relatively high pap . in our study , vasodilation occurred in most of the lobes after 20 minutes of pulsed ino , which probably contributes to the lowering of the pressure because it represents an important vascular recruitment . it occurs in well - ventilated areas because it follows , as mentioned , the ventilation , preventing v / q mismatch , and oxygen desaturation . finally , and probably , the most important observation was that the patients did feel better with less dyspnea and better exercise tolerance in the hours following the exposure to ino . our hypothesis is that heterogeneous , ventilation - driven vasodilation by pulsed ino may lead to a reduction of cardiac afterload (= larger cardiac output ) and better oxygenation ( preserved v / q ratio ) , resulting in improvement of exercise tolerance in patients with combined copd / ph . we expect that pulsed no could also be a promising treatment in other lung diseases leading to ph , such as pulmonary fibrosis . this was confirmed by channick et al.33 they showed that in a patient with pulmonary fibrosis and ph , no inhalation induced a significant improvement of arterial oxygenation and reduction in pulmonary vascular resistance . therefore , in chronic lung disease in combination with severe ph , ino could have a potential therapeutic role as a selective pulmonary vasodilator . in conclusion , using fri , we were able to show a significant vasodilation of most of the vessels after ino for a limited time period . in our patients , there was an average increase of blood volume of 7.06% after inhalation of pulsed no , without a decrease in oxygen saturation , and patients tended to feel better after the treatment . systemic treatments of ph showed no significant elevated spo2 and improved health - related quality of life . the usefulness of these systemic vasodilator agents is limited due to their side effects and poor tolerance.3436 short - term treatment with pulsed no in combination with oxygen could be a promising treatment for patients with copd and ph . further studies are needed to determine the effect of long - term continuous treatment with pulsed no and oxygen in patients with copd and ph , especially considering exercise tolerance and quality of life . there is also need to investigate the effect of pulsed no in other chronic lung diseases complicated by ph , such as pulmonary fibrosis .
riedel 's thyroiditis ( rt ) is a rare form of thyroiditis that may be related to a diverse range of clinical symptoms . it has been reported in approximately 0.06% of patients who undergo surgery for an abnormality in the thyroid gland . although it has been assumed to be one of the clinical manifestations of multifocal fibrosclerosis , its cause has not been clearly determined . as it can easily be confused with a malignant tumor , diagnosis prior to surgical excision of the thyroid gland reports of this disorder in korea are extremely rare and only a few cases with concurrent presence of rt and other thyroid diseases have so far been reported [ 2 - 4 ] . there have been no reports of rt accompanied by acute suppurative thyroiditis and micropapillary carcinoma . we report , together with a review of the literature , a single case of rt accompanied by an abscess and micropapillary carcinoma diagnosed by surgical biopsy in a 48-year - old female patient who visited our hospital complaining of a neck mass with pain accompanied by cervical lymphadenopathy . surgical biopsy was carried out because of difficulties in ruling out a malignant tumor due to the clinical characteristics . a 48-year - old woman visited our hospital due to abdominal pain and fever of 3 days ' duration . the patient began to experience pain and swelling in the anterior neck region at the time of hospitalization . the patient 's history revealed the consumption of a bottle of alcohol five times a week over the previous 20 years . the remainder of her medical history was unremarkable . at the time of consultation , the patient 's blood pressure was 130/60 mmhg , her heart rate was 68 beats / min , and her body temperature was 37.6. she was alert but had an acute ill - looking appearance . jaundice was observed in the sclera and a 3 3 cm hard mass fixed around the right anterior area of the neck , accompanied by tenderness and enlargement of adjacent cervical lymph nodes , was palpated . the patient complained of abdominal pain and had direct tenderness in the right upper quadrant of her abdomen . laboratory examinations showed a hemoglobin count of 10.7 g / dl , a leukocyte count of 11,100/mm ( neutrophils 76.0% , lymphocytes 17.0% , monocytes 3.4% , and eosinophils 1.1% ) , a thrombocyte count of 101,000/mm , an aspartate aminotransferase level of 84 iu / l , an alkaline aminotransferase level of 40 iu / l , an alkaline phosphatase level of 311 iu / l , a total protein and albumin level of 6.7/2.3 g / dl , a total bilirubin level of 6.5 mg / dl , a glucose level of 114 mg / dl , a -gt level of 141 iu / l , a prothrombin time of 20.9 seconds ( 46% ) , an erythrocyte sedimentation rate of 120 mm / hr , and a c - reactive protein level of 4.42 mg / dl . miu / l ( normal range , 0.17 to 4.05 ) , the t3 level 36.0 ng / dl ( normal range , 60.0 to 190.0 ) , the free t4 level 1.09 ng / dl ( normal range , 0.80 to 1.90 ) , the thyroglobulin antibody ( ab ) level 113.71 u / ml ( normal range , 0.00 to 100.00 ) , and the microsomal ab level 4.0 u / ml ( normal range , 0.0 to 70.0 ) . blood culture carried out at the time of hospitalization showed growth of escherichia coli and urine culture showed growth of enterococcus species , which led to treatment with piperacillin / tazobactam and amikacin . cervical computed tomography revealed a large nodular lesion of the right thyroid lobe with internal calcification and an irregular border , suggesting a malignant nodule . there was swelling and liquid collection in the surrounding soft tissue , which seemed to be due to accompanying hemorrhage or inflammation . cervical lymphadenopathy of the level ii lymph nodes on both sides and the level iii and iv lymph nodes on the right side was observed ( fig . 1 ) . positron emission tomography revealed strong fluorodeoxyglucose ( fdg ) uptake by a large nodule of the right thyroid lobe and cervical lymphadenopathy at the left level ib lymph nodes , the level ii lymph nodes on both sides , and the right level iii , iv , and v areas , with strong fdg uptake . the results suggested malignancy of the right thyroid gland and abnormal cervical lymphadenopathy . on the basis of these findings , neck ultrasonography and ultrasound - guided fine needle aspiration biopsy ( fnab ) were carried out . a 3.06 2.63 3.91 cm heterogeneous hypoechoic mass with calcification was observed in the right lobe of the thyroid gland , and fnab was performed ( fig . aspiration cytology results revealed no thyroid follicles , but fibrous tissues and spindle cells with numerous neutrophils , lymphocytes , and macrophages were observed ( fig . after antibiotic therapy for acute suppurative thyroiditis , follow - up ultrasound - guided fnab for the thyroid nodule was carried at 2 months . ultrasonographic findings revealed a 3.4 3.6 3.5 cm hypoechoic nodule accompanied by calcification in the right thyroid lobe , suggesting a malignant nodule . metastasis to the cervical lymph nodes was also suspected since the level ii lymph nodes of the right cervical region were enlarged to 1.2 cm and hyperechoic findings suggested internal calcification . no follicle cells were seen ; only a mixture of various inflammatory cells was observed . ultrasound - guided fnab was repeated a total of five times as the possibility of malignancy could not be ruled out . there was a single report of suspicion of malignancy and the other four biopsies resulted in a benign diagnosis or insufficient specimens . surgical biopsy was recommended since anaplastic carcinoma accompanied by inflammation could not be ruled out . however , since the possibility of malignancy could not be ruled out , total thyroidectomy and excision of the central cervical lymph nodes was performed . during the operation , a large hard nodule with adhesion to neighboring structures , including adjacent muscles and the internal jugular vein , the cut surface revealed a hard grayish brown nodule with fibrosis , accompanied by multifocal necrosis and calcification . histologic examination revealed loss or contraction of normal thyroid follicles of the right thyroid gland due to severe fibrosis , and many areas affected by localized abscesses , cholesterol crystallization , degenerative calcification , and infiltration of acute and chronic inflammatory cells ( fig . in addition , the characteristic findings of chronic inflammation and fibrous tissues encasing a medium - sized vein were observed ( fig . the patient is currently being treated on an outpatient basis with thyroid hormone replacement therapy ( 150 g of levothyroxine per day ) . rt is an extremely rare chronic inflammatory disorder of the thyroid gland , the cause of which is yet to be definitively defined . this disorder is mainly characterized by sudden enlargement of the thyroid gland ( goiter ) with palpation of a substantially hard mass and replacement of the normal thyroid gland parenchyma with very dense fibrous tissues . although distinguishing rt from thyroid carcinoma is important in diagnostic evaluation , it is not possible to make the diagnosis with only clinical findings . therefore , one relies on histological diagnosis at initial diagnosis since rt can be easily confused with malignant tumors , including thyroidal papillary carcinoma , lymphoma , and undifferentiated thyroid cancer , because of its clinical characteristics . in the majority of cases , it is not easy to obtain appropriate tissue samples by fnab . fnab itself is very difficult because the tissues are very hard and , even if some of the fibrotic tissue can be biopsied , there is a possibility that hidden malignant tumor cells would remain without being biopsied . therefore , surgical biopsy is carried out together with treatment , as was done in this case . the majority of surgical findings indicate that complete removal is very difficult because the stony hard fibrosis usually infiltrates the thyroid gland , respiratory tract , cervical muscles , esophagus , and nerve plexus . therefore , the risk of damage to the parathyroid and recurrent laryngeal nerve is very high . in addition , an underlying malignant tumor or simultaneous thyroid adenoma may remain , as complete excision may be impossible due to fibrosis and invasion , necessitating continuous follow - up observation after surgery . the present case involved invasion of neighboring structures , including muscles and the internal jugular vein , and surgery resulted in damage to the right parathyroid gland and right recurrent laryngeal nerve . microscopic findings in rt are characterized by the spread of fibrosis beyond the thyroid capsule and maintenance of relatively normal tissues in the noninvolved region . there may be occlusive phlebitis resulting from diffuse infiltration of the veins by lymphoid or fibrous tissue . diagnoses that need to be pathologically distinguished include the fibrosing variant of hashimoto 's thyroiditis and paucicellular variant of anaplastic carcinoma . with the fibrosing variant of hashimoto 's thyroiditis , it is also uncommon for the normal thyroid follicles to be preserved due to lymphocytic inflammation of the majority of the thyroid gland . the paucicellular variant of anaplastic carcinoma is distinguished from rt as it presents with nontypical spindle cells , occlusion of large vessels by tumor cells , and immune reaction against epithelial markers . solitary fibrous tumor is distinguished by positive findings in its reaction against cd34 and bcl-2 . the paucicellular variant of anaplastic carcinoma is distinguished by the presence of a dense fibrous stroma that displays positive findings in cytokeratin ( ck ) immunostaining against ae1 , ae3 , cam5.2 , ck7 , pan - ck ( ck5 , ck6 , ck8 , and ck17 ) , and ck19 . in the present case , acute thyroiditis is rare because the thyroid gland has a capsule and an abundant blood supply , lymph nodes , and iodine , which has antibacterial actions . although the infection route can not be definitively defined in many cases , the major routes include direct propagation as the result of spread of infection from the surrounding cervical tissues and distant propagation through the lymphatics or the blood stream . although ultrasonography , computed tomography , and magnetic resonance imaging can be helpful in diagnosis , a definitive diagnosis is achieved by gram staining and culture of pus from the abscess . treatment includes administration of antibiotics appropriate for the cultured bacteria . if there is no clinical improvement , excision or drainage of the abscess is necessary . staphylococcus aureus is the most common pathogen causing the disorder , with group a -hemolytic streptococcus the most common pathogen in the case of infants . moreover , the disorder can also be caused by infection with e. coli and pseudomonas aeruginosa ; chronic infection by treponema pallidum , tubercle bacillus , actinomycosis and salmonella typhi ; and parasitic infection by echinococcus spp . and fungal infection by aspergillus spp . . in the present case , e. coli was identified from the aspirate from the abscess . saksouk and salti described acute suppurative thyroiditis caused by e. coli in a patient with a urinary tract infection as the main source of infection . in our case , since there was cholecystitis accompanied by gall stones and e. coli in the blood culture , it was likely the infection was propagated through the blood stream . excision or drainage of the abscess was not performed due to clinical improvement after antibiotic use . rt has recently been recognized as one of the manifestations of multifocal fibrosclerosis , which is characterized by fibrosis involving multiple organ systems , such as retroperitoneal fibrosis , mediastinal fibrosis , orbital tumors , pulmonary fibrosis , sclerosing cholangitis , and fibrous mumps . in the present case , fibrosclerosis was not observed in other areas . in summary , since the radiologic findings were suggestive of a thyroid malignancy , she was initially suspected to have a malignant tumor and acute suppurative thyroiditis . however , repeated fnab did not reveal definitive findings that allowed us to diagnose a malignancy , and the patient was instead diagnosed with rt accompanied by abscesses and micropapillary carcinoma after surgical biopsy . a prudent diagnostic approach is needed for rt since differential diagnosis with other malignant tumors is clinically difficult . there is also a need for close and continuous follow - up of patients since surgical excision may be incomplete , and because of the possibility of a dormant cancer or of rt being a manifestation of multifocal fibrosclerosis , with other manifestations becoming evident later on .
osteoporosis is generally considered to be a condition affecting women , but up to 30% of fragility fractures occur in men [ 13 ] . the lifetime risk of fracture at the age of 50 years has been estimated to be 20% for men [ 1 , 4 ] . bone mineral density ( bmd ) has long been recognised as an important skeleton determinant of fracture risk , but it is becoming apparent that skeletal geometry also influences the risk . this has been most extensively studied in women at the hip , in terms of hip axis length ( hal ) , femoral neck axis length ( fnal ) , neck shaft angle ( nsa ) , and femoral neck width ( fnw ) . the role of all of these factors as independent predictors of hip fracture risk is controversial in both sexes , with studies giving conflicting results [ 5 , 6 ] . this uncertainty may have arisen partly because of differences in study design , numbers of patients studied , and also because of wide variations in geometric parameters in different countries and races [ 7 , 8 ] . given this variation it may also be necessary to generate gender - specific data , as suggested by our previous paper , which showed that men had a mean femoral nsa of 130 ( sd 3.3 , range 121138 ) , whilst women had a significantly ( p < 0.0001 ) smaller mean femoral nsa of 128 ( sd 1.7 , range 119137 ) . only one study has examined hip geometry solely in men in england and this failed to show any relationship between hal and hip fracture . however , it did not measure nsa or femoral neck width , so there is a need for further study of the role of femoral geometry in men . men with forearm fractures and vertebral fractures are at increased risk of developing hip fractures [ 10 , 11 ] , which may be due in part to altered skeletal geometry . we have therefore examined femoral neck nsa measurements in three uk case - control studies of low trauma hip , vertebral , and distal forearm fractures in men [ 1214 ] . these studies have previously demonstrated significantly lower bmd in men sustaining these fractures compared with controls , and between 42% and 83% were osteoporotic on the basis of a t - score 2.5 using male - specific reference data [ 1214 ] . it is also important to note that there can be differences in geometry between femoral neck and trochanteric hip fractures and for this reason , these fractures types need to be considered separately . the cornwall hip fracture recruited men with hip fractures of the femoral neck and trochanteric regions and so provides an opportunity to study the role of nsa in both fracture types . the full details of each of the three studies have already been published , but they will be described briefly [ 1214 ] . in all three , low trauma fractures were defined as those occurring spontaneously without trauma or following a fall from standing height or less . data were collected from the cornwall hip fracture study of men with low trauma femoral neck hip fractures . one hundred consecutive admissions of men over 50 years with low trauma hip fractures to the royal cornwall hospital in truro between 1995 and 1997 were recruited . one hundred age - matched controls were recruited concurrently from a large general practice within the catchment area of the hospital . fracture subjects were recruited during their admission , so it was only possible to perform dxa scans on 62 men with hip fracture ( 31 with femoral neck , 31 with trochanteric fractures ) and 100 control subjects . of the men with trochanteric fractures , only 16 could have their nsa measured because the rest had bilateral hip fractures , so no hip dxa could be performed . men referred to the bone clinic in newcastle upon tyne with symptomatic low trauma vertebral fractures aged 80 years or less were invited to take part in the study . the spine radiographs were reviewed to confirm the presence of at least a 20% reduction in anterior and/or posterior vertebral height . control subjects were recruited from the age - sex registers of general practitioners to match the age of the index case within two years . those with a previous diagnosis of osteoporosis were excluded . of the control subjects who agreed to take part ( 43% of those approached ) , one was selected at random to serve as the control and underwent the same clinical assessment and investigations as the patients with vertebral deformation . spinal radiographs were not taken in the control subjects however , because of the relatively high - radiation exposure involved . in total , 91 case - control pairs were recruited . a retrospective case - control study design was chosen and all men aged 4080 years who had suffered a distal forearm fracture between 1996 and 1998 were identified from the accident and emergency department records of attendance at derbyshire royal infirmary . the case notes and x - ray reports were then examined to confirm the fracture and eligibility . in this way , 147 men were identified of whom 103 responded to questionnaires and 67 agreed to dual energy x - ray absorptiometry ( dxa ) scanning . a total of 198 age - matched control subjects were selected from a preexisting local database of 692 healthy men without distal forearm fractures , so that two control subjects were matched with each man with fracture taking part in the study . in all studies , dxa was used to determine scan area ( cm ) , bmc ( g ) , and areal bmd ( g / cm ) . the lumbar spine ( l1 to l4 ) and hip ( total hip , femoral neck ) were measured . hip measurements were always taken from the left side , unless there was a fracture or joint replacement . dxa scanning was performed using either hologic qdr 1000 or qdr 2000 equipment ( hologic instruments , waltham , mass , usa ) [ 1214 ] , but there was no consistent difference in measurements obtained with the two machines . daily calibration checks were performed using the hologic spine phantom and had a coefficient of variation of 0.5% throughout the studies . in vivo precision for measurement with these systems is 1.0% at the lumbar spine ( l1l4 ) and 1.5% for the femoral neck . although the hologic 1000 machine was a pencil - beam machine demonstrating virtually no magnification error , the hologic 2000 dxa scanner included a fan beam capability and so created the potential for magnification errors . however , we have previously found the effect of possible magnification on nsa to be minimal using a fan beam scanner . subjects were all positioned on the dxa using the standardised international recommendations as described recently . for completeness , the following is extracted from the article : the patient is positioned straight on the table ( spine is straight on the image ) , not rotated ( spinous processes are centred ) and centred in the field ( roughly equal soft tissues fields on either side of the spine ) . the patient has the femur positioned straight on the table ( shaft parallel to the edge of the picture ) , with 1525 of internal rotation where possible , achieved by the use of a single positioning device , thereby presenting the long axis of the femoral neck perpendicular to the x - ray beam , providing the greatest area and the lowest bmc ( and the lowest bmd ) . this is confirmed on the scan by seeing little or none of the lesser trochanter . such standardisation of subject position should reduce error in measuring the nsa , although extreme angles of anteversion at the hip were not specifically excluded . the nsa was measured from a hologic standard dxa scan printout using a method adapted from that of faulkner et al . and quereshi et al . , 2001 a line was then drawn manually from the junction between the greater trochanter and the femoral neck down to a point in the middle of the shaft at the bottom of the scan ( figure 1 ) . the junction of these two lines gives the femoral nsa , which was measured with a long - armed protractor with 0.5 intervals ( a biomet inc . the method described gave an intraobserver error of 0.79% , interobserver error of 1.2% , and precision of 1.2% . the details of how these errors and precision were derived have been given in our previously published paper and are similar to those given in other papers in this field [ 5 , 6 , 8 , 1618 ] . statistical analysis was performed using standard statistical software packages ( graphpad prism ) and spss for windows ( spss inc . descriptive statistics were obtained and data were tested for normality using kolmogorov - smirnov test for gaussian distribution . each of the three case - control studies available had their nsas measured in men with fractures and control subjects . these were then examined separately to look for any correlations with age , height , weight , and bmd using pearson correlation coefficients . the groups were then compared via student 's t - tests ( unpaired ) to see if there was any significant difference in nsa between fracture and control subjects ( figure 2 ) . chi - squared tests were performed to compare proportions . as there were significant differences in height and weight , ancova tests were performed in order to adjust the nsa results for these covariables . table 1 summarises the anthropometric and bmd data for the three individual studies , all of which have been previously published [ 1214 ] . only the vertebral fracture study demonstrated any significant height differences between fracture and control subjects , presumably because of height loss associated with vertebral fractures . weight was significantly lower in the men with hip and vertebral fractures compared with their respective control subjects , but not in the forearm study . table 2 shows the correlations found between nsa and age , height , weight , and bmd at the lumbar spine , femoral neck , and total femur for each of the study groups . the only significant correlations identified were inverse relationships with height and lumbar spine bmd amongst control subjects in the forearm fracture study and with height alone in the control subjects in the vertebral fracture study the means , ranges , and standard deviations in each control group are all very similar ( table 3 ) . the mean nsa for men with forearm fractures was significantly smaller than that of control subjects , whereas it was significantly larger in the men with vertebral fractures . the nsa data for hip fracture subjects was first of all analysed by each fracture type to establish whether or not there was any difference between them . the femoral neck fractures had a mean of 129.8 , sd of 6.155 , and range of 111 to 143.5 compared with mean 130.6 , sd 5.228 , and range 121.5 to 139 for the trochanteric fracture group , with no significant difference between them ( p = 0.67 ) . neither was there any significant difference in nsa between the femoral neck fracture group and control subjects ( p = 0.31 ) , nor the trochanteric and control group ( p = 0.90 ) . . there was no significant difference seen between the men with hip fractures ( combined data ) compared with control subjects . combining all data showed no significant differences in nsa between fracture subjects ( mean 130 and sem 0.29 ) and control subjects ( mean 129.9 and sem 0.18 ) : p = 0.88 . the results are shown in tables 4 , 5 , and 6 and show that doing so results in no significant difference in nsa between fracture groups and control subjects , except when nsa is adjusted for height in the hip fracture group when the difference just makes significance at p = 0.02 . in all three case - control studies , bmd has been found to be significantly lower in the fracture groups than control subjects , with significantly higher proportions osteoporotic . the measurement of nsas from dxa scan printouts has produced very consistent means , ranges , and standard deviations across the studies . they are also similar to those described in our previous work in men from the newcastle thousand families study , which gave a femoral nsa of 130 and sd of 3.3 and range of 121138 . furthermore , the mean values and ranges are similar to those reported in other studies [ 5 , 6 , 8 , 1618 ] . there were few correlations between nsa and height and bmd ; those that were observed could well have been the result of multiple testing . the lack of change with age would suggest that the nsa is fixed over time . a study in finland also found no relationship between age and nsa , but did confirm that men had larger nsas than women . no significant difference in nsa could be found between those with hip fractures and control subjects and between the fracture groups and control groups as a whole . furthermore , the nsa results for the distal forearm fracture and vertebral fracture studies were conflicting , being in opposite directions . when all data were combined , there was no significant difference in nsa between those with and those without fractures . furthermore , ancova , to adjust for height and weight , resulted in the previous differences between vertebral and forearm fracture subjects and controls disappearing . the only significant difference occurred between hip fracture and control subjects after adjusting for height ( p = 0.02 ) , but there was no difference after adjusting for weight . this suggests that there is no role for nsa in predisposing to hip fractures in men from the united kingdom . 1996 ) showed that men with hip fractures have a wider pelvis , shorter hal , wider femoral necks , and larger nsas than male control subjects . a larger study by gmez alonsoet al . ( 2000 ) found that one standard deviation increase in nsa or fnw approximately doubled the risk of hip fracture in men , but there was no association with hal . these contradictions could be due to the wide geographic differences in hip geometry that have been reported [ 7 , 8 ] , and data may need to be specific for race and gender . however , a recent large chinese study published by zhang et al . , including 4067 men ( 38 with hip fractures ) across an age range from 15 to over 85 years , confirmed our findings . the nsa did not change with age and there was no significant difference in nsa between hip fracture subjects and controls . it is relatively small and it is possible that larger studies could reveal important , but smaller effects of nsa . it was also unable to assess other aspects of structure and geometry , such as fnw which may be important in determining hip fracture risk in addition to low bmd . such factors may also contribute to the known increased risk of hip fracture following vertebral or forearm fractures . the vertebral fracture study included neither vertebral morphometry nor spinal radiographs of the control subjects and so could not exclude the possibility of asymptomatic fracture and , indeed , was never designed to do so . approximately , 2025% of vertebral fractures are clinically diagnosed and therefore the control group may not have been a true control population , which may have altered the results obtained . however , there was a significant difference in height between the vertebral fracture group and control subjects . one particular strength of the hip fracture study is that all the hip fracture subjects had femoral neck fractures . there have been differences in geometry reported between trochanteric and femoral neck hip fractures [ 18 , 19 ] , and so it is important to investigate the possible geometric contributions to these fractures separately . it is worth noting that the men in the hip fracture study had a larger standard deviation than in the other groups . these men had their dxa scans performed whilst they were in hospital , and it is possible that the recent fracture made it more difficult for them to lie in the ideal scanning position . it has given consistent results in terms of means , ranges , and standard deviations in all the studies in which it was used . in our previous work , men were shown to have larger femoral nsas than women , despite their lower fracture risk . furthermore , the results of nsa measurements in the forearm , vertebral fracture , and hip fracture studies could find very little evidence to support a role for nsas even after ancova testing to adjust for height and weight as covariables . this suggests that nsa is not an important determinant of hip fracture risk in english men . other aspects of geometry and structure may be more important risk factors and need evaluation .
in most cases , alzheimer s disease ( ad ) is preceded by a prodromal stage and amnestic mild cognitive impairment ( amci ) in which an individual s memory is impaired to a greater degree than expected given the individual s age , sex , and educational background , while the individual s ability to perform the activities of daily living is preserved and the criteria for dementia are not met . a previous study reported that the conversion rate of amci to ad is 10%15% annually , compared with 1%2% among normal elderly individuals.1 structural and functional abnormalities in the hippocampus have been documented in patients with amci and ad.2,3 atrophy of hippocampal formation is a strong risk factor of ad progress.4 as the hippocampus plays a crucial role in spatial memory and navigation,5 it is generally accepted that patients with amci or ad show impairments in spatial orientation,6 navigation,7 and visuospatial short - term memory.8 such deficits may lead to failure in navigating and remembering places . however , it is difficult to measure spatial memory deficits in real situations with a classic neuropsychological battery . several studies utilized virtual reality systems to assess cognitive impairments related to degenerative changes to improve ecological validity.7,912 previous studies reported that virtual navigation performance was impaired in amci and ad and that virtual environments provide valid assessments of navigation skills that are comparable to those required in real world navigation.9,13 moreover , examination of spatial memory can be effectively performed using virtual systems , resulting in enhanced diagnostic power compared with classic neuropsychological testing.14 the virtual radial arm maze ( vram ) , virtual morris water maze , and virtual route learning are tools commonly used in assessments of spatial memory.1517 the vram , a human analogue of the radial arm maze ( ram ) test used extensively to test spatial memory in small animals such as rodents,18,19 has two merits to examine spatial working memory and reference memory simultaneously when compared to the virtual morris water maze and virtual route learning . the current vram design uses a computer human interface , and human subjects use monitors and joysticks to control their movements inside the virtual reality in which the classic design of ram is implemented . the vram has been successfully used in human studies on sex differences in these two types of spatial memory,20 on working memory load in elderly patients,21 and on hippocampal activity.22 the hippocampus and prefrontal cortex of rodents and humans are involved in spatial navigation.22,23 evidence from experiments with rodents indicates that memory functions involve information processing in the hippocampus and prefrontal cortex.24,25 however , spatial reference and working memory systems use different neuronal networks;26 the prefrontal cortex is involved in working memory , whereas the hippocampal region is involved in reference memory.12 a study in rodents also reported that deficits in the two types of spatial function have different patterns ; reference memory deficits are common as a function of aging , but working memory deficits are not.27 as in rodents , spatial reference memory in humans is mediated by the hippocampus,28 whereas spatial working memory is more closely related to the frontal cortex.29,30 this study aimed to identify spatial memory impairment in patients with amci and ad using the vram and to determine whether the vram can differentiate between the two types of memory deficits . beginning with the onset of amci , patients with ad typically show early changes in the hippocampus.31 thus , we expected that both amci and ad patients would have reference memory deficits attributable to hippocampal atrophy.32 ad pathology is also associated with spatial working memory problems as measured by the spatial span backward test.31 this may be related to the fact that prefrontal cortical atrophy is more prominent in ad than in amci.33 therefore , we expected that spatial reference memory deficits would appear in both amci and ad patients and working memory loss would be found only in patients with ad . we also followed amci participants for 5 years to find a virtual navigation predictor of progression from amci to ad.1 all procedures in this study were in accordance with the ethical standards of the responsible committee on human experimentation and with the helsinki declaration of 1975 , as revised in 1983 . eligibility criteria for participation were 1 ) age 60 years or older and 2 ) no known history of head trauma , brain tumor , stroke , mental retardation , or any severe medical , neurological , or psychiatric illness affecting cognitive functioning other than ad . patients with mild depression were included only if their scores on the short form of the geriatric depression scale were lower than 10.34 all participants were assigned to one of three groups : normal control ( nc ) ( n=20 ) , amci ( n=20 ) , or mild ad ( n=20 ) . the nc group had no memory complaints , scored in the normal range on standardized neuropsychological tests , and had no neurological abnormalities . amci patients met the criteria for amci initially proposed by petersen:1 1 ) memory complaints , preferably corroborated by an informant ; 2 ) memory impairment relative to age- and education - matched healthy individuals ( below 1.0 standard deviation ) ; 3 ) intact general cognitive functioning ; 4 ) largely intact activities of daily living ; and 5 ) absence of dementia . ad was diagnosed according to the national institute of neurological and communicative diseases and stroke / alzheimer s disease and related disorders association criteria for probable ad,35 and subjects were mildly demented , scoring 0.5 or 1 on the clinical dementia rating ( cdr ) global scale.36 after 5 years , in eleven of the 20 amci patients , ad was newly developed ( amci converters ) , seven still had amci ( amci nonconverters ) , and two were lost to follow - up . a trained psychologist administered the korean mini - mental state examination ( mmse),37,38 cdr , and neuropsychological memory and visuospatial tests to the subjects . visuospatial construction and memory were assessed with the simplified rey figure test ( srft).39 working memory was examined using the spatial span forward and backward tasks to validate the vram . a trained psychiatrist made the clinical diagnoses in consultation with a consensus conference at which the clinical and neuropsychological data were reviewed . an original virtual maze program , which included a maze with six arms and treasures at the distal end of three arms , was used as the vram test tool ( figure 1 ) . participants were told that they were in a virtual room with six arms extending from a middle area . the virtual room had various colored objects and visual cues to indicate the relative directions , and the room remained unchanged throughout each trial . although participants were instructed to find the three treasures as quickly as possible , no time limit was imposed . after discovering all three treasures , the trial ended , and participants returned to the center of the maze to begin the next trial . this test measured working memory errors by the number of times a subject reentered the same arm ; reference memory errors were measured by the number of times a subject reentered the arms with no rewards.22 distance traveled and time required to find all rewards during each trial were also recorded . all subjects were visually and verbally informed that the test would occur in a virtual room . the apparatus for the vram consisted of a desktop computer with a color monitor and a joystick . all subjects participated in pretrial training before the task . trained psychologists who did not participate in neuropsychological tests guided the subjects to ensure they were completely familiar with the virtual environment , the rules of the game , and the manipulation of joysticks . irrespective of disease severity , all subjects were clearly able to perform the tasks without any problems . five years after the vram test , the amci participants were surveyed and categorized into amci converters and amci nonconverters . the original vram trial results previously gathered the mean age , years of education , and mmse scores were compared using analysis of variance ( anova ) of the three groups . time latency , distance , number of working memory errors , and number of reference memory errors in the five trials of the vram test were compared using repeated measures anova with the bonferroni adjustment for multiple testing . we used kendall s tau - b to analyze the correlation between the vram results ( working and reference memory errors , time , and distance traveled ) and other neuropsychological memory tests to examine the concurrent validity of the vram . all procedures in this study were in accordance with the ethical standards of the responsible committee on human experimentation and with the helsinki declaration of 1975 , as revised in 1983 . eligibility criteria for participation were 1 ) age 60 years or older and 2 ) no known history of head trauma , brain tumor , stroke , mental retardation , or any severe medical , neurological , or psychiatric illness affecting cognitive functioning other than ad . patients with mild depression were included only if their scores on the short form of the geriatric depression scale were lower than 10.34 all participants were assigned to one of three groups : normal control ( nc ) ( n=20 ) , amci ( n=20 ) , or mild ad ( n=20 ) . the nc group had no memory complaints , scored in the normal range on standardized neuropsychological tests , and had no neurological abnormalities . amci patients met the criteria for amci initially proposed by petersen:1 1 ) memory complaints , preferably corroborated by an informant ; 2 ) memory impairment relative to age- and education - matched healthy individuals ( below 1.0 standard deviation ) ; 3 ) intact general cognitive functioning ; 4 ) largely intact activities of daily living ; and 5 ) absence of dementia . ad was diagnosed according to the national institute of neurological and communicative diseases and stroke / alzheimer s disease and related disorders association criteria for probable ad,35 and subjects were mildly demented , scoring 0.5 or 1 on the clinical dementia rating ( cdr ) global scale.36 after 5 years , in eleven of the 20 amci patients , ad was newly developed ( amci converters ) , seven still had amci ( amci nonconverters ) , and two were lost to follow - up . a trained psychologist administered the korean mini - mental state examination ( mmse),37,38 cdr , and neuropsychological memory and visuospatial tests to the subjects . visuospatial construction and memory were assessed with the simplified rey figure test ( srft).39 working memory was examined using the spatial span forward and backward tasks to validate the vram . a trained psychiatrist made the clinical diagnoses in consultation with a consensus conference at which the clinical and neuropsychological data were reviewed . an original virtual maze program , which included a maze with six arms and treasures at the distal end of three arms , was used as the vram test tool ( figure 1 ) . participants were told that they were in a virtual room with six arms extending from a middle area . the virtual room had various colored objects and visual cues to indicate the relative directions , and the room remained unchanged throughout each trial . although participants were instructed to find the three treasures as quickly as possible , no time limit was imposed . after discovering all three treasures , the trial ended , and participants returned to the center of the maze to begin the next trial . this test measured working memory errors by the number of times a subject reentered the same arm ; reference memory errors were measured by the number of times a subject reentered the arms with no rewards.22 distance traveled and time required to find all rewards during each trial were also recorded . all subjects were visually and verbally informed that the test would occur in a virtual room . the apparatus for the vram consisted of a desktop computer with a color monitor and a joystick . all subjects participated in pretrial training before the task . trained psychologists who did not participate in neuropsychological tests guided the subjects to ensure they were completely familiar with the virtual environment , the rules of the game , and the manipulation of joysticks . irrespective of disease severity , all subjects were clearly able to perform the tasks without any problems . five years after the vram test , the amci participants were surveyed and categorized into amci converters and amci nonconverters . the original vram trial results previously gathered were compared between the two groups for any significant group effect . the mean age , years of education , and mmse scores were compared using analysis of variance ( anova ) of the three groups . time latency , distance , number of working memory errors , and number of reference memory errors in the five trials of the vram test were compared using repeated measures anova with the bonferroni adjustment for multiple testing . we used kendall s tau - b to analyze the correlation between the vram results ( working and reference memory errors , time , and distance traveled ) and other neuropsychological memory tests to examine the concurrent validity of the vram . the sample consisted of 60 elderly individuals : 20 with ad , 20 with amci , and 20 nc subjects . demographic data and mean mmse scores are presented in table 1 . the groups had similar mean ages ( f=0.6 , p=0.53 ) and sex distributions ( p=0.81 ) , but they differed significantly in years of education ( f=5.8 , p<0.05 ) and mmse scores ( f=38.6 , p<0.05 ) . post hoc analysis revealed that those in the ad group were less educated and had lower mmse scores compared with those in the nc and amci groups ( p<0.05 ) . repeated measures anovas ( figure 2 ) revealed a significant main effect of number of trials on working ( f=8.0 , df=4 , partial =0.37 , p<0.05 ) and reference ( f=20.0 , df=4 , partial =0.60 , p<0.05 ) memory errors . additionally , we found a significant effect of group on working ( f=12.0 , df=2 , partial =0.30 , p<0.05 ) and reference ( f=17.7 , df=2 , partial =0.38 , p<0.05 ) memory errors . according to the post hoc analysis , amci and nc participants committed a comparable number of working memory errors ( p=0.1 ) , but both groups committed fewer working memory errors ( p<0.05 ) than the ad subjects . amci subjects committed more reference memory errors than nc subjects ( p<0.05 ) and committed a similar number of reference memory errors ( p=0.4 ) to ad subjects . we observed a significant main effect of trial on distance traveled to find the rewards ( f=20.3 , df=4 , partial =0.60 , p<0.05 ) ; hence , all three groups traveled shorter distances to find the rewards as the trials progressed . a significant main effect of group on distance traveled to find the rewards ( f=17.8 , df=2 , partial =0.39 , p<0.05 ) was also observed . specifically , nc subjects found the rewards after traveling shorter distances than amci subjects ( p<0.05 ) , and amci subjects found the rewards after traveling shorter distances than ad subjects ( p<0.05 ) . finally , the data reflected a significant main effect of trial on time latency to find the rewards ( f=20.0 , df=4 , partial =0.60 , p<0.05 ) . moreover , we found a significant group effect of latency ( f=15.8 , df=2 , partial =0.36 , p<0.05 ) , revealing that nc subjects found the rewards more quickly than amci subjects ( p<0.05 ) , whereas the time to find the rewards did not differ between amci and ad subjects ( p=0.18 ) . after following patients who were tested 5 years ago , values of the latency , travel distance , and number of working and reference memory errors were compared between the amci converter and amci nonconverter groups . despite the small sample , the amci converter group made significantly more reference memory errors on the vram than the amci nonconverter group ( figure 3 , p<0.05 ) . however , the groups did not significantly differ regarding latency ( p=0.08 ) , travel distance ( p=0.53 ) , and working memory errors ( p=0.1 ) . table 2 shows the correlations between number of working and reference memory errors in vram trials , and neuropsychological test results of spatial span forward and backward , srft copy , srft immediate recall , and srft delayed recall . the numbers of working and reference memory errors on the vram had significant linear correlations with each other and with scores on all neuropsychological tests except the spatial span forward . the sample consisted of 60 elderly individuals : 20 with ad , 20 with amci , and 20 nc subjects . demographic data and mean mmse scores are presented in table 1 . the groups had similar mean ages ( f=0.6 , p=0.53 ) and sex distributions ( p=0.81 ) , but they differed significantly in years of education ( f=5.8 , p<0.05 ) and mmse scores ( f=38.6 , p<0.05 ) . post hoc analysis revealed that those in the ad group were less educated and had lower mmse scores compared with those in the nc and amci groups ( p<0.05 ) . repeated measures anovas ( figure 2 ) revealed a significant main effect of number of trials on working ( f=8.0 , df=4 , partial =0.37 , p<0.05 ) and reference ( f=20.0 , df=4 , partial =0.60 , p<0.05 ) memory errors . additionally , we found a significant effect of group on working ( f=12.0 , df=2 , partial =0.30 , p<0.05 ) and reference ( f=17.7 , df=2 , partial =0.38 , p<0.05 ) memory errors . according to the post hoc analysis , amci and nc participants committed a comparable number of working memory errors ( p=0.1 ) , but both groups committed fewer working memory errors ( p<0.05 ) than the ad subjects . amci subjects committed more reference memory errors than nc subjects ( p<0.05 ) and committed a similar number of reference memory errors ( p=0.4 ) to ad subjects . we observed a significant main effect of trial on distance traveled to find the rewards ( f=20.3 , df=4 , partial =0.60 , p<0.05 ) ; hence , all three groups traveled shorter distances to find the rewards as the trials progressed . a significant main effect of group on distance traveled to find the rewards ( f=17.8 , df=2 , partial =0.39 , p<0.05 ) was also observed . specifically , nc subjects found the rewards after traveling shorter distances than amci subjects ( p<0.05 ) , and amci subjects found the rewards after traveling shorter distances than ad subjects ( p<0.05 ) . finally , the data reflected a significant main effect of trial on time latency to find the rewards ( f=20.0 , df=4 , partial =0.60 , p<0.05 ) . moreover , we found a significant group effect of latency ( f=15.8 , df=2 , partial =0.36 , p<0.05 ) , revealing that nc subjects found the rewards more quickly than amci subjects ( p<0.05 ) , whereas the time to find the rewards did not differ between amci and ad subjects ( p=0.18 ) . after following patients who were tested 5 years ago , values of the latency , travel distance , and number of working and reference memory errors were compared between the amci converter and amci nonconverter groups . despite the small sample , the amci converter group made significantly more reference memory errors on the vram than the amci nonconverter group ( figure 3 , p<0.05 ) . however , the groups did not significantly differ regarding latency ( p=0.08 ) , travel distance ( p=0.53 ) , and working memory errors ( p=0.1 ) . table 2 shows the correlations between number of working and reference memory errors in vram trials , and neuropsychological test results of spatial span forward and backward , srft copy , srft immediate recall , and srft delayed recall . the numbers of working and reference memory errors on the vram had significant linear correlations with each other and with scores on all neuropsychological tests except the spatial span forward . the present study found differences in the spatial memory abilities of nc , amci , and ad groups . participants with amci made more spatial reference memory errors than nc subjects , but their spatial working memory seemed intact . ad subjects made more errors in both spatial working and reference memory than did nc subjects . the 5-year follow - up analysis showed that the amci converter group made more spatial reference memory errors before developing dementia than the amci nonconverter group . amci subjects traveled shorter distances on the vram to find the rewards than ad subjects , and were as slow as ad subjects in finding the rewards . spatial working memory functions have been found to be controlled by the prefrontal cortical area , which is damaged in the progress of ad,32,33 and that spatial reference memory functions are affected by hippocampal atrophy that emerges early in the prodromal amci stage.28,31 a y - maze study showed that spatial working memory may be preserved longer than spatial reference memory in transgenic mice expressing ad pathology.2 therefore , our results suggest that the loss in spatial reference memory may happen earlier during the amci stage , before the spatial significant working memory loss associated with the progress of ad appears . in addition , the current results ( figure 2 ) indicate that spatial reference memory is significantly more impaired in amci patients who develop ad within 5 years than in those who do not . vram test results may be used to detect amci which can progress on to ad . this impairment in locomotion is reportedly caused by a general deterioration in visual attention and visual processing speed,40 as well as by deficits in global cognitive ability and perceptual speed.41 patients with amci also show attention deficits and process visual stimuli more slowly , although these impairments are less severe in amci than in ad.42 ad and amci patients are unable to disengage attention and use a visual cue to produce an alerting effect.43 consistent with previous studies , amci and ad subjects showed increased time latency , reflecting a decline in visual stimulus processing and attention . interestingly , amci subjects traveled shorter distances , but with a similar latency when compared to ad patients . this may be indicative of intact spatial working memory in amci which helped to prevent reentry into previously entered rooms despite decline in the ability to process visually the landmarks in the vram . the results of the vram had linear correlations with those of the spatial span backward and srft tests . the statistical significance suggests that the vram s ability to detect spatial memory deficits may be comparable to that of neuropsychological tests . however , the spatial working and reference memory errors in the vram tests were not significantly correlated with those in the spatial span forward task ( p>0.05 ) , which assesses passive short - term memory . whereas the spatial span backward test involves more active attention processes,44 the spatial span previous studies have indicated that passive short - term memories are more resilient and are less likely to be impaired in amci and mild ad,31 which is why the spatial span forward test can not examine spatial impairments in amci and ad , and it was not correlated with the vram results . due to the small number of amci patients available for follow - up , significant differences in other spatial memory functions between ad converters and nonconverters may not be observed . in addition , although statistically significant , the linear correlations between vram results and the neuropsychological tests ( table 2 ) were not strong . a larger sample size will improve the quality of statistical data in future investigations . we also assumed that we could completely separate the two different types of spatial memories by counting the number of reentries into emptied arms and selections of wrong arms . however , to test this hypothesis , future research should provide brain imaging analyses to find different brain activation during the occurrence of reference memory and working memory errors . amci subjects differed from ad subjects in their intact spatial working memory , whereas both amci and ad subjects demonstrated impaired spatial reference memory , suggesting that the vram can help to distinguish among deficits associated with normal aging , amci , and ad . additionally , the vram results regarding reference memory errors may be useful as a clinical marker of possible future development of ad among current amci subjects , as shown in the 5-year follow - up . our study shows that the vram can be used to examine impairments in spatial working and reference memories in the early stage of ad .
heart failure ( hf ) is a common complication of myocardial infarction ( mi ) , which may develop early or late and persist , resolve or recur . a growing proportion of patients with mi are aged > 65 years . older patients are at greater risk of developing hf and have a poorer prognosis. surprisingly , the complex pattern and timing of the development and resolution of hf and the importance of such distinctions has not been quantified in relationship to age . , understanding the drivers of morbidity and mortality after mi is important , given the great difference in mortality rates reported in clinical trials of mi compared to epidemiological studies . improved understanding about which patients are at risk and the nature of the risk could help focus attention on patients at greater need , to ensure that they receive appropriate therapy and that they are targeted for recruitment into clinical trials , which currently have rather low event rates . treatment can only help patients who are at risk of complications that the treatment aims to prevent . two hospitals in hull and the east riding of yorkshire ( uk ) are sole providers of acute cardiac services for about 560,000 people . mi 's during 1998 were identified from the hospitals information department and confirmed by case note review . the case records of all patients were reviewed to identify use of loop diuretics and if so whether this was due to symptoms or signs of hf . the occurrence of major events , such as recurrent mi , and stroke were recorded . at least two of the following five criteria were used to confirm a diagnosis of mi : ( 1 ) prolonged cardiac chest pain ; ( 2 ) increases in biomarkers ( in 1998 , usually creatinine kinase ( ck ) or ck - mb mass ) ; ( 3 ) electrocardiographic changes of mi or new - onset left bundle branch block ; ( 4 ) sudden unexpected death ; and ( 5 ) autopsy evidence of mi . heart failure was defined either as signs and symptoms consistent with that diagnosis ( principally breathlessness and signs of fluid retention ) resulting in treatment with loop diuretics . use of loop diuretics for the treatment of hypertension or renal failure was not included in the definition of hf . criteria for left ventricular systolic dysfunction ( lvsd ) were left ventricular ejection fraction ( lvef ) < 40% or a qualitative report of moderate or severe lvsd . patients were categorised into three age groups : ( 1 ) < 65 years ; ( 2 ) 6575 years and ( 3 ) > 75 years . consistent with european society of cardiology guidelines , resolution of heart failure was defined as the withdrawal of diuretics without the recurrence of symptoms . data were entered into a microsoft access database and analysed using spss inc . , version 13.0 ( uk , ltd . ) . continuously distributed data are presented as median and inter - quartile range ( iqr ) . groups of patients with and without hf were compared by the chi - squared test . kaplan - meier ( k - m ) curves were generated to illustrate patients ' overall survival , and in subgroups . k - m curves were compared by the log - rank test on the appropriate degrees - of - freedom . cox regression was used to look at mortality , from which hazard ratios ( hrs ) and 95%cis were calculated . the cox regression model is semi - parametric in the sense that no assumption concerning event - free survival times is necessary . the cox regression model is based on the assumption that the effect of a risk factor , expressed as a hr , is constant over time . we did not build a model using automated selection methods but rather on biological variables relevant to heart failure . heart failure status was categorised into six groups : ( 1 ) no hf at any time ( this was the reference group for statistical comparisons ) ; ( 2 ) persistent hf ( phf ) , patients with hf on the index admission and persisting at follow up until death or end of follow - up ; ( 3 ) late resolution hf ( lrhf ) , patients with hf on the index admission that resolved only subsequent to discharge ; ( 4 ) recurrent hf ( rhf ) , patients with hf on the index admission that resolved prior to discharge but recurred during follow - up ; ( 5 ) transient hf ( thf ) on the index admission that resolved prior to discharge and did not recur prior to death or end of follow - up ; and ( 6 ) late - onset hf ( lohf ) , patients who did not develop hf on the index admission but who later developed hf during follow - up . two hospitals in hull and the east riding of yorkshire ( uk ) are sole providers of acute cardiac services for about 560,000 people . mi 's during 1998 were identified from the hospitals information department and confirmed by case note review . the case records of all patients were reviewed to identify use of loop diuretics and if so whether this was due to symptoms or signs of hf . the occurrence of major events , such as recurrent mi , and stroke were recorded . at least two of the following five criteria were used to confirm a diagnosis of mi : ( 1 ) prolonged cardiac chest pain ; ( 2 ) increases in biomarkers ( in 1998 , usually creatinine kinase ( ck ) or ck - mb mass ) ; ( 3 ) electrocardiographic changes of mi or new - onset left bundle branch block ; ( 4 ) sudden unexpected death ; and ( 5 ) autopsy evidence of mi . heart failure was defined either as signs and symptoms consistent with that diagnosis ( principally breathlessness and signs of fluid retention ) resulting in treatment with loop diuretics . use of loop diuretics for the treatment of hypertension or renal failure was not included in the definition of hf . criteria for left ventricular systolic dysfunction ( lvsd ) were left ventricular ejection fraction ( lvef ) < 40% or a qualitative report of moderate or severe lvsd . patients were categorised into three age groups : ( 1 ) < 65 years ; ( 2 ) 6575 years and ( 3 ) > 75 years . consistent with european society of cardiology guidelines , resolution of heart failure was defined as the withdrawal of diuretics without the recurrence of symptoms . data were entered into a microsoft access database and analysed using spss inc . , version 13.0 ( uk , ltd . ) . continuously distributed data are presented as median and inter - quartile range ( iqr ) . groups of patients with and without hf were compared by the chi - squared test . kaplan - meier ( k - m ) curves were generated to illustrate patients ' overall survival , and in subgroups . k - m curves were compared by the log - rank test on the appropriate degrees - of - freedom . cox regression was used to look at mortality , from which hazard ratios ( hrs ) and 95%cis were calculated . the cox regression model is semi - parametric in the sense that no assumption concerning event - free survival times is necessary . the cox regression model is based on the assumption that the effect of a risk factor , expressed as a hr , is constant over time . we did not build a model using automated selection methods but rather on biological variables relevant to heart failure . heart failure status was categorised into six groups : ( 1 ) no hf at any time ( this was the reference group for statistical comparisons ) ; ( 2 ) persistent hf ( phf ) , patients with hf on the index admission and persisting at follow up until death or end of follow - up ; ( 3 ) late resolution hf ( lrhf ) , patients with hf on the index admission that resolved only subsequent to discharge ; ( 4 ) recurrent hf ( rhf ) , patients with hf on the index admission that resolved prior to discharge but recurred during follow - up ; ( 5 ) transient hf ( thf ) on the index admission that resolved prior to discharge and did not recur prior to death or end of follow - up ; and ( 6 ) late - onset hf ( lohf ) , patients who did not develop hf on the index admission but who later developed hf during follow - up . of 1,012 patients with a death or discharge diagnosis of acute mi in 1998 , 116 were excluded from further analysis because they were transferred from another region or because mi could not be confirmed . this left 896 patients for analysis , of whom 311 ( 35% ) were < 65 years , 297 ( 33% ) 6575 years and 288 ( 32% ) were > 75 years ( table 1 ) . survival status was known for all patients by december 2005 , apart from 16 , 8 and 6 from each age - group , respectively . st - segment elevation myocardial infarction ( stemi ) occurred in 193 ( 62% ) , 174 ( 59% ) and 151 ( 53% ) cases . about 15% had a history of hf preceding the index event , rising from 7% in those aged < 65 years to 25% in those aged > 75 years . during the index admission , younger patients were more often treated with aspirin , statins , beta - blockers , intravenous nitrate , heparin and thrombolysis . older patients were more likely to receive loop diuretic and digoxin ( p < 0.001 ) ( table 1 ) . overall , 75 ( 24% ) patients < 65 years , 170 ( 57% ) aged 6575 years , and 235 ( 82% ) > 75 years had died by december 2005 ( figure 1 ) . figure 2 shows the overall proportion of patients that developed hf at any time during follow - up and their categorisation according to persistence , remission and timing of development of hf in different age groups . during the index hospitalization , 24 patients ( 8% ) < 65 years , 68 ( 23% ) patients 6575 years , 107 patients ( 37% ) > 75 years died with about 80% of deaths being associated with evidence of heart failure . transient heart failure was observed in 26 ( 32% ) patients < 65 years , in 27 ( 19% ) patients 6575 years , and in 21 ( 11% ) patients > 75 years , but had resolved by discharge with cessation of diuretic therapy . heart failure was present at discharge in 37 ( 12% ) patients < 65 years , 63 ( 21% ) patients 6575 years and 82 ( 28% ) patients > 75 years , which had preceded admission in approximately one third of cases in each age group . the differences for na are exaggerated because of the relative large sample sizes between the three groups , and the relatively low standard deviations ( in other words , this is a statistical quirk ) . thirty - three cases newly diagnosed as diabetic on index admission ; p - value for st calculated between three groups ( ste , no ste and other ( lbbb ) and pace ) ; treatment any time until 31 december 2005 ; evidence of left ventricular function during index admission or shortly after ; three patients age < 65 years and one 6575 years lost follow - up ; eight patients age < 65 years , two with 6575 years and two in those > 75 years lost follow - up ; three patients age < 65 years , two with 6575 years lost follow - up . arb : angiotensin receptor blockers ; cabg : coronary artery bypass grafting ; ck : creatine kinase ; ecg : electrocardiogram ; hf : heart failure ; lbbb : left bundle branch block ; lvef : left ventricular ejection fraction ; lvsd : left ventricular systolic dysfunction ; mi : myocardial infarction ; ste : st- segment elevation ; ptca : percutaneous transluminal coronary angioplasty . ( a ) : flow diagram showing the sequence of development of hf and relationship with recurrent ischemic episodes and mortality over approximately 6 years in patients less than 65 years old which admitted with an acute mi during 1998 . ( b ) : flow diagram showing the sequence of development of heart failure and relationship with recurrent ischemic episodes and mortality over approximately 6 years in patients 6575 years old which admitted with an acute mi during 1998 . ( c ) : the sequence of development of heart failure and relationship with recurrent ischemic episodes and mortality over approximately 6 years in patients more than 75 years old which admitted with an acute mi during 1998 . ( a ) : > 65 years ; ( b ) : 6575 years and ( c ) : > 75 years . hf : heart failure ; mi : myocardial infarction ; thf : transient heart failure . amongst patients aged < 65 years , 6575 years and > 75 years with persistent heart failure at discharge , crude mortalities at six years were 41% , 70% and 76% , respectively . amongst patients with transient heart failure during the index admission , it recurred in 46% , 56% and 67% and of these 23% , 56% and 81% died in each age group , respectively . amongst patients who did not have heart failure at discharge , 25% , 41% and 50% subsequently developed hf and of these 26% , 62% and 76% died in each age group , respectively . thus of 271 , 221 and 175 patients aged < 65 years , 6575 years and > 75 years who survived to discharge and were not lost to follow - up , 50 ( 18% ) , 102 ( 46% ) and 126 ( 72% ) subsequently died , of whom 35 ( 70% ) , 93 ( 91% ) and 107 ( 85% ) first developed hf ( figures 3a , 3b ) . a report on lv function during or shortly after the index admission was available in 228 ( 83% ) , 175 ( 81% ) and 104 ( 60% ) surviving patients in the three age groups and in 16 , 24 and 31 patients who died during the index admission ( table 1 ) . lvsd was associated with a greater likelihood of developing heart failure and a worse prognosis . of patients who died after the index admission , 23 ( 45% ) , 67 ( 66% ) and 78 ( 61% ) patients died during a re - admission to hospital in each of the three age groups ( table 2 ) . little detailed information was available for out - of - hospital deaths but review of existing data suggested that most were unexpected and probably sudden . patients with hf during the index admission had the poorest survival across all age groups ( figure 3a and b ) particularly if phf . the increased risk conferred by phf was marked for patients aged 6575 years and less pronounced for patients aged > 75 years , most of whom developed hf and had a poor outcome even if they were not reported to develop hf ( table 3 ) . ( a ) : prognosis amongst patients discharged after the index myocardial infarction in different age groups ( > 65 years , 6575 years and > 75 years ) with any hf ( persistent or transient ) and those who never developed hf ; ( b ) : kaplan - meier curves showing prognosis amongst patients discharged after the index myocardial infarction with and without transient or persistent heart failure according to different age group ( > 65 years , 6575 years and > 75 years ) . for statistical comparisons one patient with age > 75 years had missing data during last admission ; with age 6575 years one patients died of self - poisoning , with age < 65 years one patient had three vessel disease and was waiting for cabg , one patient had three vessel disease and was waiting for ptca and another had lad disease but were not suitable for surgery and one patient 6575 year old had severe pulmonary hypertension ; severe hf during one month prior to death of whom two had missed hf ( chest x - rays report were pulmonary oedema after death ) ; lvsd in last cardiac imaging prior to death p - values not calculated owing to small cell numbers . cabg : coronary artery bypass grafting ; hf : heart failure ; lad : left anterior descending ; lvsd : left ventricular systolic dysfunction ; ptca : percutaneous transluminal coronary angioplasty ; scd : sudden cardiac death . amongst patients aged < 65 years , 6575 years and > 75 years with persistent heart failure at discharge , crude mortalities at six years were 41% , 70% and 76% , respectively . amongst patients with transient heart failure during the index admission , it recurred in 46% , 56% and 67% and of these 23% , 56% and 81% died in each age group , respectively . amongst patients who did not have heart failure at discharge , 25% , 41% and 50% subsequently developed hf and of these 26% , 62% and 76% died in each age group , respectively . thus of 271 , 221 and 175 patients aged < 65 years , 6575 years and > 75 years who survived to discharge and were not lost to follow - up , 50 ( 18% ) , 102 ( 46% ) and 126 ( 72% ) subsequently died , of whom 35 ( 70% ) , 93 ( 91% ) and 107 ( 85% ) first developed hf ( figures 3a , 3b ) . a report on lv function during or shortly after the index admission was available in 228 ( 83% ) , 175 ( 81% ) and 104 ( 60% ) surviving patients in the three age groups and in 16 , 24 and 31 patients who died during the index admission ( table 1 ) . lvsd was associated with a greater likelihood of developing heart failure and a worse prognosis . of patients who died after the index admission , 23 ( 45% ) , 67 ( 66% ) and 78 ( 61% ) patients died during a re - admission to hospital in each of the three age groups ( table 2 ) . little detailed information was available for out - of - hospital deaths but review of existing data suggested that most were unexpected and probably sudden . patients with hf during the index admission had the poorest survival across all age groups ( figure 3a and b ) particularly if phf . the increased risk conferred by phf was marked for patients aged 6575 years and less pronounced for patients aged > 75 years , most of whom developed hf and had a poor outcome even if they were not reported to develop hf ( table 3 ) . ( a ) : prognosis amongst patients discharged after the index myocardial infarction in different age groups ( > 65 years , 6575 years and > 75 years ) with any hf ( persistent or transient ) and those who never developed hf ; ( b ) : kaplan - meier curves showing prognosis amongst patients discharged after the index myocardial infarction with and without transient or persistent heart failure according to different age group ( > 65 years , 6575 years and > 75 years ) . for statistical comparisons see table 3 . one patient with age > 75 years had missing data during last admission ; with age 6575 years one patients died of self - poisoning , with age < 65 years one patient had three vessel disease and was waiting for cabg , one patient had three vessel disease and was waiting for ptca and another had lad disease but were not suitable for surgery and one patient 6575 year old had severe pulmonary hypertension ; severe hf during one month prior to death of whom two had missed hf ( chest x - rays report were pulmonary oedema after death ) ; lvsd in last cardiac imaging prior to death p - values not calculated owing to small cell numbers . cabg : coronary artery bypass grafting ; hf : heart failure ; lad : left anterior descending ; lvsd : left ventricular systolic dysfunction ; ptca : percutaneous transluminal coronary angioplasty ; scd : sudden cardiac death . this analysis shows that the development of heart failure after a mi increases steeply with age , that most patients who die subsequent to a mi will first develop heart failure and that heart failure is a powerful adverse prognostic factor . patients aged < 65 years were least likely to get heart failure but half developed it over the subsequent six years and 70% of deaths in this age group occurred subsequent to the onset of heart failure . for patients aged 6575 years , 73% developed heart failure during follow - up and 91% of deaths in this group occurred subsequent to the development of hf . in patients aged > 75 years , 87% developed heart failure but the prognosis was poor whether or not overt heart failure developed . few of those with documented substantial lvsd after a myocardial infarction escaped death or the development of heart failure over the subsequent six years . however , about half of patients in whom substantial lvsd had been excluded still went on to develop heart failure , of whom a large proportion died . thus the prevention and management of heart failure rather than lvsd may be the most important therapeutic target in patients with heart failure . * with reference to no hf : heart failure ; hr : hazard ratio ; mi : myocardial infarction ; phf : persistent heart failure during the index admission ; thf : transient hf during the index admission . this cohort of patients was enrolled prior to the widespread adoption of primary angioplasty and before national audits were introduced to improve the quality of care . studies show that thrombolysis and primary percutaneous angioplasty can reduce myocardial damage leading to improved long - term recovery of cardiac function and reduced mortality. this should reduce the incidence of heart failure , although evidence in support of this hypothesis is scant . ace inhibitors , angiotensin receptor blockers , , aldosterone receptor antagonists , beta - blockers and statins were not used optimally by contemporary standards . greater use might have assisted recovery in ventricular function , reduced the development of heart failure and improved prognosis . thus , our data should not be perceived as an accurate description of outcome in contemporary patients but rather likely outcome if modern standards are not applied . further cohorts should be enrolled to assess contemporary populations , recognising these must still be many years out of date if 5-year outcome is to be reported . those lucky enough to reach the catheter laboratory probably have a better prognosis than those who do not , partly due to case - selection . hopefully , improvements in care have improved the prognosis of patients with myocardial infarction . however , a repeat survey in our hospital conducted in 2005 , with much higher uptake of guideline - indicated therapy , revealed a three year mortality which was still in excess of 30% , suggesting that the prognosis of mi in epidemiologically representative cohorts of patients , not just those who get to the catheter laboratory , remains poor . the median age in our cohort in 1998 was 70 years and 35% were women . this had changed little by 2005 and is similar to that reported by the myocardial infarction national audit programme ( minap ) in the uk in 20032005 ( mean age 69 years and 36% women ) . the euro heart survey of acute coronary syndromes reported a mean age of just 63 years and 29% women amongst patients with an st elevation mi and 66 years and 36% in those with mi but no st elevation . in contemporary clinical trials of acute mi , such as the platelet inhibition and patient outcomes ( plato ) study , the median age was only 62 years , only 15% were 75 years old and 28% were women . the mean age of these patients was 61 years and only 11% were aged 75 years . in the triton - timi 38 study , the median age was 61 years and only 13% of patients were aged > 75 years . overall mortality was 3% over a median follow - up of 15 months , patients aged > 75 years were more likely to reach the primary end - point ( cardiovascular death , mi or stroke ) ( 18.3% vs. 10.6% in those assigned to clopidogrel ) . these outcomes compare to in - patient and one year mortalities in our epidemiological cohorts of 22% and 31% in 1998 and 11% and 19% in 2005 . , the difference in outcome between observational and trial data - sets could reflect differences in care but may also reflect differences in case ascertainment and selection . old , frail patients with multiple comorbidities may be excluded by the protocol or may decline to participate in trials . alternatively , investigators , for a variety of reasons including compassion and the fact that managing such cases consumes more research time and resources , may avoid enrolling frail , elderly patients . a low threshold for the detection of mi with the use of more sensitive troponin assays may also lead to an apparent improvement in prognosis , as small mis will generally have a better prognosis than large ones . however , it is also possible that compassionate clinicians decide that palliative care is appropriate and that it is no longer appropriate to try to modify the prognosis in some older patients . the prognosis of younger patients enrolled in many contemporary trials of acute coronary syndrome is now so good it may be difficult to improve . clinical trials specifically amongst older patients would be valuable as they are at high risk , both in terms of prognosis and side effects of treatment . , however , disease in older patients may be more resistant to modification by therapy . older patients have more co - morbid conditions such as af , conducting system disease , respiratory disease , renal dysfunction , anaemia and , worst of all , heart failure . in a sense , age is a surrogate for the drivers of an adverse outcome . patients aged 6575 years are at intermediate risk and this may be where the greatest therapeutic gains occur . it may be difficult to reduce risk in a group already at low risk , whereas in patients aged > 75 years , an effective therapy may still not be effective enough to make a meaningful difference in outcome . identifying and managing modifiable risk is key and it may be best to target intermediate risk to achieve the greatest benefit . systematic attempts were not made to withdraw diuretics , therefore we may have over- estimated the persistence of hf . however , patients who receive loop diuretics and who have cardiovascular disease clearly have a poor prognosis whether or not they have a low ejection fraction . ultimately , the diagnosis of hf relies on a doctor 's skill in assessing patients in the light of appropriate investigations . the development of hf precedes death in the great majority of patients who die within six years of an mi , especially amongst patients aged > 65 years . improved prevention and management of hf and its important co - morbidities may improve outcome . systematic attempts were not made to withdraw diuretics , therefore we may have over- estimated the persistence of hf . however , patients who receive loop diuretics and who have cardiovascular disease clearly have a poor prognosis whether or not they have a low ejection fraction . ultimately , the diagnosis of hf relies on a doctor 's skill in assessing patients in the light of appropriate investigations . the development of hf precedes death in the great majority of patients who die within six years of an mi , especially amongst patients aged > 65 years . improved prevention and management of hf and its important co
the university of minnesota biocatalysis / biodegradation database ( um - bbd , ) contains compound , enzyme , reaction and pathway information for microbial catabolism of primarily anthropogenic materials . it has been available on the web for over 10 years , and has grown from 4 to almost 150 pathways . as it starts its second decade , the um - bbd contains information on over 900 compounds , over 600 enzymes , nearly 1000 reactions and about 350 microorganism entries . its data content and methods , including data format , update and access , have been reported previously ( 14 ) . along with pathway data , the um - bbd now includes biochemical periodic tables and a biodegradation pathway prediction system ( pps ) . its biochemical periodic tables have grown to include biological information for almost all stable , non - noble - gas elements , and an about the biochemical periodic tables web page has been created ( ) . the pps ( ) is now an internationally recognized , open system for predicting microbial catabolism of organic compounds , described in more detail below . since its last report ( 4 ) , the um - bbd has grown overall 20% . it has added about 200 reactions and compounds and 150 enzymes . enzyme entries grow more slowly than reactions , not only because some new reactions are catalyzed by existing enzymes , but also because some are not well - characterized enough for their enzymes to be entered . now that our pathways span a wide representation of microbial catabolism , we primarily limit additions to pathways that represent new carbon skeletons or novel metabolism , and spend more time and resources keeping existing information up - to - date . as further examples of the last , links on compound pages to the national toxicology program ( ) , and on pathway pages and microorganism entries to the american type culture collection ( ) , were updated during this period . rules for microbial biotransformation of organic functional groups , derived from um - bbd data , are developed . users input a chemical structure , the pps determines the functional groups it contains , and the compound is metabolically transformed in silico using pps rules . the pps displays the resulting compounds , the user selects one , the cycle repeats and a predicted biodegradation pathway grows ( 5,6 ) . an about the pps web page has been developed ( ) . over the past 3 years for example , rule bt0024 , ester alcohol + carboxylate , is also known as lactone hydroxyacid . rules can be now be searched on by name of substrate or product , or a complete list of all rules can be browsed . rule web pages list all um - bbd reactions that exemplify each rule ; and um - bbd reaction pages list the rules they exemplify . a um - bbd reaction may exemplify up to three rules , in series . as the number of rules increases and each rule increases in complexity , the average number of biotransformations shown to the user at each prediction cycle also increases . for example , the pps initially predicted six biotransformations for benzyl alcohol [ figure 8 in ref . users increasingly complain of too many choices. to address this complaint , with support from the 6th eu framework assessing large - scale environmental risks with tested methods ( alarm ) project ( ) , on may 67 , 2005 , a predictbt workshop was held to prioritize um - bbd rules ( ) . the rules available in april 2005 were given to biodegradation content experts who each scored subsets of them as follows : 1 , very likely ; 2 , likely ; 3 , neutral ; 4 , unlikely ; 5 , very unlikely to occur under standard aerobic conditions [ 25c , soil ( moderate moisture ) or water , neutral ph , no toxic or competing chemicals ] . rules with a wider range of scores were discussed at the workshop ; participants were able to reach consensus on all of them . users may now see the color - coded score for each predicted biotransformation and optionally chose to see only the first three of the five groups , the ones most likely to occur under standard aerobic conditions ( figure 1 ) . if a user starts with benzene and chooses the most probable aerobic reaction at each step , ring opening is predicted on the third step , in a pathway generated as if a personal biodegradation consultant guided each choice . participants also recommended that displays of predicted compounds in the um - bbd indicate those predicted compounds in the um - bbd , since this may guide user choices . a new cpd button is that indication ( shown in figure 1 for the first and fourth predicted compounds ) . that button links to the um - bbd web page for that compound , and , from that , to information on its known reactions and catabolic pathways . the original system was created , in part , using the jchem software ( now jchembase ) ( 7 ) from chemaxon , inc . one limitation of the system was that rules were implemented as java function calls ( 5 ) , not readily understood by content experts . chemaxon later introduced reactor software , which provides a graphic user interface for rule creation . rules can now be created by non - programmers , and readily reviewed by content experts and general users . all new rules and modifications of existing rules are carried out in reactor ; existing rules are being converted to reactor as time permits . rules that have been moved to reactor now display graphics on the rule page ( figure 2 ) . for example , we first encoded the formation of aromatic dihydrodiols in several specific rules ( 6 ) . now there is one more general rule for most of them ( figure 2 ) , since , as we state in the public comment to this rule , aromatic hydrocarbon dioxygenases produce an activated dioxygen species that is thought to be sufficiently reactive to potentially functionalize most , if not all , aromatic ring carbon atoms . the um - bbd has always been freely available on the web and that will continue for the foreseeable future . however , some users have expressed interest in a stand - alone version that can be used behind a firewall or in other environments where the internet is not accessible . in 1999 , um - bbd users voted um - bbd on cdrom as what their institutions would be most willing to pay to use ( ) . such a stand - alone version , including the pps ( but not the biochemical periodic tables ) is being developed with support from lhasa limited , leeds , uk . lhasa limited developed meteor ( 8) , stand - alone windows software for predicting mammalian detoxification metabolism , using a functional group approach similar to that used in the pps . lhasa limited is supporting transfer of um - bbd microbial biodegradation rules into the meteor framework , to create meteor pps ( mepps ) . since 2000 , the european bioinformatics institute has mirrored the um - bbd , as part of the ebi srs server ( 9 ) . srs files are updated in synchrony with the um - bbd ; they omit the biochemical periodic tables and the pps . the mepps system described above under prediction , derived from the pps , contains a static version of the um - bbd . in principle , mepps um - bbd files could be updated synchronously with the master um - bbd . however , mepps is stand - alone software to be used at multiple sites , possibly behind a firewall . timing of updates has not yet been determined , and may be less frequent . in june 2000 , kyoto encyclopedia of genes and genomes ( kegg ) ( 10 ) began to add um - bbd information to other metabolic pathways . this is a derivative work , not a mirror , since um - bbd information is reformatted to kegg standards , only a subset of um - bbd information is used , and several um - bbd pathways may be included in one kegg graphic . presently , about one - third ( 53/146 ) of um - bbd pathways are included . kegg documents and provides links to the um - bbd pathways that are included in each kegg graphic ( see , section 1.11 , xenobiotics ) . kegg does not update its um - bbd - derived pathways in synchrony with the um - bbd . metarouter ( 11 ) is a static derivative , based on information taken from the um - bbd in 2002 . it is a proof - of - concept for alternative display and enhancement of um - bbd information . catabol ( 12 ) , a commercial biodegradability prediction system , uses um - bbd information for approximately half the pathways it contains ( ovanes mekenyan , personal communication ) . with implementation of graphical display of pps rules , development of a stand - alone version , and improvement guidance for pps users , the next decade should see the pps , and the um - bbd on which it is based , find increasing use by national and international government agencies , commercial organizations and educational institutions . the um - bbd is now approaching its teen years . as an adolescent , in the next stage of growth it will find new interests , explore new surroundings and develop new friends and colleagues . the next decade will be the deciding point : can it make the transition to maturity and independence ? the complete page appears if demo is chosen from the initial pps page ( ) . predicted compounds 1 and 4 are in the um - bbd ; these are the only displays that include a cpd button . that button links to their um - bbd compound pages , and , from there , to reaction and pathway pages . excerpt from the web page for bt0005 , a rule that has been moved to reactor . more than one pattern is used for this rule , a link to a graphic showing all of them is to its right . benzyl alcohol triggers both patterns for this rule at two different locations , producing predicted compounds 5 through 8 in figure 1 . examples button at the bottom will link to the appropriate static um - bbd rule web page ( and from that to static reaction , enzyme , compound and pathway pages ) once the prototype is complete .
preserving the vitality of traumatically injured and mechanically exposed immature teeth is one of the major treatment aims in endodontics . partial pulpotomy ( cvek pulpotomy ) and deep ( cervical ) pulpotomy techniques are the choices of treatment for immature permanent teeth with exposed pulps.1,2 these techniques can maintain pulp vitality and provide continuation of tooth root development in association with the formation of a hard tissue bridge across the exposed pulp surface.3 calcium hydroxide ( ch ) materials have long been used as the agents of choice for treating exposed pulp , due to the short - term antimicrobial effect and the stimulation of hard - tissue bridge formation.4 coagulation necrosis produced by releasing hydroxyl ions from ch materials has been believed to induce bridge formation at the exposed area.4,5 higher calcium levels and alkalinity provided by ch were also shown to lead to increased bone morphogenetic protein-2 expression and solubilization of bioactive molecule tissue growth factor-1 from dentin tissue.6,7 tgf-1 and bmp-2 may play roles in dentin bridge formation.8 moreover , calcium ions in association with necrotic cell debris may contribute to the formation of dystrophic calcification , to which fibronectin can bind and thereby initiate differentiation of odontoblast - like cells.9 mineral trioxide aggregate ( mta ) has been used as an alternative agent to ch materials in direct pulp capping and pulpotomy treatments . ch is one of the major hydration products during setting reactions of mta , and ch is soluble from mta in decreasing rates over time.10 calcium and oh ion release from mta may be essential for pulp tissue healing with bridge formation , which is similar to ch materials.8 mta generally showed better sealing ability as a root - end filling material than conventional zinc - oxide eugenol cements , and it induced the formation of more and thicker dentin bridges than ch.1114 clinical reports also demonstrated very successful results such as the maintenance of pulp vitality over longer periods of time and the continuation of root formation with mta as a pulpotomy agent.1517 in recent years , the use of mta has been popular in pulpotomy of permanent teeth showing symptoms of reversible and irreversible pulpitis and in complicated crown fractures of immature teeth and mature teeth.18,19 contrarily , the high solubility , high price and dis - coloring effect of both gray and white forms are among the disadvantages of mta , when used as a pulpotomy agent.15,20 the present case report demonstrates long - term clinical performance of mta pulpotomies in six immature permanent teeth . four patients with complicated crown fractures of 5 maxillary immature central incisor teeth were referred to the department of endodontics , istanbul university , between 2005 and 2006 . all 5 teeth were treated with deep pulpotomy using gray mta ( proroot mta ; dentsply tulsa dental , tulsa , ok , usa ) . another immature mandibular first molar that was sensitive to cold stimulation and exposed mechanically during cavity preparation was also included in this report . after completion of dental history records , all teeth were locally anesthetized and a rubber dam was positioned . exposed pulp tissue was removed to the pulp canal orifice with a sterile diamond round bur in high - speed handpiece with copious saline irrigation . hemorrhage was controlled first with 2 ml of saline irrigation and then by putting cotton pellets soaked with 5% naocl on pulp tissue for 1-minute . mta was first placed with a spatula - shaped hand instrument and then wet cotton pellets were used to adapt it onto the exposed pulp area . wet cotton pellets were put on mta ; the dentist waited for at least 1-minute to stop hemorrhage and then another piece of new mixed mta was inserted into the cavity . at layer of mta at least 3 mm thick the cavity was sealed with a wet cotton pellet and zinc - oxide eugenol cement ( kalzinol , de trey , dentsply , konstanz , germany ) . three days later , the temporary fillings were removed and restored with a composite filling ( supreme , 3 m espe , dental products , mn , usa ) . one patient came to our clinic at the year 2006 ( 10 months after pulpotomy ) with signs of acute apical periodontitis , and root canal treatment was performed ( tab . vitality tests and clinical diagnostic tests were applied , and periapical radiographs were also taken at the follow - ups . a periapical lesion around the right central incisor of patient # 5 ( the patient with 2 fractured incisors , figures . 1 , an apexification procedure was followed using ch for 9 months by renewing it at 1 week , 1 month , 3 months and then every 3 months . at the 9-month visit , the tooth was filled with laterally condensed gutta - percha and a sealer ( ah plus ; dentsply de - trey , konstanz , germany ) . the radiographic healing was assessed as uncertain at the 2012 follow - up ( figure 4 ) . all composite restorations on maxillary incisors of this patient were renewed at the 2012 follow - up ( figure 5 ) . the remaining 4 teeth ( including exposed molar and left central incisor of patient # 5 ) were assessed as vital cases at the 2010 and 2011 follow - ups ( mean time of follow - up was 55 months at 2011 ) . no signs of periapical inflammatory changes were present clinically or radiographically in any of these cases . there were radiographic signs of apical root completion in these cases . dentin bridge formations were detected clearly adjacent to mta in 3 fractured maxillary incisors radiographically ( figures 68 ) . there were no radiographic signs of pulp tissue obliteration and internal resorption in all 4 cases . all composite restorations showed insufficient marginal adaptation and color changes on marginal enamel walls at the follow - ups . four patients with complicated crown fractures of 5 maxillary immature central incisor teeth were referred to the department of endodontics , istanbul university , between 2005 and 2006 . all 5 teeth were treated with deep pulpotomy using gray mta ( proroot mta ; dentsply tulsa dental , tulsa , ok , usa ) . another immature mandibular first molar that was sensitive to cold stimulation and exposed mechanically during cavity preparation was also included in this report . after completion of dental history records , all teeth were locally anesthetized and a rubber dam was positioned . exposed pulp tissue was removed to the pulp canal orifice with a sterile diamond round bur in high - speed handpiece with copious saline irrigation . hemorrhage was controlled first with 2 ml of saline irrigation and then by putting cotton pellets soaked with 5% naocl on pulp tissue for 1-minute . mta was first placed with a spatula - shaped hand instrument and then wet cotton pellets were used to adapt it onto the exposed pulp area . wet cotton pellets were put on mta ; the dentist waited for at least 1-minute to stop hemorrhage and then another piece of new mixed mta was inserted into the cavity . at layer of mta at least 3 mm thick the cavity was sealed with a wet cotton pellet and zinc - oxide eugenol cement ( kalzinol , de trey , dentsply , konstanz , germany ) . three days later , the temporary fillings were removed and restored with a composite filling ( supreme , 3 m espe , dental products , mn , usa ) . one patient came to our clinic at the year 2006 ( 10 months after pulpotomy ) with signs of acute apical periodontitis , and root canal treatment was performed ( tab . vitality tests and clinical diagnostic tests were applied , and periapical radiographs were also taken at the follow - ups . a periapical lesion around the right central incisor of patient # 5 ( the patient with 2 fractured incisors , figures . 1 , an apexification procedure was followed using ch for 9 months by renewing it at 1 week , 1 month , 3 months and then every 3 months . at the 9-month visit , the tooth was filled with laterally condensed gutta - percha and a sealer ( ah plus ; dentsply de - trey , konstanz , germany ) . the radiographic healing was assessed as uncertain at the 2012 follow - up ( figure 4 ) . all composite restorations on maxillary incisors of this patient were renewed at the 2012 follow - up ( figure 5 ) . the remaining 4 teeth ( including exposed molar and left central incisor of patient # 5 ) were assessed as vital cases at the 2010 and 2011 follow - ups ( mean time of follow - up was 55 months at 2011 ) . no signs of periapical inflammatory changes were present clinically or radiographically in any of these cases . there were radiographic signs of apical root completion in these cases . dentin bridge formations were detected clearly adjacent to mta in 3 fractured maxillary incisors radiographically ( figures 68 ) . there were no radiographic signs of pulp tissue obliteration and internal resorption in all 4 cases . all composite restorations showed insufficient marginal adaptation and color changes on marginal enamel walls at the follow - ups . four immature teeth ( including the molar case ) treated with mta were healthy after a mean time recall 55 months at the year 2011 . in the control radiographs of these 4 cases , there was complete formation of root apices and visible dentin bridge formation in three incisor cases . these successful cases may indicate that mta could induce pulp healing with dentin bridge formation and maintain root development . there are several mta pulpotomy case reports involving immature root formation in the literature.1518 karabucak et al15 observed that mta pulpotomy was successful in two immature central incisors after 12 and 18 months of a clinical trial . el meligy and avery16 compared ch and mta pulpotomies in 30 immature permanent teeth for one year . they found that all 15 mta cases were successful , while 2 ch cases displayed periapical inflammation . whitherspoon et al17 reported that 5 of 6 immature central maxillary incisors that were treated with mta pulpotomy due to complicated crown fractures were diagnosed as healed after 13 to 15 months . barrieshi - nusair and qudeimat18 reported that all 7 immature permanent cases treated with mta pulpotomy were successful after 2 years . according to these clinical reports , mta pulpotomy has a superior success rate in developing teeth with open apices . in the present case report the primary cause of pulp inflammation after vital pulp treatment is known as bacterial infection.21 early contamination associated with treatment delay and later contamination with microleakage are the ways for bacteria to cause inflammation and necrosis in complicated crown fractures . there is no time limit for the pulpotomy of fractured immature cases when healthy pulp is clinically present.22 for unsuccessful cases 3 and 6 , the level of pulpotomy may not have been deep enough to reach healthy tissue for recovery . materials used during pulpotomy , such as naocl and mta , may have put additional pressure on pulp tissue in these cases . the distribution of mta particles during hemorrhage may also exceed the clean - up capacity of pulp tissue . such a complication following pulpotomy specifically in the anterior teeth should be accounted as a clinical failure of treatment . discoloration effect of gray mta as a pulpotomy agent in the crowns was shown and white mta was developed because of this clinical complication . however , significant tooth discoloration was also reported after the use of white mta.23 belobrov and parashos23 observed that most of the discoloration was inside white mta and did not penetrate into dentin . they questioned and did not encourage the use of white mta for vital therapy in the esthetic zone . lenherr et al24 asserted that the infiltration of blood components into the porosities within mta may be the prime cause of discoloration . the other possible reasons for tooth discoloration may be bismuth oxide , magnesium oxide and ferric oxide ingredients in mta powder.25,26 composite fillings also showed poor marginal adaptation , which may indicate the presence of coronal leakage around the fillings during service . restorations should be replaced when a possible microleakage is diagnosed clinically , in order to maintain the vitality of pulpotomized cases . mta may be used as an alternative pulpotomy agent in immature teeth with pulp exposure to stimulate pulp healing with dentin bridge formation and complete root formation .
as one of the most remarkable findings over the past 15 years , rna interference ( rnai ) is an endogenous process in which small noncoding rnas , including small interfering rnas ( sirnas ) and micrornas ( mirnas ) , post - transcriptionally regulate gene expressions by binding to their complementary mrnas . due to its unique roles in regulating the stabilities and functions of mrnas , rnai has emerged as a promising alternative for the treatment of various diseases and attracted substantial attention . once inside cells , sirnas are unwound by an atp - dependent helicase and the antisense strand is incorporated into the rna - induced silencing complex ( risc ) . subsequently , the antisense strand guides the risc to its complementary mrnas in a very specific way and triggers the degradation of target mrnas . unlike sirnas , mirnas encoded in the genome are transcribed into primary mirnas ( pri - mirnas ) in the nucleus . the pri - mirnas are then processed by ribonuclease drosha to form 75 nucleotide ( nt ) long hairpin precursor mirnas ( pre - mirnas ) , which are translocated to the cytoplasm . the dicer cleaves the pre - mirnas to form mature mirnas , which are duplex rnas . the mirna strand ( also termed guide strand ) is then separated from its complementary strand and incorporated into the risc , followed by binding to its target mrnas to suppress translation or trigger degradation of the mrnas . in general , sirna and mirna / mirna mimics are similar in nature and activity except their origin and specificity . sirnas are artificial double - stranded rnas of 1921 nt in length , while mirnas are endogenous single - stranded rnas of 2125 nt . endogenous mirnas may be either downregulated or upregulated in a pathological condition and can be brought back to normal level by mirna replacement therapy or mirna inhibition therapy . sirnas are always exogenous and need to be delivered into cell cytoplasm to silence an overexpressed disease gene . a single sirna forms a perfect match to its complementary mrna and only induces the degradation of its target mrna . on the contrary , a single mirna may target hundreds of mrnas that can form imperfect matches . unlike exogenous sirnas instead , synthetic mirna mimics ( rna duplexes containing the guide strand of the mirna ) are always used in mirna replacement therapy . although both sirnas and mirnas have been extensively studied as novel therapeutics for a wide range of diseases , the large molecular weight , anionic surface charges , instability in blood circulation , and intracellular trafficking to the risc after cellular uptake have hindered the translation of these rnas from bench to clinic . as a result , a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding rnas . among them , cationic peptides have emerged as a promising type of carrier due to their inherent ability to condense negatively charged rnas , ease of synthesis , controllable size , and tunable structure for tailoring physicochemical properties and targetability of the cargo . in general , three approaches have been used for cationic peptide - mediated rna delivery : covalent conjugation of cationic peptides to one strand of rna duplex ; noncovalent complexation of cationic peptides with rna ; and inclusion of cationic peptides as a condensing agent in a lipid or polymeric carrier . moreover , peptides can also be used as targeting ligands in rna delivery systems . direct covalent conjugation is the easiest strategy but less successful because of technical difficulties in synthesizing the cationic peptide sirna conjugates and neutralization of the positive charges , which are vital for membrane translocation and cellular uptake . some of the cationic peptide conjugated sirnas fail to improve gene silencing effect in vivo compared to unmodified sirna . it is possibly due to insufficient amount of cationic peptides , which fail to effectively condense negatively charged sirnas in this approach . in contrast , noncovalent complexation of cationic peptides with rna exhibits a significant gene silencing effect in vitro and in vivo . peptides used in such a strategy are usually composed of two domains : a hydrophilic domain and a hydrophobic domain . the hydrophilic domain contains positively charged amino acids , such as ariginine ( arg ) , lysine ( lys ) , and histidine ( his ) , to provide at least a net positive charge of + 8 . the positive charge allows the condensation of rna and enables multiple hydrogen bondings with anionic cell membrane to facilitate cellular uptake . the hydrophobic domain contains tryptophan ( trp ) and phenylalanine ( phe ) residues , which enhance interaction with the lipid bilayer of cells . moreover , one terminal extreme of the cationic peptides can be modified with hydrophobic moieties , such as cholesterol , stearic acid , and cholic acid to enhance hydrophobicity . the other terminal of the peptide can be conjugated with relatively hydrophilic moieties , such as polyethylene glycol ( peg ) . these constructs can form micelle - like structure after mixing with sirna and efficiently deliver them to target cells . to enhance stability and circulation time , a number of block copolymers of peptides , such as mpeg2000-pla3000-b - r15 , pei - g-(pll - b - peg ) , and mpeg - plga - b - pll [ where plga is poly(lactic - co - glycolic acid ) ] , alternatively , cationic peptides such as protamine can be used as the condensing component of nanocomplex to encapsulate negatively charged rnas . mirna and sirna can also be delivered using plasmid vector to achieve long - term activity . however , delivery of these plasmid forms falls within the purview of dna delivery and therefore beyond the scope of this article . in this review , we will focus on three major types of cationic peptides , including poly(l - lysine ) ( pll ) , protamine , and cell penetrating peptides ( cpp ) , as well as peptide targeting ligands that have been extensively used in rna delivery . the delivery strategies , applications , and limitations of these cationic peptides in sirna / mirna delivery will be discussed . although most of the rna delivery systems mentioned in this article are initially designed for sirna , they can also be used for mirna delivery because of the similar size and chemical properties between sirna and mirna mimics . poly(l - lysine ) , also known as alpha poly(l - lysine ) or pll , is one of the first nucleic acid carriers reported back in 1989 . pll can be synthesized by a living polymerization of n - carboxyanhydrides ( nca ) , which provides narrow chain length distributions and the ability to obtain high molecular weight poly(peptide ) polymers . various molecular weights ranging from 500 da to more than 100 kda of pll are commercially available . among them , pll ranging from 2.4 to 30 kda have been exploited for sirna delivery , and pll with the molecular weight > 70 kda is mainly recommended for enhancing cell adhesion to solid surface . naturally , polylysine appears as -poly - l - lysine ( or epl ) , which is produced by bacterial fermentation . the amide bond of epl is formed between the carboxyl terminal and the epsilon - amino groups . epl contains similar positive charges as pll and has been used in condensing nucleic acids . pll is a well optimized cationic peptide for condensation of dna molecules by electrostatic interaction between positively charged amino acids of pll and the phosphate backbone of dna . however , the molecular weight and topology of dna molecules are very different from those of small noncoding rna and other small oligonucleotides . therefore , the findings from dna complexation with cationic polymers , such as pll , can not be directly extrapolated to sirna and mirna . in fact , many factors , including polymer molecular weight , salt concentration , ph , charge ratio , and mixing order , can affect the complexation of sirna with pll . for example , zheng et al . have compared complexation characteristics of pll with a long double - stranded dna ( dsdna ) and a 21nt double - stranded oligonucleotide ( ds - oligo ) which is structurally similar to a 21nt sirna against firefly luciferase gene in the same condition . the ds - oligo forms a compact rod shape structure with high scattering intensity in complexation with pll . on the contrary , the dsdna forms a coil conformation with pll in aqueous media . these conformations are governed by charge density , rigidity , and chain length of the nucleic acids . the coiled structure of dsdna prevents an ideal match of each base pair with pll , leading to the formation of a loose structure . the linear , flexible , and shorter chain length of the ds - oligo interacts with pll in a more ordered manner that allows the formation of a compact structure . however , even though the ds - oligo forms a dense complex , the scatter density gradually decreases with incubation time due to slow dissociation of the complex . dissociation of the ds - oligo pll complex exposes oligonucleotides to external medium , leading to reduction of the transfection efficiency of these complexes . by contrast , long chains of dna entangle together and stabilize the complexes with pll . although pll has been extensively used and characterized as a cationic carrier for sirna delivery , there are few reports of successful sirna delivery by simple complexation of sirna with linear pll alone . the potential reasons behind this may be toxicity , poor endosomal release , and nonspecific binding of linear pll to serum proteins . to circumvent these limitations , several derivatives of pll have been developed to improve the efficacy of small noncoding rna delivery ( table 1 ) . one possible reason for the insufficient transfection efficacy of pll is its inability to be released from endosomes ( table 1 ) . pll complex is retained in endosomes and then transported to lysosomes , where it is digested by hydrolase enzymes . this might be attributed to the absence of fusogenic groups to facilitate endosomal release of sirna . consequently , various endosomal releasing residues , such as fusogenic peptides , chloroquine , and histidine , have been conjugated to pll to overcome this problem . the imidazole ring of histidine is a weak base that is positively charged at endosomal ph ( 6 ) and facilitates endosomal disruption by the proton sponge mechanism . for example , a reducible copolypeptide ( rcpp ) composed of a histidine - rich peptide ( hrp ) and a nuclear localization sequence ( nls ) peptide was developed for sirna delivery . in this system , three lysine residues were utilized to condense sirna , and six histidine residues were inserted to promote endosomal release of the complex . in another study , branched histidine - lysine rich ( hk ) peptides containing four ( h3k4b and h2k4b ) or eight ( h3k8b ) terminal branches were synthesized and compared for effective delivery of sirna targeting -galactosidase ( -gal ) expressed in svr - bag4 cells . the sirna complex made with the h3k8b peptide showed silencing activity up to 80% . the presence of positive charges on the surface of pll / sirna complexes is believed to enhance cell internalization . however , interaction of the positively charged pll / sirna complexes with negatively charged serum proteins may cause undesired aggregation and res uptake , which consequently decrease the therapeutic outcome of sirna . modification of pll with polysaccharides , peg , or other water - soluble polymers is the major strategy to enhance the systemic circulation profile of pll / sirna complexes by decreasing nonspecific interaction with blood components . such particles contain a cationic fragment in the inner core that helps to condense nucleic acids . the uncharged hydrophilic peg outer layer helps to reduce particle size , cytotoxicity , and nonspecific interaction with blood components , leading to prolonged systemic circulation . however , shielding of the cationic core with peg may inhibit the interaction of these complexes with the negatively charged cell membrane and eventually decrease cellular uptake and endosomal release . to overcome these obstacles , sirna delivery systems containing cleavable peg spacers the cleavable spacer allows easy detachment of peg from carriers at the site of destination . cleavable linkers such as ph - sensitive , reduction - sensitive ( disulfide ) , and enzyme - sensitive linkers have been used to tether cleavable peg in polymeric and lipid based nanocarriers , and similarly can also be utilized for peptide - based sirna delivery systems . in recent years , pll - based triblock copolymers composed of a cationic core , a hydrophobic moiety , and an amphiphilic peg chain have been designed for sirna delivery ( table 1 ) . the order of pll , peg , and hydrophobic core can be adjusted to achieve the desired property . synthesized a pll derivative in which the backbone was modified with cholic acid on one side and peg on another side with a ph - sensitive linker , benzoic imine . shell structure containing a hydrophobic cholic acid in the core and a hydrophilic segment , where sirna is condensed on the shell surface hanging with peg . the presence of a ph - cleavable linker allows the release of peg and decreases shielding in the vicinity of tumor cells , where the ph is slightly acidic . the cationic micelles show significant inhibition of a reporter gene and tumor growth in a mouse model of prostate cancer . due to its amphiphilic nature , the pll triblock copolymer ( hydrophobic core - pll - peg ) can self - assemble into micelles prior to sirna encapsulation . these micelles consist of three layers : a hydrophobic core , pll / sirna in the middle layer , and peg in the outer corona . because of the covalent conjugation of the hydrophobic core to pll and peg , a dense layer of the pll / sirna complex is formed around the compressed hydrophobic core , leading to relatively smaller particles ( micelle - polyplex ) as compared to an sirna / pll - peg physical mixture without a hydrophobic core , which is called a polyplex . moreover , formation of the hydrophobic core prior to sirna addition allows the pll / sirna complex to localize on the surface of the hydrophobic core , and the peg layer is squeezed out to form the third layer . however , sirna encapsulation in the micelle formulation is lower than that of the polyplexes . a limited thickness of the peg layer on the corona of micelles exhibits a higher zeta potential of the complex that does not vary with n / p ( the molar ratio of the amine groups of a cationic peptide to the phosphate groups of an rna ) ratio . in contrast , simple physical mixing of pll - peg and sirna shows an increase in zeta potential with increase in n / p ratios , probably due to the availability of sirna / pll complexes near the surface . to compare the advantage of the micellar structure over polyplexes , a triblock polymer of monomethoxy poly(ethylene glycol)-poly(-caprolactone)-poly(l - lysine ) ( mpeg - pcl - pll , abbreviated m ) with variable length pcl and a diblock polymer without pcl block ( mpeg - pll , abbreviated p ) were synthesized to form micelle and polyplex structures , respectively . both the polymers the study compared the effect of the hydrophobic segment ( pcl and its different lengths ) on particle size , zeta potential , stability , and sirna delivery efficiency . m polymers show small particle sizes because the hydrophobic pcl core is more compressible . the particles size depends only on the length of the pcl core and not on the n / p ratio used for sirna condensation . as a result , m moreover , the zeta potential of micelles made with these polymers was higher compared to p polymers due to the association of the sirna / pll complex on the pcl surface , which was covered uniformly with the mpeg layer . both the p and m polymers showed similar sirna condensation ability confirmed by gel electrophoresis , suggesting that only the pll fragment is responsible for sirna binding through electrostatic interactions . . it could be due to the loose architecture and large volume of the p polymer lipoplex , which hinder cellular uptake of the sirna . this finding is in accordance with a recent report in which cholesterol was conjugated to a lysine containing peptide to form a micelle structure before complexation with sirna . the cholesterol modified peptide demonstrated much higher condensation capability and transfection efficacy compared to the peptide carrier without cholesterol . the length of pll in pll - peg block polymers is also important in determining sirna delivery efficacy . for instance , ambardekar et al . investigated the effect of pll molecular weight on a delivery system containing nuclease - resistant cholesterol - sirna ( 3 end of the sense strand is modified with cholesterol ) , and a block polymer of pll - peg ( 5 kda ) in which the pll molecular weight was variable from 10 to 50 kda . increasing the pll block length from 10 to 50 kda decreased the minimum n / p ratio required to form complex with sirna . compared to unmodified sirna , cholesterol modified sirnas required lower n / p ratio at the same pll block length . in addition , an increase in pll chain length also enhanced sirna loading and serum stability . moreover , the pll - peg / sirna complexes inhibited the target gene in a pll length - dependent manner in primary breast tumors after iv administration . the reason behind these observations could be that longer pll chain protects sirna from nuclease degradation in systemic circulation and consequently enhances the bioavailability . pll based amphiphilic polymers can also be used for codelivery of sirna and hydrophobic anticancer drugs to synergetically impede tumor growth . for instance , codelivery of the anticancer drug docetaxel ( dtx ) and bcl-2-targeting sirna in a micelle made by a triblock copolymer of poly(ethylene glycol)-poly(l - lysine)-poly(l - leucine ) ( peg - pll - plleu ) was investigated . plleu served as the hydrophobic core to entrap dtx , while sirna were condensed with pll via electrostatic interaction . the resulting formulation exerted enhanced antitumor activity with a small dose of dtx in mcf-7 xenograft murine model . the therapeutic effect of the codelivery system was significantly higher compared to an individual dose of sirna or dtx alone . the earlier - developed g2 and g3 generation dendritic plls have shown efficient gene transfer into cos-7 and bhk cells , respectively , and attracted scientists to develop higher generation dendrimers such as g5 and g6 . these novel hyperbranched plls showed enhanced transfection efficiency similar to lipofectin or superfact transfection reagent . however , the absence of primary amine in the interior of these dendrimers minimizes the endosomal disruption property and consequently decreases the sirna release in the cytoplasm . therefore , dendritic pll have been investigated in combination with endosome - disrupting agents to improve the transfection efficacy of sirna . for example , inoue et al . used endo - porter , a weakly basic amphiphilic peptide , to enhance the capability of kg6 ( a sixth - generation pll dendrimer ) for efficient delivery of sirna . in addition , dendritic analogues of pll can also be modified in terminal ends with histidine and arginine for effective endosomal disruption . one strategy to improve the biodegradability and endosomal release of sirna from pll is to introduce reducible disulfide bonds that can cross - link low molecular weight pll . the resulting pll polymers can be degraded into small fragments via reduction of disulfide bonds in the cytoplasm . this enhances the release of nucleic acid cargo in the cytoplasm without addition of chloroquine or other endosomolytic agents and eventually increases the silencing effects . for instance , compared to a high molecular weight pll ( 20,900 da ) , a 3200 da reducible linear pll showed significantly higher transfection efficiency with less cell cytotoxicity in several cell lines . similarly , a reducible polycation consisting of histidine and polylysine ( termed his rpc ) was evaluated for sirna delivery to avoid the use of endosomolytic agents , such as chloroquine , and enhance biodegradation . cys - his3-lys3-his3-cys ( his3 rpc ) and cys - his6-lys3-his6-cys ( his6 rpc ) were synthesized by oxidative polycondensation ( 59 kda and 113 kda , respectively ) and compared with cys - lys10-cys ( 65 kda ) . on the other hand , histidine residues enabled the buffering capacity of polymer in endosomal ph without the use of any endosomolytic agents . as a result , interestingly , when these his6 rpcs were evaluated for silencing of p75 gene , almost 100% knockdown was observed , akin to oligofectamine but higher than pei . in addition , his6 rpc mediated silencing of gfp gene was significantly higher than that of commercially available jetpei . in another study , stevenson et al . investigated the effect of the molecular weight of his6 rpcs on the knockdown effect of sirna . his6 rpcs with four different molecular weights ( 38 , 44 , 80 , and 114 kda ) were synthesized by controlling the time of oxidative polycondensation reaction . only the 80 kda his6 rpc exerted a higher condensation of sirnas with smaller particle size of 80 nm . the rpcs can efficiently silence stably expressed egfp in liver cell lines . on the contrary , moreover , incorporation of a hepatocyte - specific peptide sequence derived from the plasmodium falciparum circumsporozite protein ( hnmpndpnrnvdenanansayc ) exhibited an enhanced knockdown effect of his6 rpcs in hepatocytes but not nonliver cells . the well - defined structures of msns allow for controlled loading and release of entrapped sirnas . other advantages include good chemical and physical stability , nontoxicity , biocompatibility , higher drug - loading efficiency , and controllable drug release . loading and release kinetics of sirna in msns can be adjusted by modulating the pore size , shape , surface properties , and surface area of the msns . because msns are anionic in nature , surface modification with cationic peptides or polymers is therefore required to deliver small noncoding rnas ( table 1 ) , for example , a large pore mesoporous silica nanoparticle ( lp - msn ) functionalized with pll through covalent immobilization . compared to unmodified or amino modified msns , the pll modified msns show far more efficiency in delivering sirna into cancer cells and silence the oncogenes . protamine is an fda approved , naturally occurring peptide of 5000 da obtained from sperm of salmon and certain other species of fish . protamine sulfate injection , usp , is a sterile , nonpyrogenic , isotonic solution of protamine sulfate used as heparin antagonist in humans . it acts as a heparin antidote by forming a stable salt with heparin , which results in loss of anticoagulant activity of both the protamine and heparin . numerous mechanisms have been proposed for adverse reactions caused by protamine , including thromboxane generation , inhibition of carboxypeptidase , histamine release , complement activation , and immunological reactions . in addition , increase in vasodilator factors , such as nitic oxide , and depression of myocardial function , including bradycardia , leads to hypotension in patients treated with protamine . moreover , direct toxic effects of protamine on the phospholipid membranes result in thrombocytopenia and leukopenia . the presence of high arginine content ( 67% ) in protamine and inherent characteristics to condense negatively charged dna in sperm has been extensively exploited in gene delivery . a peptide containing a higher content of arginine ( r ) promotes nucleus entry through nuclear pore complexes ( npc ) . in line with these observations , protamine shows dna uptake in the nucleus due to the presence of six consecutive arginine residues in its backbone . in recent years , to reduce immunological toxicity mediated by native protamine , several low molecular weight protamines have been synthesized for sirna delivery . recently , protamine has been utilized by our group to enhance sirna condensation . a complex containing streptavidin , sirna targeting poly(rc ) binding protein 2 ( pcbp2 ) , and cholesterol ( ssc ) was formed by noncovalent interaction between biotin ( present in sirna and cholesterol ) and streptavidin . the resulting complexes were stable in the serum but unable to enter cells due to the absence of positively charged component to neutralize sirna . the incorporation of protamine in the ssc complex results in smaller size and higher cellular uptake in hepatic stellate cells ( hscs ) . in another study , we conjugated protamine to the lncap specific peptide kyl ( kylaypdsvhiw ) to condense a fitc - labeled sirna and showed high cellular uptake in the cells . several approaches including sirna / protamine complexed within liposomes , and antibody - protamine fusions , , have been investigated for effective sirna delivery ( table 2 ) . liposome is the most commonly used nucleic acid delivery system . to efficiently entrap small rnas in liposomes , cationic agents , such as protamine , are usually used to enhance the condensation and encapsulation of small rnas in liposome - based systems ( table 2 ) . for example , -7 integrin - targeted liposomes were prepared for the delivery of cyclin d1 ( cyd1 ) sirna . cyd1 governs the proliferation of normal and malignant cells , and its overexpression is observed in the colon during inflammatory bowel disease . liposomes were prepared using neutral phospholipid to avoid toxicity associated with cationic lipids . the liposomes were stabilized with surface decoration of hyaluronan , which was then conjugated with -7 integrin - targeted antibodies . sirna was encapsulated by rehydration of the liposomes in the presence of protamine / sirna complex . the resulting nanocarriers showed more than 80% sirna loading while maintaining nanoscale dimensions . in vivo administration of the liposomal formulations exhibited significant knockdown of cyd1 in leukocytes and similarly , a sigma receptor - targeted liposomal formulation of sirna against human survivin was prepared . sigma receptors are membrane - bound proteins overexpressed in various human tumor cells , including breast cancer , prostate cancer , and nsclc , and exert higher binding affinity to benzamide derivatives such as anisamide . to prepare a targeted delivery system , sirna and calf thymus dna ( 1:1 weight ratio ) these complexes were further coated with cationic liposomes consisting of cholesterol and dotap ( 1:1 molar ratio ) to form liposome - polycation - dna ( lpd ) nanoparticles . consequently , to enhance the systemic circulation and targetability of the resulting lpd nanoparticles , peg conjugated with anisamide on the terminal end was tethered on the nanoparticle surface . calf thymus dna in the lpd nanoparticles is essential to increase the delivery efficiency ( 2080% ) of particles while reducing particle size up to 1030% . higher protamine concentrations altered the net surface charge of protamine - dna / sirna complex to slightly positive and therefore decreased the interaction with cationic liposomes and lowered the encapsulation efficiency of sirnas . compared to lpd functionzlized with peg alone , these anisamide functionalized nanocarriers showed enhanced sirna uptake and survivin mrna knockdown in sigma receptor - overexpressing cells h1299 . in another study , an rgd - targeted lpd sirna delivery system was prepared following the procedure developed by li et al . to knock down vegfr-2 ( also referred to as fetal liver kinase-1 ( flk-1 ) in angiogenic cells ) . to target angiogenic cells , a cyclic arg - gly - asp ( rgd ) peptide that specifically binds to integrins expressed on tumor - associated endothelial cells the rgd peptide modified formulation showed enhanced uptake and silencing of vegfr-2 in two endothelial cell lines . tissue- and cell - specific delivery of small noncoding rnas is a key obstacle to their therapeutic applications . one way to deliver small rnas to target cells is to make fusion proteins of protamine with targeting antibodies ( table 2 ) . protamine in these fusion proteins helps in sirna condensation because of its cationic nature , while antibodies allow cell - specific targeting . several proteins , antigens , and receptors , such as human integrin lymphocyte function associated antigen-1 ( lfa-1 ) , epidermal growth factor receptor family member erbb2 ( her2 ) , prostate - specific membrane antigen ( psma ) , and hiv envelope proteins , are overexpressed in specific cells and have been utilized as targets for sirna delivery using the protamine antibody fusion strategy . for instance , the recombinant fusion protein of protamine to her-2 specific single - chain fragmented antibodies ( scfvs ) ( named f5-p ) successfully delivered polo - like kinase 1 ( plk1 ) sirnas into her2(+ ) breast cancer cell lines and primary human cancers in orthotopic breast cancer models . silencing of the target gene induced apoptosis of her2(+ ) breast cancer cell lines . when injected intravenously , the f5-p / plk-1-sirna complex showed significant accumulation in orthotopic her2(+ ) breast cancer xenografts , leading to suppressed plk1 gene expression and tumor cell apoptosis . in another study , a fusion protein containing the heavy chain of a fab fragment ( f105 ) specific for the hiv envelope protein gp160 and protamine ( named f105-p ) was constructed to deliver sirna to hiv - infected cells or cells expressing exogenous hiv envelope glycoprotein gp160 ( hiv env ) . to investigate the targeting efficacy of the f105-p / sirna complex in hiv env expressing jurkat cells , fitc - sirna were transfected either alone or with the unmodified f105 antibody or with f105-p . as a result , the f105-p / sirna complex showed significantly higher uptake only in hiv env positive cells . interestingly , sirna alone or simple mixture with unmodified antibody ( without protamine ) showed negligible cellular uptake . similarly , intravenous or intratumoral injection of the f105-p / sirna complex was carried out in mice bearing hiv env expressing b16 melanoma cells . a cocktail of sirnas targeting the cell cycle ( c - myc ) , apoptosis ( mdm2 ) , and angiogenesis ( vegf ) significantly reduced tumor growth only in hiv env positive tumors but not in normal tissue or in envelope - negative tumors . intravenous injections of the complex also showed higher accumulation and inhibition of malicious genes in tumors . protamine , although effective in nucleic acid delivery , may have adverse effects such as mild hypotension to severe or ultimately fatal cardiac arrest and immunological responses . this has led researchers to develop nontoxic low molecular weight protamine ( lmwp ) . lmwps are nontoxic arginine - rich peptides derived from native protamine by enzymatic digestion with thermolysin . briefly , digestion is carried out by incubation of thermolysin with protamine for 30 min at room temperature , and peptide fragments are separated using a heparin affinity column . five peptides , thermolysin - digested segment of protamine ( tdsp ) 15 , are obtained from the process . among them , tdsp4 containing a mixture of 2 tridecyl peptides with sequences of vsrrrrrggrrrr and asrrrrrggrrrr and tdsp5 ( vsrrrrrrggrrrr ) maintain the heparin - neutralizing ability . however , due to the similarity of their structures to tat4757 peptides , only tdsp5 has been used for sirna and protein delivery . ( table 2 ) in addition , these lmwps have been suggested to be clinically safe delivery carriers , as neither an antigenic nor a mutagenic response was elicited when tested on a dog model . sirna delivery using lmwps was as effective as the tat4757 peptide , a known potent cpp . choi et al . used these lmwps for the delivery of sirna against vegf . in an in vitro experiment on carcinoma cells , high cytoplasmic accumulation of fluorescently tagged sirna was observed within a short period of time , leading to significant downregulation of vegf . intraperitoneal injection of the lmwp / sirna complexes also delivered the sirna into tumors , knocked down vegf expression , and eventually inhibited tumor growth . in addition , the lmwp / sirna complex did not induce the expression of cytokines including interleukin ( il)-12 and interferon ( ifn)- , suggesting good safety in animals . in another proof - of - concept study , lmwp was used to enhance brain delivery of nanoparticles . in this context , poly(ethylene glycol)-poly(lactic acid ) ( peg - pla ) nanoparticles were modified with thiolated lmwp . the resulting nanoparticles exhibited significantly enhanced cellular accumulation in 16hbe140 cells without cytotoxicity . further intranasal administration of coumarin-6-loaded lmwp surface functionalized nanoparticles showed significantly higher fluorescence accumulation in the rat cerebellum , cerebrum , olfactory tract , and olfactory bulb compared to nonfunctionalized nanoparticles . this study clearly suggested that brain delivery of nanoparticles can be enhanced by surface functionalization with lmwp . moreover , this strategy can be employed for the brain delivery of sirna and diagnostic and other therapeutic agents . lmwp was also investigated for the delivery of mir-29b to human stem cells to induce osteogenic differentiation . arginine rich lmwp ( vsrrrrrrggrrrr ) was synthesized to condense human mirna-29b sequence ( sense : 5-uagcaccauuugaaaucaguguu ) . the size of the resulting particles is small ( 3050 nm ) and dependent on the n / p ratios . to confirm the functional activity of the mir-29b on osteoblastic differentiation in hmscs , real - time rt - pcr was employed to evaluate the expression of osteogenic gene markers such as col1a1 , alp , runx2 , opn , ocn , and taz . as a result , except col1a1 , mrna levels of all osteogenic markers increased at 48 h , which was higher than that observed by using lipoplex delivery system of the same mirna . in 1988 , two independent groups found that the transcription trans - activating ( tat ) protein of hiv-1 can enter cells by crossing the cell membrane . later on , the first cpp , penetratin ( pantp , rqikiyfqnrrmkwkk ) , was identified from the third helix homeodomain of the drosophila antennapedia protein . the minimal tat sequence ( ygrkkrrqrrr ) that mediates cellular uptake was also identified . since then , several cationic and/or amphiphilic cpp peptides containing 530 amino acids with the ability to cross the cell membrane and deliver attached cargo have been discovered . the most commonly used cpps include transportan , vp22 , model amphipathic peptide ( map ) , and synthetic arginine - rich peptides . the uptake mechanism for cationic cell - penetrating peptides has been extensively studied but is not fully understood . early studies demonstrated that fluorophore - conjugated cpp was predominantly taken up in a receptor- , energy- , and temperature - independent manner . for example , when using confocal microscopy , the cell fixation protocol using methanol could cause artifactual redistribution of fluorescent signals from cell membrane to cytosol and nucleus . furthermore , quantitative methods such as flow cytometry may have overestimated the uptake rate because they failed to distinguish between extracellular association and internalized fluorescence signals . since then , studies on internalization mechanism were mostly conducted in live cells instead of fixed cells , and a more thorough washing step , using trypsin or heparin , was introduced to remove membrane bound fluorescent cpps prior to flow cytometry analysis . now it is widely accepted that both endocytotic and nonendocytotic pathways are involved in the cellular uptake of cpps . in both cases , internalization begins with the interaction of peptides and extracellular matrix , such as electrostatic interactions , hydrophobic interactions , and hydrogen bonds . positively charged cpps strongly associate with the plasma membrane by binding to polysulfated and negatively charged cell - surface heparin proteoglycans , including syndecans and glypicans , which are commonly linked with specific core proteins via a glca - gal - gal - xyl - ser linkage . another common interaction is the electrostatic interaction between positively charged residues of the peptides and the anionic - charged phospholipid head groups . in the case of arginine - rich peptides , such as r9 , the binding affinity of the peptides to heparin proteoglycan is greater than to phospholipid head groups . chemical modification of the peptide with stearic acid or cholesterol could increase the hydrophobicity and affinity to lipid bilayers . it has also been postulated that peptides containing arginines are favorable for counterion - mediated membrane translocation . guanidinium groups in arginine tend to form bidentate hydrogen bonds with anions to reduce charge repulsion with adjacent arginines . the features of cargo ( size and type ) and loading method ( covalent or noncovalent binding ) can influence the internalization mechanism . many cpps that are attached to a large cargo or complexed with nucleic acid are taken up via endocytotic pathway . endo - porter is a histidine- and leucine - containing , cationic amphipathic peptide that is able to deliver sirna and morpholino - rna into cells when noncovalently bound to these nucleic acids . although endocytotic pathway inhibitors , such as cytochalasin d and dynasore , do not block the endo - porter - mediated knockdown , lower temperature ( 4 c ) does abolish the gene silencing effect , suggesting that the internalization of endo - porter / sirna complex is an energy - dependent process . similarly , gene silencing effect of the mpg peptide / sirna complex and cholesteryl peptide micelle / sirna complex is completely or partially reversed at lower temperature ( 4 c ) , indicating that endocytosis may be the primary pathway accounting for the functional delivery of sirna . some peptides , such as penetratin , can be translocated through the lipid bilayer of unilamellar vesicles without the assistance of any cell membrane proteins . one of the most popular models is inverted micelle formation . in this model , the cationic peptide first associates with the plasma membrane via electrostatic interaction to transiently form an inverted micelle . the inverted micelle structure allows cpps to cross the hydrophobic environment of the phosphate lipid tail and release the cargo into the cytoplasm . alternatively , cationic peptide / sirna complexes may insert into the membrane and form a transient transmembrane -structure to allow the complex to pass through cells . in general , due to the highly dynamic structure of cell membrane , the interaction of cpps with cell surface at the molecular level has been studied in the presence of a lipid system to mimic the cell membrane . in aqueous medium , cpp forms a negligible amount of secondary structures , which transforms into alpha- or beta - structures in the presence of lipid medium . these secondary structures in the presence of a lipid system ( mimicking the cell membrane ) are often oriented in such a way that favors cpp translocation through the cell membrane . the inherent ability of cpps to enhance cellular uptake and translocate to different intracellular compartments , such as the nucleus , mitochondria , and cytoplasm , has been utilized extensively in sirna delivery . approaches ranging from electrostatic noncovalent complexes to the synthesis of cpp sirna conjugates and cpp - functionalized nanoparticles have been developed for sirna delivery ( table 3 ) . sirna covalent conjugate enables well - defined one to one conjugation with the flexibility of incorporating a cleavable linker between the sirna and cpp ( table 3 ) . in addition , covalent conjugation also ensures that the cpp sirna conjugate remains intact in the systemic circulation . the direct conjugation approach may , however , minimize or completely abolish the effectiveness of cpp by neutralizing its positively charged amino acids , which are vital for cellular translocation . to avoid these obstacles to some extent , less negatively charged nucleic acid analogues , such as peptide nucleic acid ( pna ) or phosphorodiamidate morpholino oligonucleotides ( pmos ) , have been investigated . cpp s steric hindrance , the effective dose ratio between cpp and sirna , the stability / cleavability of the linker , and the intracellular localization of sirna after uptake are other important factors that need to be considered before using this approach . although the covalent conjugation approach is not widely used for sirna delivery , there are a few studies showing effective sirna delivery using this approach . for instance , muratovska et al . conjugated penetratin and transportan to thiol - containing sirnas targeting luciferase or green fluorescent protein ( gfp ) transgenes . the resulting disulfide bond containing conjugates showed reduction of the target genes in several mammalian cell lines . moreover , the silencing effect of the cpp / sirna conjugate was equivalent to or better than that of cationic liposomes . at an optimal charge or molar ratio , the nanoscale cpp / sirna complexes can enter cells by interacting with proteoglycans on the cell surface . these noncovalent complexes are easy to prepare and therefore can be formed on a large scale in a cost - effective manner ( table 3 ) . for instance , the mpg peptide ( galflgflgaagstmgawsqpkkkrkv ) has been used for sirna delivery using the noncovalent complexation approach . mpg is a fusion peptide derived from the nuclear localization signal ( nls ) of sv40 large t antigen and hiv-1 gp41 protein . the presence of the nls in these peptides allows sirna accumulation in the nucleus , but cationic charges enable sirna condensation . however , replacement of a single lysine with serine in the cationic domain of the peptide resulted in sirna delivery only to the cytoplasm , which demonstrated a stronger silencing effect compared to the unmutated sequence . in another study , the mpg peptide was utilized for the delivery of mirna-122 ( mir 122 ) mimic and inhibitor into primary mouse liver hepatocytes , liver cell lines and caenorhabditis elegans . the targetability of cpps can be improved by attaching a peptide targeting ligand . for example , to achieve tumor selectivity , a six amino acid peptide ( a1 ) with high affinity for vascular endothelial growth factor receptor-1 ( vegfr1 ) was fused with the tat peptide ( termed tat - a1 ) . the resulting tat - a1 exhibited higher sirna delivery efficacy in cancer cells compared to tat alone . some cationic proteins contain canonical double stranded rna ( dsrna ) binding motifs , which can bind to dsrna with high affinity but not to double stranded dna . protein kinase r ( pkr ) is one of the well - studied dsrna binding proteins . it contains two dsrna binding domains ( drbd ) : an n - terminal domain ( kd = 3.8 10 mol / l ) and a c - terminal domain ( kd = 2 10 mol / l ) . recently , pkr - drbd was fused to the tat peptide for sirna delivery ( named ptd - drbd ) ( table 3 ) . the ptd - drbd showed impressive sirna delivery in many primary and transformed cells , including human embryonic stem cells , human umbilical vein endothelial cells , and t cells . however , the propensity of tat to interact with serum proteins such as glycosaminoglycans may retard their application in iv administration . moreover , nonspecific cell uptake by tat may also induce several side effects . to overcome these hurdles , the tat sequence was replaced with cell - homing peptides or receptor ligands to achieve cell - specific delivery of sirna . geoghegan et al . have developed a fusion protein consisting of two drbd domains ( 2 drbd ) and three repeats of the b2 peptide sequence ( ghkvkrpkg ) in place of the tat peptide . the b2 peptide sequence , identified by phage display against recombinant transferrin receptor ( tfr ) , showed enhanced tfr mediated uptake . the resultant b2 - 2 drbd / sirna complexes significantly reduced the expression of a housekeeping gene , hypoxanthine - guanine phosphoribosyltransferase ( hprt ) , in hela cells . the silencing effect was further increased by the addition of chloroquine ( an endosomal acidification inhibitor ) , suggesting endosomal entrapment of the b2 - 2 drbd sirna complex . cpps , along with fusogenic and membrane - disruptive peptides , have been linked to the surface of nanoscale sirna systems , including lipid nanoparticles , polymer nanoparticle , and inorganic material - based nanoparticles , to enhance their cellular uptake . prepared a micelle using the amphiphilic block copolymers poly(ethylene glycol ) ( mpeg)/polycaprolactone ( pcl ) conjugated with tat peptide via a disulfide linkage ( mpeg - pcl - ss - tat ) for effective delivery of a vegf sirna . in this system , tat was used for sirna condensation , while the disulfide bond was introduced for rapid dissociation by glutathione in the cell cytoplasm . the resulting 100200 nm mpeg - pcl - ss - tat / sirna complexes were safe and exerted good silencing activity in vitro ( s-180 sarcoma cells ) and in vivo . intravenous injection of these micelles ( mpeg - pcl - ss - tat / sirna ) exhibited a significantly stronger antitumor effect in s-180 tumor - bearing mice compared to mpeg - pcl - ss - tat / control . in another study , the resulting covalent conjugate was utilized for sirna delivery to neuronal cells ( neuro-2a ) . these nanoparticles showed safe and effective sirna delivery with higher reduction of the target ataxin-1 gene compared to nanoparticles made only with chitosan . apart from rna condensation , peptides have also been used as targeting ligands in rna delivery systems . several approaches , such as phage display technology and one - bead one - compound ( oboc ) combinatorial bead library method , for example , qin et al . identified a lncap specific peptide using the m13 phage display peptide library ( ph.d.-12 ) . four rounds of biopanning were carried out with lncap cells after precleaning on pc-3 cells to remove nonspecific peptides . as a result , a lncap cell specific peptide ligand kylaypdsvhiw ( also termed kyl peptide ) was identified . the kyl peptide conjugated protamine successfully delivered fitc - labeled sirna into lncap cells . table 4 summarizes some of the peptide targeting ligands that have been adopted for rna delivery . cyclic rgd ( crgd ) is a widely utilized peptide targeting ligand for various therapeutic agents including small noncoding rnas . crgd is the targeting ligand of the v3 integrin , which plays important roles in the regulation of cell differentiation , progression , proliferation , and apoptosis . more importantly , the v3 integrin is overexpressed in various cancer cells and promotes cancer cell growth and metastasis . as a result for example , a crgd decorated poly(lactic - co - glycolic acid ) ( plga ) nanoparticle containing microrna-132 ( mir-132 ) was developed to transfect cultured endothelial cells before transplantation , thereby sensitizing the cells to endogenous growth factors . in another study , a rgd - peg decorated polycation liposomes ( pcls ) containing tetraethylenepentamine ( tepa ) was developed for efficient sirna delivery . gene silencing of the nanocomplexes was first optimized using a luciferase sirna ( siluc2 ) in b16f10-luc2 murine melanoma cells stably expressing the luciferase 2 gene . later on , the silencing activity was improved by grafting cholesterol on the 3 end of the sirna sense strand that allows better retention in the liposomes . this improved delivery system exhibited higher efficiency against metastatic b16f10-luc2 tumors in a mouse model . peptides have also been used as brain - specific targeting ligands for sirna delivery . for example , manjunath et al . demonstrated that a 29 amino acid peptide derived from rabies virus glycoprotein ( rvg ) can specifically bind to the nicotinic acetylcholine receptor ( achr ) on neuronal cells . the chimeric peptide consisting of the rvg peptide and nine arginines was mixed with sirna and successful delivered the sirna to neuronal cells in a dose - dependent manner . moreover , the same chimeric peptide can not deliver sirna into achr - negative hela cells , indicating the specificity of the chimeric peptide for neuronal cells . in another study , the rvg peptide was decorated on the surface of the sirna / trimethylated chitosan ( tmc ) complexes through bifunctional peg for brain delivery of sirna . the rvg peptide modified sirna / tmc mpeg complexes showed significantly higher uptake in achr - positive neuro-2a cells as well as in mouse brain compared to unmodified complexes . moreover , sirna encapsulated in these complexes exhibited potent knockdown of the bace1 gene , a therapeutic target in alzheimer s disease . one major challenge in the clinical transition of rna therapeutics is the development of an efficient rna delivery system with a broad therapeutic window . therefore , low toxicity of peptide - based carriers is critical for its successful application in rna therapy because the carrier peptide is mainly responsible for both efficacy and toxicity . the toxicity of a peptide - based carrier depends on the amine type , arrangement , molecular weight , and number of cationic charges per monomer unit . in general , polypeptides are safe because of the presence of polyamide backbone that can be degraded in the body by proteolytic enzymes . since l - amino acids are the components of naturally occurring polypeptides , the cellular proteolytic machinery does not recognize polypeptides made of d - amino acids . it has been observed that the cpp sequences containing d - amino acids induce higher toxicity than parent l - peptides due to enhanced stability against proteolytic enzymes present in intracellular environment . recently synthesized numerous poly(amide)-based polymers using the n - carboxy anhydride ( nca ) polymerization method and studied the structure activity relationships ( sar ) of these sirna delivery carriers . the fully d - isomer polymers pa block ( d - orn : d - phe ) and poly(d - ornithine ) homopolymer ( pdo ) were stable up to 2 h in the presence of protease cocktail , indicating negligible degradation of d - isomer polymers . on the other hand , the pa block ( d - orn : l - phe ) showed little degradation , while the pa block ( l - orn : d - phe ) showed modest degradation , indicating that l - orn has better degradability as compared to l - phe . the pa block ( l - orn : l - phe ) and plo ( l - orn ) exhibited the highest degradation rate in the presence of protease cocktail . similarly , a significant difference was observed in the plasma pk of the c - mesyl conjugated nondegradable d - isomer pdo versus the degradable l - isomer plo . a very slow elimination rate of pdo from the plasma was observed as compared to plo . for example , peptide nucleic acids ( pna ) show nephrotoxicity after being conjugated to amphipathic peptide containing ala , leu , and lys . however , addition of arg sequence in the peptide does not show such toxicity arg containing peptides show good splicing redirection in targeted adipose tissues even at a low dose of 2.5 mg / kg . despite the great promise of small noncoding rnas in treating various diseases , the effort of translating rna therapeutics from bench to bedside has been hampered by several obstacles , such as formulation variations , aggregation in systemic circulation , nonspecific binding , and endosomal entrapment . a great variety of lipids , polymers , and peptides have been investigated in rna delivery . among them , peptides have attracted unique attention due to their ease of synthesis , controllable size , multiple functionalities , and tunable structure . peptides can be used in an rna delivery system as a cationic component , a cell penetrating component , a targeting ligand , or the hydrophobic portion of an amphiphilic carrier . while using peptides alone as a carrier may not be enough to efficiently deliver rnas into cells , peptides can definitely be used as an essential part of a multicomponent rna delivery system . for example , cationic peptides can be utilized as the condensing component to form a nanocomplex with rnas . peptide targeting ligands can also be used to modify an rna delivery system to achieve targeted delivery . therefore , combination of different strategies targeting each of the barriers is necessary to explore the safe and effective delivery of rnas using peptide - based carriers . nonspecific binding and stability of peptides in systemic circulation could be a potential hurdle for any peptide - based delivery systems . as a result , careful fabrication of the delivery system and even pegylation are needed to guarantee the effectiveness of peptides . possible immune response is another potential problem for some of the peptides used in rna delivery . however , these are general problems associated with any peptide - based drug delivery systems , and therefore many strategies have been developed to overcome these problems . toxicity or therapeutic window of rna therapeutics is another important issue that scientists need to consider during drug development . it is critical to use the minimum amount of peptide or polymer carriers in rna therapeutics to avoid any possible toxicity associated with these carriers , thus leading to a broad therapeutic window . moving forward , we believe that peptides will continue playing critical roles in a significant portion of rna delivery systems .
to become permanently immune to some illness , you must either catch it or be vaccinated against it . vaccine is an antigenic substance prepared from the causative agent of a disease or a synthetic substitute , used to provide immunity against disease . during vaccination , a harmless version of a germ is introduced to the body and the immune system responds by producing antibodies to attack the intruder . thereafter , a memory of this invasion remains so that the immune system can quickly recognize and neutralize disease causing agents when they appear [ figure 1 ] . different types of vaccines weaker or attenuated viruses to generate immunitylive viruses that have been attenuated ( weakened or altered so as not to cause illness)inactivated or killed organisms or virusesinactivated toxins ( for bacterial diseases where toxins generated by the bacteria , and not the bacteria themselves , cause illness)segments of the pathogen ( this includes both subunit and conjugate vaccines ) . weaker or attenuated viruses to generate immunity live viruses that have been attenuated ( weakened or altered so as not to cause illness ) inactivated or killed organisms or viruses inactivated toxins ( for bacterial diseases where toxins generated by the bacteria , and not the bacteria themselves , cause illness ) segments of the pathogen ( this includes both subunit and conjugate vaccines ) . as dermatologists herein the commonly used and experimental vaccines of relevance to dermatology have been elucidated in a simplified manner [ tables 1 and 2 ] . vaccines of dermatological significance vaccines dose , indications , effects and side effects advances in genetics , immunology and molecular biology has helped researchers to make vaccines with safer alternatives . newer vaccines for preventing and treating hiv , hsv , vaccines against acne and malignant melanoma etc have completed phase 1 trial successfully , so hopefully in future we can not only prevent but also fight and cure these diseases .
type 2 diabetes ( t2d ) is a multifactorial disease , caused by a complex interplay between genetic and nongenetic risk factors . compelling evidence has identified increasing age , higher body mass index ( bmi ) , impaired fasting glucose , impaired glucose tolerance , higher glycated hemoglobin ( hba1c ) level , and metabolic syndrome as important t2d risk factors ( table 1 ) [ 110 ] . for example , metabolic syndrome poses an eight times higher t2d risk and is present in more than 40% of the individuals over 50 years of age . the high impact and frequency make these risk factors suitable candidates for targeting preventive interventions , such as medication , weight loss , and increased physical activity that can slow down or even reverse the disease process [ 11 , 12].table 1risk factors for type 2 diabetesrisk factorpopulationfrequency ( % ) diabetes risk ( % ) rrage ( y ) 0 to 44general us population61.3 1.7 1 45 to 6425.912.27.2 65 to 746.819.911.7 75 + 6.117.910.5sex femalegeneral us population50.7 5.9 1 male49.36.61.1bmi ( kg / m ) < 25us adults ages 20 years32.0 [ 4 , 5]8 1 25 to < 3034.2151.9 30 to < 3519.5232.9 35 to < 408.6334.1 405.7435.4ifg / igt normoglycemicnondiabetic us adults ages 18 years ( frequency)65.4 na 1 igt only5.44.46.45.5 ifg only19.46.19.27.5 ifg+igtglobal cohorts ( diabetes risk and rr)9.8101512.1hba1c ( % ) < 5.0nondiabetic middle - aged adults from 4 us communities8.6 6 0.5 5.0 to < 5.544.6121 5.5 to < 6.033.2211.9 6.0 to<6.59.3444.5 6.54.37916.5metabolic syndrome nous adults ages 50 years56.5 4.1 1 yes43.534.08.3values reported are prevalences unless otherwise indicatedunless referenced , values are calculated from the values depicted in the column diabetes riskannualized incidence of diabetesannualized relative riskcumulative 15-year incidence of diagnosed diabetesmultivariable adjusted hazard ratio of 15-year risk for each absolute increase in 1 percentage point of glycated hemoglobinbmi body mass index ; hba1c glycated hemoglobin ; ifg impaired fasting glucose ; igt impaired glucose tolerance ; na not available ; rr relative risk risk factors for type 2 diabetes values reported are prevalences unless otherwise indicated unless referenced , values are calculated from the values depicted in the column diabetes risk annualized incidence of diabetes annualized relative risk cumulative 15-year incidence of diagnosed diabetes multivariable adjusted hazard ratio of 15-year risk for each absolute increase in 1 percentage point of glycated hemoglobin bmi body mass index ; hba1c glycated hemoglobin ; ifg impaired fasting glucose ; igt impaired glucose tolerance ; na not available ; rr relative risk in the past 5 years , genome - wide association studies have identified and replicated over 40 single nucleotide polymorphisms ( snps ) that predispose to t2d [ 13 , 14 ] . however , the effect sizes of the associated variants are very modest , with per allele odds ratios ranging from 1.05 to 1.35 . even the strongest susceptibility variant , rs7903146 in the tcf7l2 gene , is a weaker predictor of t2d risk than most nongenetic risk factors . evidently , the low effect sizes make single genetic risk factors unsuitable for targeting preventive interventions , but there is increasing interest in investigating the extent to which genetic risk factors combined can improve the prediction of the disease . an improvement in the early identification of high - risk groups is warranted because t2d imposes a great burden on human health and health care systems [ 15 , 16 ] . an estimated 285 million people worldwide have diabetes and this number is expected to increase by more than 50% in the next 20 years if no preventive strategies are implemented . to identify high - risk individuals , guidelines for t2d prevention advocate the use of clinical risk scores as primary screening tools , followed by blood glucose measurements to detect individuals with impaired fasting glucose , impaired glucose tolerance , or metabolic syndrome . examples of commonly used risk scores include the findrisc ( finnish diabetes risk score ) and the diabetes risk calculator [ 18 , 19 ] . the findrisc score is based on age , bmi , waist circumference , use of antihypertensive medication , history of elevated blood glucose , daily physical activity and daily intake of fruits or vegetables , and the diabetes risk calculator on age , waist circumference , gestational diabetes , height , race / ethnicity , hypertension , family history of diabetes , and exercise . the area under the receiver operating characteristic curve ( auc ) is a commonly used measure to indicate the predictive ability ; the auc indicates the discriminative accuracy of a prediction model . to generate the curve , on the x - axis 1-specificity the auc value represents the probability that the predicted risk of a random patient is higher than that of a random nonpatient . when predicted risks of individuals who will develop the disease are always higher than the risks of those who will not develop the disease , the auc is 1.0 . when their risks are higher for 50% of the random pairs , the auc is 0.50 , equaling the predictive performance of tossing a coin . the auc was 0.65 in men and 0.66 in women for the findrisc score predicting impaired fasting glucose , impaired glucose tolerance , or undiagnosed diabetes , and 0.72 and 0.75 for detecting metabolic syndrome . the auc of the diabetes risk calculator was 0.70 for detecting impaired fasting glucose , impaired glucose tolerance , or undiagnosed diabetes . these modest auc values indicate that many people who will develop t2d are not identified as being at increased risk by these risk scores , and that many that will not develop the disease are labeled as increased risk . although offering lifestyle modification programs to individuals who will not develop t2d may do no harm and may even provide other benefits by reducing the risk of other diseases , not recognizing the many who will develop diabetes would clearly be missed opportunities to reduce the serious burden of disease . some clinical risk models that include invasive measurements showed higher auc values for detecting individuals who will develop t2d . an example is the framingham risk score including age , sex , obesity , hypertension , parental history of diabetes , low levels of high - density lipoprotein cholesterol , elevated triglyceride levels , and impaired fasting glucose . the auc of this risk model was 0.85 for predicting t2d in middle - aged adults . however , inclusion of invasive measurements that can change over time in clinical risk models might be inconvenient at the population level and these models still leave room for improvement . recent studies have investigated the predictive ability of risk models that include genetic variants only or genetic variants added to clinical risk factors . a study that investigated a genetic risk score based on 34 diabetes - associated variants showed a significant association of the risk score with risk of developing diabetes [ 22 ] . this risk was attenuated by lifestyle interventions , also in individuals in the highest genetic risk quartile , suggesting that detecting individuals at high risk of developing t2d based on genetic variants and offering them lifestyle modification programs is useful . in this paper , we review genetic risk prediction studies from a methodologic perspective by focusing on factors in the choice of study design and population that may have impacted the observed predictive ability . the number of studies that investigate the predictive ability of genetic variants in t2d has increased rapidly ( table 2 ; [ 23 , 24 , 2538 , 39 , 4042 , 43 ] ) . these studies assessed risk models that were based on genetic variants only or on a combination of both genetic and nongenetic variants . the table shows that the number of snps included in the genetic models has increased from 3 in 2005 to 40 in 2011 . the models show considerable overlap in the genetic variants that were considered , but there also are many differences . since its discovery , all but one of the studies had included tcf7l2 and the majority additionally investigated pparg , cdkn2a / b , kcnj11 , igf2bp2 , slc30a8 , and hhex - ide - kif11 . yet , most other snps were included in one or two models only [ 43 ] . most clinical models included at least age , sex , and bmi , but they differed in the other factors that were added , such as blood pressure , family history of t2d , and fasting plasma glucose level.table 2genetic risk prediction studies in t2dstudyno of polymorphismsclinical risk factorsauc geneticauc clinicalauc combineddesignage ( mean , years)sex ( % men)bmi ( mean , kg / m)european balkau et al . men2fpg , smoking status , wc , ggtna0.850.85prospective cohort50/47100/10027.5/25.1women2fpg , bmi , fh , tgna0.920.91prospective cohort52/470/029.2/23.7lyssenko et al . 10age , sex , bmi , fh , smoking , alcohol intake , pana0.780.79nested case malmo11age , sex , bmi , fh , bp , tg , fpg0.630.740.75prospective cohort45.564.924.3botnia11age , sex , bmi , fh , bp , tg , fpg , hdl , wc0.680.790.80prospective cohort44.945.525.6cauchi et al . 15age , sex , fh , pa , whr , triacylglycerol / hdl ratio0.590.860.87cross - sectional60.7/52.867/4630.4/25.5fontaine - bisson et al . 18age , sex , bmi , fh , fpg , sbp , hdl , tg0.580.900.90prospective cohort42.14725.6sparso et al . findrisc19age , bmi , wc , pa , fh , diet , antihypertensive medication , previously known high glucose0.550.730.73cross - sectional4574100/100nafindrisc+19findrisc , tg , hdl , adiponectin , alt0.550.770.77cross - sectional4574100/100naschulze et al . 20age , wc , height , history of ht , pa , smoking , consumption of red meat , whole - grain bread , coffee and alcohol , glucose , hba1c , tg , hdl , ggt , alt , hs - crpna0.900.90prospective case - cohort54.6/49.458.7/36.930.4/25.9talmud et al . [ 39]cambridge score20age , sex , bmi , drug treatment , fh , smoking status0.550.720.73prospective cohort51.0/49.072.9/72.827.5/24.7framing - ham offspring score20age , sex , bmi , parental history of t2d , hdl , tg , fpg0.550.780.78prospective cohort51.0/49.072.9/72.827.5/24.7de miguel - yanes et al . 40age , sex , fh , bmi , fpg , sbp , hdl , tg0.610.900.91prospective cohort464726.0 asian miyake et al . control61.2/57.452/4124.0/23.6values provided are for participants with and without t2d , respectively , when two values are reported and for the total population when one value is reported . for prospective cohort studies , values are means unless otherwise indicatedmedianadjusted for sexrangealt alanine aminotransferase ; auc area under the receiver operating characteristic curve ; bmi body mass index ; bp blood pressure ; fh family history of type 2 diabetes ; findrisc finnish diabetes risk score ; fpg fasting plasma glucose ; ggt -glutamyltransferase ; hba1c glycated hemoglobin ; hdl high - density lipoprotein cholesterol ; hs - crp high - sensitivity c - reactive protein ; ht hypertension ; na not available ; pa physical activity ; sbp systolic blood pressure ; tg triglycerides ; t2d type 2 diabetes ; wc waist circumference ; whr waist - hip ratio(adapted from mihaescu et al . [ 43 ] ) genetic risk prediction studies in t2d values provided are for participants with and without t2d , respectively , when two values are reported and for the total population when one value is reported . for prospective cohort studies , descriptive data from baseline examinations are given . values are means unless otherwise indicated alt alanine aminotransferase ; auc area under the receiver operating characteristic curve ; bmi body mass index ; bp blood pressure ; fh family history of type 2 diabetes ; findrisc finnish diabetes risk score ; fpg fasting plasma glucose ; ggt -glutamyltransferase ; hba1c glycated hemoglobin ; hdl high - density lipoprotein cholesterol ; hs - crp high - sensitivity c - reactive protein ; ht hypertension ; na not available ; pa physical activity ; sbp systolic blood pressure ; tg triglycerides ; t2d type 2 diabetes ; wc waist circumference ; whr waist - hip ratio ( adapted from mihaescu et al . [ 43 ] ) table 2 shows that , almost without exception , the genetic risk models had lower auc values than the clinical models . the auc values for the genetic models ranged from 0.55 to 0.68 and for the clinical models from 0.61 to 0.92 . table 2 also shows that the addition of genetic factors either did not or only marginally improved the auc beyond that of the clinical risk models . the differences in the predictive ability of clinical risk models are explained by how many and which risk factors are included in the model and by differences in study design and study population . this is nicely illustrated by three studies that had investigated largely the same 18 genetic variants . the aucs of the genetic risks models in these studies were similar ( 0.580.60 ) , but the aucs of the clinical models were 0.66 , 0.78 , and 0.90 [ 3335 ] . the clinical models with auc values of 0.66 and 0.78 included age , sex , and bmi , but the model with an auc value of 0.90 also included t2d family history , fasting plasma glucose , systolic blood pressure , high - density lipoprotein cholesterol , and triglycerides . the excellent predictive ability was likely due to the inclusion of fasting plasma glucose , as individuals with impaired fasting glucose have a very high risk of developing t2d ( table 1 ) . table 2 shows that auc values tend to be higher when more risk factors are included in the model , particularly when fasting plasma glucose was included . yet , also the two studies that both investigated age , sex , and bmi in the clinical model had markedly different auc values ( 0.66 and 0.78 ) . the difference in these auc values was likely explained by differences in the study design and population . the auc of 0.66 was obtained in a prospective cohort study , the rotterdam study , and the auc of 0.78 in a case control study , consisting of case and control subjects from the godarts ( genetics of diabetes audit and research tayside study ) . participants in the rotterdam study were older and less often men ( table 2 ) , but the two populations predominantly differed in bmi . the mean bmi of the cases in the godarts study was higher than the mean bmi of cases in the rotterdam study ( 31.5 vs 28.0 kg / m ) . also , the difference in mean bmi between cases and controls was much larger in the godarts study compared with the rotterdam study ( 4.6 vs 2.0 kg / m ) . in general and by definition , the predictive ability of risk models is higher when there are larger differences between cases and controls on the risk factors included in the risk model . along the same lines , study design and population characteristics may have influenced the observed auc values of the other clinical models , and also of auc values of the genetic risk models . the auc values of the genetic risk models ranged from 0.55 to 0.68 , a range that was much smaller than that of the clinical models . similar as for the clinical risk models and given that all snps approximately have the same low effect size , one would expect better predictive ability for models that included a higher number of snps , but figure 1 shows that this was not observed for the studies listed in table 2 . the differences in the auc values of the genetic risk scores can not be explained by the number of polymorphisms included in the risk models . in fact , the highest genetic auc ( 0.68 ) was found for a model that included 11 snps , and the lowest for a model that included these exact 11 snps plus an additional 8 others . the explanation for the absence of this relationship is likely in the low effect sizes of the genetic variants . a higher number of snps only yields a slightly higher auc , a combined effect that could easily be outweighed by the influence of other factors , such as study design and study population.fig . 1the area under the receiver operating characteristic curve ( auc ) versus the number of single nucleotide polymorphisms included in the genetic risk models the area under the receiver operating characteristic curve ( auc ) versus the number of single nucleotide polymorphisms included in the genetic risk models genetic risk prediction models have been investigated in prospective cohort studies , in case control studies and in cross - sectional studies , and in study populations that differed in age , sex , and bmi ( table 2 ) . these methodologic aspects may have impacted the observed auc values in a similar way as they impact the auc values of the clinical models . first , clinical and demographic characteristics of the study population may have influenced the observed predictive ability of the genetic risk models . there are two ways in which these characteristics may impact the predictive ability : the clinical and demographic characteristics of the study population itself and the differences in these characteristics between patients and nonpatients . table 2 describes mean age and bmi and the percentage of men in published genetic risk prediction studies for t2d . mean age varied from 42.1 to 68.9 years , mean bmi from 23.4 to 29.1 kg / m , and the percentage of men from 0% to 100% . it is often hypothesized that genetic risk factors may be more predictive in populations in which nongenetic t2d risk factors are not yet present ( eg , in younger or normal weight cohorts ) , but auc values of the genetic models were not markedly higher when populations were younger , had lower bmi , or had a lower percentage of men . however , because of the heterogeneity between the studies and their relatively small number , conclusions must be drawn with caution . moreover , one study that had investigated the predictive performance in two age categories ( < 50 years vs 50 years ) did find higher auc values for the genetic risk score in younger people ( auc 0.66 vs 0.59 ) . the observation that a stratified analysis within a single study did show differences in predictive ability suggests that the absence of a clear relation of age , bmi , and sex with auc values across studies is likely explained by the presence of other differences between the studies . the other way in which clinical and demographic characteristics of the study population impact the predictive ability of risk models is through differences in these characteristics between patients and nonpatients . this specifically holds for characteristics that are included as risk factors in the prediction models , and for characteristics that are associated with these risk factors . evidently and by definition , the presence of risk factors will differ between patients and nonpatients , but the difference can also be enlarged as a result of selection procedures . for example , patients who are recruited through hospitals may have more unfavorable risk profiles than patients randomly selected from the total patient population . consequently , differences in risk factors between hospital - based cases and population - based controls will be larger and the impact of these risk factors on the predictive ability higher . for the studies listed in table 2 , differences in mean age ranged from 6.2 to 16.9 years , in mean bmi from 0.3 to 5.5 kg / m , and differences in the percentage of men from 0.1% to 21.8% . figure 2 shows that larger differences in mean age and bmi between patients and nonpatients were associated with higher auc values for the clinical risk models , and , although less apparent , lower auc values for the genetic models . no relation was observed between clinical auc values and the percentage of men included in the studies , but this may be because male sex only marginally increases t2d risk compared with age and bmi ( table 1).fig . 2the area under the receiver operating characteristic curve ( auc ) of the genetic and clinical models in relation to differences in mean age , percentage of men , and mean body mass index ( bmi ) between patients and controls the area under the receiver operating characteristic curve ( auc ) of the genetic and clinical models in relation to differences in mean age , percentage of men , and mean body mass index ( bmi ) between patients and controls a second methodological aspect that may impact the predictive ability of risk models is study design . genetic risk prediction studies are preferably conducted in prospective follow - up studies , but cross - sectional and case control studies have been used as well ( table 2 ) . the impact of study design on auc values of t2d risk prediction models is in part related to the impact of population characteristics . selection procedures for cases and controls may affect differences in clinical and demographic characteristics between patients and nonpatients . case control studies may demonstrate auc values that deviate from those observed in prospective cohort and cross - sectional studies when cases and controls are recruited from different sources . another way in which study design may impact the predictive ability of risk models is length of follow - up in prospective cohort studies . longer follow - up increases the likelihood that clinical t2d risk factors change over time , and that as a result their baseline values will be less predictive for the development of disease , resulting in prediction models with lower auc . the length of follow - up of the studies listed in table 2 varied from 6 to 25 years . again , the number of prospective cohort studies was too small to investigate the impact of follow - up duration , but one study investigated the predictive ability in quintiles of follow - up time . this study demonstrated that the auc of the clinical risk model decreased with increasing duration of follow - up , whereas the auc of the genetic risk model increased . from the first to the fifth quintile , the clinical auc value decreased from 0.75 to 0.67 and the genetic auc value increased from 0.57 to 0.62 . the differences in the predictive ability of clinical risk models are explained by how many and which risk factors are included in the model and by differences in study design and study population . this is nicely illustrated by three studies that had investigated largely the same 18 genetic variants . the aucs of the genetic risks models in these studies were similar ( 0.580.60 ) , but the aucs of the clinical models were 0.66 , 0.78 , and 0.90 [ 3335 ] . the clinical models with auc values of 0.66 and 0.78 included age , sex , and bmi , but the model with an auc value of 0.90 also included t2d family history , fasting plasma glucose , systolic blood pressure , high - density lipoprotein cholesterol , and triglycerides . the excellent predictive ability was likely due to the inclusion of fasting plasma glucose , as individuals with impaired fasting glucose have a very high risk of developing t2d ( table 1 ) . table 2 shows that auc values tend to be higher when more risk factors are included in the model , particularly when fasting plasma glucose was included . yet , also the two studies that both investigated age , sex , and bmi in the clinical model had markedly different auc values ( 0.66 and 0.78 ) . the difference in these auc values was likely explained by differences in the study design and population . the auc of 0.66 was obtained in a prospective cohort study , the rotterdam study , and the auc of 0.78 in a case control study , consisting of case and control subjects from the godarts ( genetics of diabetes audit and research tayside study ) . participants in the rotterdam study were older and less often men ( table 2 ) , but the two populations predominantly differed in bmi . the mean bmi of the cases in the godarts study was higher than the mean bmi of cases in the rotterdam study ( 31.5 vs 28.0 kg / m ) . also , the difference in mean bmi between cases and controls was much larger in the godarts study compared with the rotterdam study ( 4.6 vs 2.0 kg / m ) . in general and by definition , the predictive ability of risk models is higher when there are larger differences between cases and controls on the risk factors included in the risk model . along the same lines , study design and population characteristics may have influenced the observed auc values of the other clinical models , and also of auc values of the genetic risk models . the auc values of the genetic risk models ranged from 0.55 to 0.68 , a range that was much smaller than that of the clinical models . similar as for the clinical risk models and given that all snps approximately have the same low effect size , one would expect better predictive ability for models that included a higher number of snps , but figure 1 shows that this was not observed for the studies listed in table 2 . the differences in the auc values of the genetic risk scores can not be explained by the number of polymorphisms included in the risk models . in fact , the highest genetic auc ( 0.68 ) was found for a model that included 11 snps , and the lowest for a model that included these exact 11 snps plus an additional 8 others . the explanation for the absence of this relationship is likely in the low effect sizes of the genetic variants . a higher number of snps only yields a slightly higher auc , a combined effect that could easily be outweighed by the influence of other factors , such as study design and study population.fig . 1the area under the receiver operating characteristic curve ( auc ) versus the number of single nucleotide polymorphisms included in the genetic risk models the area under the receiver operating characteristic curve ( auc ) versus the number of single nucleotide polymorphisms included in the genetic risk models genetic risk prediction models have been investigated in prospective cohort studies , in case control studies and in cross - sectional studies , and in study populations that differed in age , sex , and bmi ( table 2 ) . these methodologic aspects may have impacted the observed auc values in a similar way as they impact the auc values of the clinical models . first , clinical and demographic characteristics of the study population may have influenced the observed predictive ability of the genetic risk models . there are two ways in which these characteristics may impact the predictive ability : the clinical and demographic characteristics of the study population itself and the differences in these characteristics between patients and nonpatients . table 2 describes mean age and bmi and the percentage of men in published genetic risk prediction studies for t2d . mean age varied from 42.1 to 68.9 years , mean bmi from 23.4 to 29.1 kg / m , and the percentage of men from 0% to 100% . it is often hypothesized that genetic risk factors may be more predictive in populations in which nongenetic t2d risk factors are not yet present ( eg , in younger or normal weight cohorts ) , but auc values of the genetic models were not markedly higher when populations were younger , had lower bmi , or had a lower percentage of men . however , because of the heterogeneity between the studies and their relatively small number , conclusions must be drawn with caution . moreover , one study that had investigated the predictive performance in two age categories ( < 50 years vs 50 years ) did find higher auc values for the genetic risk score in younger people ( auc 0.66 vs 0.59 ) . the observation that a stratified analysis within a single study did show differences in predictive ability suggests that the absence of a clear relation of age , bmi , and sex with auc values across studies is likely explained by the presence of other differences between the studies . the other way in which clinical and demographic characteristics of the study population impact the predictive ability of risk models is through differences in these characteristics between patients and nonpatients . this specifically holds for characteristics that are included as risk factors in the prediction models , and for characteristics that are associated with these risk factors . evidently and by definition , the presence of risk factors will differ between patients and nonpatients , but the difference can also be enlarged as a result of selection procedures . for example , patients who are recruited through hospitals may have more unfavorable risk profiles than patients randomly selected from the total patient population . consequently , differences in risk factors between hospital - based cases and population - based controls will be larger and the impact of these risk factors on the predictive ability higher . for the studies listed in table 2 , differences in mean age ranged from 6.2 to 16.9 years , in mean bmi from 0.3 to 5.5 kg / m , and differences in the percentage of men from 0.1% to 21.8% . figure 2 shows that larger differences in mean age and bmi between patients and nonpatients were associated with higher auc values for the clinical risk models , and , although less apparent , lower auc values for the genetic models . no relation was observed between clinical auc values and the percentage of men included in the studies , but this may be because male sex only marginally increases t2d risk compared with age and bmi ( table 1).fig . 2the area under the receiver operating characteristic curve ( auc ) of the genetic and clinical models in relation to differences in mean age , percentage of men , and mean body mass index ( bmi ) between patients and controls the area under the receiver operating characteristic curve ( auc ) of the genetic and clinical models in relation to differences in mean age , percentage of men , and mean body mass index ( bmi ) between patients and controls a second methodological aspect that may impact the predictive ability of risk models is study design . genetic risk prediction studies are preferably conducted in prospective follow - up studies , but cross - sectional and case control studies have been used as well ( table 2 ) . the impact of study design on auc values of t2d risk prediction models is in part related to the impact of population characteristics . selection procedures for cases and controls may affect differences in clinical and demographic characteristics between patients and nonpatients . case control studies may demonstrate auc values that deviate from those observed in prospective cohort and cross - sectional studies when cases and controls are recruited from different sources . another way in which study design may impact the predictive ability of risk models is length of follow - up in prospective cohort studies . longer follow - up increases the likelihood that clinical t2d risk factors change over time , and that as a result their baseline values will be less predictive for the development of disease , resulting in prediction models with lower auc . the length of follow - up of the studies listed in table 2 varied from 6 to 25 years . again , the number of prospective cohort studies was too small to investigate the impact of follow - up duration , but one study investigated the predictive ability in quintiles of follow - up time . this study demonstrated that the auc of the clinical risk model decreased with increasing duration of follow - up , whereas the auc of the genetic risk model increased . from the first to the fifth quintile , the clinical auc value decreased from 0.75 to 0.67 and the genetic auc value increased from 0.57 to 0.62 . in this review , we showed that study design and population characteristics may have affected the observed predictive performance of risk models . auc values of the clinical risk models were higher and , although weaker , auc values of the genetic risk models were lower when there were larger differences in age and bmi between cases and controls . this observation has important implications for the design and health care relevance of genetic risk prediction studies . the predictive ability of risk models is preferably investigated in prospective cohort studies , but in practice often only case control or cross - sectional designs are available . because clinical risk factors , particularly the difference in risk factors between cases and controls , impact auc values , it is expected that auc values for genetic risk models obtained in case control or cross - sectional studies may be valid when the distribution of these risk factors does not differ from prospective studies . for case control studies , this means that the selection of cases and controls is not affected by these risk factors . in case of selection , transparency about the methods is important to enable a correct interpretation of the scientific and health care relevance of the results . for this reason , the grips ( genetic risk prediction studies ) statement , a recently published guideline for the reporting of genetic risk prediction studies , recommends to describe eligibility criteria for participants , and sources and methods of selection of participants [ 44 ] . the observed impact of population characteristics implies that it is important to assess the predictive ability of risk scores in representative samples of the population in which the model is ultimately applied to get valid estimates of their performance in that population . the question then is : which populations do we want to target for the prevention of t2d ? evidently , these may include individuals with metabolic syndrome or overweight , but for genetic prediction this may particularly concern young individuals who have not developed clinical risk factors . to date none of the t2d risk prediction studies have been conducted in younger populations ; all studies were conducted in populations who on average were older than 40 years of age , two even in populations over 60 years of age [ 33 , 41 ] . the study that best approximates the desired study population has been conducted in a population with a mean age of 42 years , a mean bmi of 25.6 kg / m , and an almost equal number of men and women . given the observed differences in auc values , we must conclude that we do not know whether genetic variants are useful in predicting t2d risk in younger populations . none of the studies so far has started from a health care perspective when investigating the predictive ability of t2d risk models . there is increasing interest in investigating the value of genetic risk factors in the prediction of t2d risk . in this review , we demonstrated that the choice of study design and predominantly the choice of study population impact the observed predictive ability of risk models . for this reason it is important that the planning of future genetic risk prediction studies in t2d starts from a health care perspective by asking in which population we want to predict t2d risk . it is the answer to this question that determines the population in which the predictive ability should be assessed and that determines whether the results of the study ultimately can be informative and change health care practice .
the proposed scientific , medical , and technical applications of nanomaterials have been greatly increased recently . nanomaterials have unique physicochemical qualities compared to micromaterials in terms of size , surface structure , solubility , and aggregation . thus , the reduction in particle size from micro- to nanoscale might be beneficial for many industrial and scientific applications . however , nanomaterials have potential toxicities not found in micromaterials , which makes it essential to understand the biological activity and potential toxicity of the former . high dosage of manganese ( mn ) can be toxic , but it is crucial for maintaining the proper function and regulation of many biological processes . mn is a constituent of many enzymes involved in fat and protein metabolism and is utilized by various antioxidant enzymes such as mn superoxide dismutase ( mnsod ) and glutamine synthetase . additionally , this important element is involved in immune function , regulation of blood sugar , production of cellular energy , reproduction , digestion , bone growth , carbohydrate metabolism , and blood clotting . there are many manganese applications in different fields such as steel and non - steel alloy production companies , battery manufacture , colorant , pigments , ferrites , welding fluxes , fuel additives ( methylcyclopentadienyl manganese tricarbonyl ) , catalysts , and metal coating . manganese oxides have also been significant in the environmental remediation , mri diagnosis , and drug and pharmaceutical industries . manganese oxide ( mno2)-nps are promising materials that are used as contrast agents for magnetic resonance imaging ( mri ) , drug delivery , and ionization - assisting reagent in mass spectroscopy . mn is also present in nanotechnological applications such as semiconductor nanocrystals , zns , and mn2 + nanoflowers ( three - dimensional synthetic nanostructures , growing in a flower- or a tree - like shape ) . an increase in the production and use of manganese oxide nps may enhance the probable risk of occupationally exposed humans and the environment . occupational exposure to mn can result in neurological disorder , called manganism , and is similar to parkinson disease . some patients were reported to receive long - term mn - supplemented parenteral nutrition , hypermanganesaemia and altered magnetic resonance imaging ( mri ) scans ( similar to those observed in the case of manganism ) . in fact , one report suggested that even short - term total pn therapy with mn - supplementation might cause mn toxicity in patients with obstructive jaundice , followed by an increase in the blood mn concentration as a result of reduced biliary flow . since mno2 is used as a substrate for synthesis of other mn - containing compounds , therefore , a higher rate of contamination of mno2 in the environment is reported . in comparison with other forms of mn particles , , various groups have reported toxicological studies on mno2 nanoparticles , both in vitro and in vivo . these results have mainly focused on their neurotoxicity , pulmonary toxicity , hepatotoxicity , cytotoxic effects , inflammatory response , and genotoxicity . based on a previous report , change in mno2 particle size affects mn distribution and clearance from cns . chronic administration of mno2 nano- and microparticles were also associated with manganese accumulation in hepatic tissue and liver injury . in the present study , a 14-week repeated subcutaneous dose toxicity of mno2 nano- and microparticles in this experimental study , 105 male albino wistar rats ( pasteur institute , tehran , iran ) weighing 14010 g were housed in an air - conditioned colony room on a12-hour light / dark cycle ( 21 - 23c , humidity of 30 - 40% ) and supplied with standard diet and tap water ad libitum . procedures involving animals and their care were conducted in conformity with the nih guidelines for the care and use of laboratory animals . mno2 microparticles ( figure 1 ) used in this research were purchased from merck company , germany . in practice , 20 ml of kmno4 ( 0.2 mm / lit ) were mixed with 16 ml mno4 ( 0.125 mm / lit ) for 5 minutes . the resulting mixture was taken directly into a steel autoclave with teflon cover and kept for 16 h at 160c and then was cooled at room temperature . the resulting brown product was collected , washed with distilled water and ethanol 3 times , and dried with the hot air current 80c for 12 h. the resulting particles were scrutinized by an electron microscope to ensure that they were 25 to 85 nanometers in size ( figure 2 ) . a scanning electron micrograph of mno2 microparticles . a scanning electron micrograph of mno2 nanoparticles . rats were randomly divided into three groups , namely ( i ) control group received normal saline ( 1 ml / kg bw , sc ) for 14 weeks , ( ii ) mno2 nanoparticles group received mno2 nanoparticles ( 100 g / kg in saline , sc ) every two weeks for 14 weeks , and ( iii ) mno2 microparticles group received mno2 microparticles ( 100 g / kg in saline , sc ) every two weeks for 14 weeks . five rats were chosen from each group every two weeks and were deeply anesthetized with ether ( merck ) . blood sampling was provided directly from the animal heart and the spurting blood was collected in clean centrifuge tubes and allowed to clot for an hour at room temperature . it was then centrifuged at a rate of 12,000 revolutions per minute ( rpm ) for 10 min . the serum levels of glucose were measured by glucose oxidase method kit ( pars azmoon , tehran , iran ) using blood chemical analyzer ( vitalab selectra e , uk ) and its total cholesterol and triglycerides by enzymatic colorimetric , ldl , hdl were measured using standard biochemical kits by enzymatic cholesterol assay ( pars azmoon , tehran , iran ) . in this experimental study , 105 male albino wistar rats ( pasteur institute , tehran , iran ) weighing 14010 g were housed in an air - conditioned colony room on a12-hour light / dark cycle ( 21 - 23c , humidity of 30 - 40% ) and supplied with standard diet and tap water ad libitum . procedures involving animals and their care were conducted in conformity with the nih guidelines for the care and use of laboratory animals . mno2 microparticles ( figure 1 ) used in this research were purchased from merck company , germany . , with some modification . in practice , 20 ml of kmno4 ( 0.2 mm / lit ) were mixed with 16 ml mno4 ( 0.125 mm / lit ) for 5 minutes . the resulting mixture was taken directly into a steel autoclave with teflon cover and kept for 16 h at 160c and then was cooled at room temperature . the resulting brown product was collected , washed with distilled water and ethanol 3 times , and dried with the hot air current 80c for 12 h. the resulting particles were scrutinized by an electron microscope to ensure that they were 25 to 85 nanometers in size ( figure 2 ) . a scanning electron micrograph of mno2 microparticles . a scanning electron micrograph of mno2 nanoparticles . rats were randomly divided into three groups , namely ( i ) control group received normal saline ( 1 ml / kg bw , sc ) for 14 weeks , ( ii ) mno2 nanoparticles group received mno2 nanoparticles ( 100 g / kg in saline , sc ) every two weeks for 14 weeks , and ( iii ) mno2 microparticles group received mno2 microparticles ( 100 g / kg in saline , sc ) every two weeks for 14 weeks . five rats were chosen from each group every two weeks and were deeply anesthetized with ether ( merck ) . blood sampling was provided directly from the animal heart and the spurting blood was collected in clean centrifuge tubes and allowed to clot for an hour at room temperature . it was then centrifuged at a rate of 12,000 revolutions per minute ( rpm ) for 10 min . the serum levels of glucose were measured by glucose oxidase method kit ( pars azmoon , tehran , iran ) using blood chemical analyzer ( vitalab selectra e , uk ) and its total cholesterol and triglycerides by enzymatic colorimetric , ldl , hdl were measured using standard biochemical kits by enzymatic cholesterol assay ( pars azmoon , tehran , iran ) . the rats body weight gain during the 14 weeks of treatment ( figure 3 ) showed some difference between the groups . the body weight gain of animals treated with nanoparticles was continuous during the whole treatment , and significantly ( p<0.05 ) increased compared to the untreated control group during weeks 10 to 14 after injection . the weight gain of rats receiving the same dose of microparticles during weeks 8 to 14 was significantly ( p<0.05 ) lower than the control group . comparison of body weight of rats ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in body weight between groups . * control vs. nanoparticle and microparticle groups ; # nanoparticle vs. microparticle groups . the results of serum glucose level in groups 14 weeks after injections are shown in figure 4 . mno2 micro- and nanoparticles injection significantly ( p<0.01 ) increased the blood glucose level in all weeks . however , the same treatment had no effect on triglycerides concentrations , compared to the control group ( figure 5 ) . comparison of blood glucose level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in blood glucose level between groups . * control vs. nanoparticle and microparticle groups , # : nanoparticle vs. microparticle groups . comparison of blood triglyceride level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in triglyceride between groups . * control vs. nanoparticle and microparticle groups , # : nanoparticles . table 1 shows the effect of manganese particles toxicity on cholesterol level . the cholesterol level in mno2 nanoparticles group was initially decreased and then significantly increased in weeks 4 , 8 and after week 14 compared to control . in mno2 microparticles group , cholesterol level had fluctuation compared with the control group . at first , it presented a decrease and then it significantly increased until week 10 and after week 14 . comparison of blood cholesterol level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks control vs. nanoparticle and microparticles groups ; nanoparticles vs. microparticle groups . repeated measurement test showed a significant ( p<0.05 ) difference in ldl level between groups mno2 nano- and microparticles significantly ( p<0.01 ) decreased the hdl level until week 8 . however , mno2 nanoparticles increased the hdl level at week 14 , which was significantly more than the control group ( table 2 ) . comparison of blood hdl level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks control vs. nanoparticle and microparticles groups ; nanoparticles vs. microparticle groups . repeated measurement test showed a significant ( p<0.05 ) difference in hdl between groups in mno2 nanoparticles groups , ldl alterations in weeks 2 and 4 were near to the control group , and then in most weeks , it was significantly less than the control group . the ldl level in mno2 microparticles groups significantly ( p<0.01 ) decreased , compared to controls ( table 3 ) . comparison of blood ldl level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks control vs. nanoparticle and microparticle groups ; nanoparticle vs. microparticle groups . the rats body weight gain during the 14 weeks of treatment ( figure 3 ) showed some difference between the groups . the body weight gain of animals treated with nanoparticles was continuous during the whole treatment , and significantly ( p<0.05 ) increased compared to the untreated control group during weeks 10 to 14 after injection . the weight gain of rats receiving the same dose of microparticles during weeks 8 to 14 was significantly ( p<0.05 ) lower than the control group . comparison of body weight of rats ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in body weight between groups . * control vs. nanoparticle and microparticle groups ; # nanoparticle vs. microparticle groups . the results of serum glucose level in groups 14 weeks after injections are shown in figure 4 . mno2 micro- and nanoparticles injection significantly ( p<0.01 ) increased the blood glucose level in all weeks . however , the same treatment had no effect on triglycerides concentrations , compared to the control group ( figure 5 ) . comparison of blood glucose level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in blood glucose level between groups . * control vs. nanoparticle and microparticle groups , # : nanoparticle vs. microparticle groups . comparison of blood triglyceride level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks . repeated measurement test showed a significant ( p<0.05 ) difference in triglyceride between groups . * control vs. nanoparticle and microparticle groups , # : nanoparticles . table 1 shows the effect of manganese particles toxicity on cholesterol level . the cholesterol level in mno2 nanoparticles group was initially decreased and then significantly increased in weeks 4 , 8 and after week 14 compared to control . in mno2 microparticles group , cholesterol level had fluctuation compared with the control group . at first , it presented a decrease and then it significantly increased until week 10 and after week 14 . comparison of blood cholesterol level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks control vs. nanoparticle and microparticles groups ; nanoparticles vs. microparticle groups . repeated measurement test showed a significant ( p<0.05 ) difference in ldl level between groups mno2 nano- and microparticles significantly ( p<0.01 ) decreased the hdl level until week 8 . however , mno2 nanoparticles increased the hdl level at week 14 , which was significantly more than the control group ( table 2 ) . comparison of blood hdl level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during 14 weeks control vs. nanoparticle and microparticles groups ; nanoparticles vs. microparticle groups . repeated measurement test showed a significant ( p<0.05 ) difference in hdl between groups in mno2 nanoparticles groups , ldl alterations in weeks 2 and 4 were near to the control group , and then in most weeks , it was significantly less than the control group . the ldl level in mno2 microparticles groups significantly ( p<0.01 ) decreased , compared to controls ( table 3 ) . comparison of blood ldl level of rat ( n=6 ) in mno2 microparticles or nanoparticles ( 100 g / kg ) treated groups during the 14 weeks control vs. nanoparticle and microparticle groups ; nanoparticle vs. microparticle groups . as the results of the present study indicated , body weight gain of the animals treated with mno2 nanoparticles significantly increased compared to microparticle groups in which a significant decrease was observed . the present investigation also demonstrated that exposure to micro- and nanoparticles of mno2 induced significant hyperglycemia effect in rats . it is important to understand the cause of changes in body weight gain and glucose level and their correlation induced by mno2 particles . hyperglycemia disorder is caused by the relative deficiency of insulin secretion and varying degrees of insulin resistance and is characterized by high circulating glucose . several pathogenic pathways are activated in diabetes among which reactive oxygen species ( ros ) , generated by high glucose levels , are responsible for metabolic abnormalities and chronic complications . moreover , any imbalance in the production and scavenging of ros leads to excessive levels of either molecular oxygen or ros . oxidative stress. mno2 nanoparticles have a higher oxidation power in comparison with other forms of mn particles . proposed that manganese is transported to organs rich in mitochondria ( in particular the liver , pancreas , and pituitary ) where it is rapidly concentrated . the ability of mno2 nanoparticles in generating ros and induction of lipid peroxidation , restore the imbalances in the antioxidants and liver enzymes responsible for the cell dysfunction and destruction ; and might lead to tissue injury and hyperglycemia in our test groups . since the k - atpase has a significant role in insulin secretion of the pancreas ; hyperglycemia indicates that insulin secretion process may be affected by mno2 . it has been reported that activities of total , na / k , mg and ca / atpases are significantly inhibited in a dose - dependent manner in rats brain after exposure to mno2-nps . huang et al . observed a significant decrease in the activities of na / k , mg and ca / atpases in hepatocyte mitochondria after 30 days of i.p . there has been no study , until now , which has investigated the effects of mno2 nanoparticles in blood glucose . in the present study , mno2 nanoparticle showed to be quite effective in lipid metabolism by the decreased ldl and hdl fraction and the increased plasma cholesterol without a concomitant increase in triglycerides . in comparison to controls , rats exposed to the evidence for mno2-induced disruptions in lipid metabolism is shown in the increase of cholesterol and decrease of hdl and ldl levels in plasma without a concomitant increase in triglycerides . the various forms of lipids can not dissolve in the blood and must be transported to / and from the cells by low - density and high - density lipoproteins . high - density lipoprotein cholesterol ( hdl - c ) tends to carry cholesterol away from the arteries back to the liver . as a result hdl enables lipids like cholesterol and triglycerides to be transported within the water - based bloodstream . hdl particles are able to remove cholesterol from within artery atheroma and transport it back to the liver for excretion or re - utilization , which is the main reason for calling that cholesterol carried within hdl particles ( hdl - c ) good cholesterol ( despite the fact that it is exactly the same as that cholesterol in ldl particles ) . those with higher levels of hdl - c seem to have fewer problems with cardiovascular diseases while those with low hdl - c cholesterol levels increase the rate of heart disease . when ldl particles are within the blood vessel walls and oxidized by free radicals , they appear harmless . in previous studies , it has been reported that the administration of other metals such as lead and cadmium to experimental animals affects lipid metabolism . the histopathological studies at our laboratory have revealed the toxic effects of nanoparticles on the liver and kidney organs . mno2 exposure produced pronounced hepatic histopathology ; evidenced by histological alternations in the liver , including focal necrosis with hepatocyte vacuolization and swelling , pyknotic nuclei , and dilation of central vein and sinusoids . it is reported that nanoparticles interact with proteins and enzymes and interfere with the antioxidant defense mechanism , leading to ros generation causing apoptosis and necrosis . a previous study reported a significant increase in dna damage in leukocytes , micronuclei and chromosomal aberrations in bone marrow cells after exposure to mno2-nps and mno2-mps . in addition , dna damage and ros production were reported in the liver organ when mncl2 was given in drinking water to male wistar rats for 30 consecutive days . in rats at 5 , 10 , and 20 mg / kg b.w daily for 3 months , showed a significant increase in mitochondrial dna damage in the rat brain and liver . the mechanisms responsible for the genotoxicity of nps involve oxidative stress , which causes redox imbalance within cells usually as a result of an increase in intracellular ros . similarly , oral administration of mncl2 ( 20 mg / ml ) for 30 days increased the activities of hepatotoxicity biomarkers such as ast , alt , and ldh levels compared to the control in male wistar rats . recently , many studies have been conducted on the application of ( mno2)-nps in mri and drug delivery . however , their toxic effects can not be ignored . in the case of probable toxic effect the toxicity of repeated subcutaneous injection of manganese nanoparticles ( 25 - 85 nm ) in a rat was studied comparatively with manganese microparticles ( 3 m ) . both particles induced hyperglycemia and alteration of serum lipid profile in male wistar rats . therefore , it can be concluded that both particles adversely affect the serum lipid profile and glucose level . this study was designed to achieve its objectives as mentioned above . however , the potential limitation of the study was changes in manganese in serum and the synthesis mno2 nanoparticles . this can be addressed in future studies to elucidate the role of oxidative stress by measurement ( gsh and antioxidant enzymes , e.g. sod ) . the results of the present study suggest that mno2 nano- and microparticles induced pancreas toxicity , providing further details of the molecular mechanism underlying mno2 toxicity .
across the flowering plants ( the angiosperms ) , bilaterally symmetrical ( zygomorphic ) flowers are thought to have evolved many times independently from radially symmetrical ( actinomorphic ) ancestors . transitions to bilateral flower symmetry have been associated with the evolution of specialized pollinators and have been crucial in the diversification of flowering plants . zygomorphic flowers have dorsal ( adaxial ) organs that are morphologically different from ventral ( abaxial ) ones ( figure 1a ) . bilaterally symmetrical corollas ( petal whorls ) help promote the approach of pollinators from one particular orientation . in addition , the dorsalmost and/or ventral stamens are often aborted , leaving only a rudimentary stamen ( staminode ; figure 1a ) . this can facilitate access to the remaining stamens by pollinators or increase the specificity of pollen deposition during pollinator visits . independent recruitment of cyc - like genes for the evolution of floral zygomorphy and stamen reduction . ( a ) images and diagrams of flowers of several lamiales lineages , illustrating the diversity in stamen reduction . antirrhinum , mohavea , veronica and gratiola are members of the plantaginaceae ; opithandra is a member of the gesneriaceae . shading indicates the approximate expression of at least one cyc - like tcp homolog in each of these lineages ; x indicates the presence of a staminode . in veronica , gratiola and opithandra , at least one other close paralog of the cyc - like gene whose expression is illustrated has a highly divergent pattern of expression . in mohavea and opithandra , expression correlates with additional stamen reduction compared with antirrhinum . in veronica and gratiola , there is no correlation between cyc - like gene expression and additional stamen reduction . in veronica , staminodes are absent in the dorsal and ventral flower regions where stamen loss is inferred . ( b ) three of many independent transitions from radial floral symmetry to bilateral symmetry across the core eudicot lineage are indicated in bold . functional studies of a. majus , l. japonicus , p. sativum and i. amara have demonstrated that developmental genetic pathways using cyc - like tcp genes have been independently recruited to establish bilateral flower symmetry . groundbreaking comparative studies over the past few years have demonstrated that cycloidea ( cyc)-like genes , which belong to the class ii tcp family of transcription factors , have been recruited multiple times to pattern dorsal flower identity in core eudicot lineages that have independently evolved zygomorphic flowers ( reviewed in ; figure 1b ) . until recently , cyc - like genes had been mostly thought to be related to the control of dorsal and lateral floral organ development . their work contributes significantly to the growing body of evidence that changes in the expression and/or function of tcp genes have been a powerful tool , recruited multiple times , to generate novel floral morphologies . specific effects vary depending on the tissue in which the genes are acting . not surprisingly , their activity is tightly controlled , both spatially and temporally , as subtle alterations in their regulation usually lead to noticeable phenotypic effects that are , in most cases , deleterious . however , some of these regulatory changes have been maintained during evolution , probably by natural selection , giving rise to adaptive novel traits such as corolla zygomorphy and stamen abortion ( reviewed in ) . in antirrhinum majus ( snapdragon , family plantaginaceae ) , cyc and its close paralog dichotoma ( dich ) are expressed early in the dorsal domain of the flower meristem , where they limit the rate of cell proliferation and primordium initiation . later , they continue to be expressed in dorsal petals to control their size and shape and in the dorsalmost stamen primordium , where they cause abortion of this organ to form a staminode ( figure 1a ) . cyc - like genes have been recruited several times independently during angiosperm evolution to carry out this function ( reviewed in ) . a possible explanation for the repeated co - option of cyc - like genes comes from studies in arabidopsis , a species with radially symmetrical flowers . the arabidopsis cyc - like gene , like cyc in snapdragon , is expressed dorsally in floral meristems , even though the meristems are destined to form radially symmetrical flowers . this suggests that ancestral species with radially symmetrical flowers may have had cyc - like genes dorsally expressed in flower meristems . this incipient asymmetry could then have been recruited several times independently , by changes in timing of expression and/or interactions with target genes , to generate bilaterally symmetrical flowers ( reviewed in ) . evidence for the independent recruitment of cyc - like genes for the development of floral zygomorphy comes largely from studies in the core eudicot lineages fabales and brassicales . bilateral flower symmetry is a prominent condition within the pea family , leguminoseae ( fabales , figure 1b ) . in two emerging model legume species , lotus japonicus and pisum sativum , gene expression and functional analyses both implicate cyc - like genes in the control of bilateral flower symmetry . in these species , cyc - like gene expression is restricted to dorsal or dorsal plus lateral regions of developing flowers , similar to cyc expression in snapdragon . more compelling than the correlation between cyc - like gene expression and zygomorphy are the functional data that demonstrate a role for cyc - like genes during dorsal flower development . specifically , ectopic or reduced expression of cyc - like genes in l. japonicus and p. sativum disrupts wild - type patterns of dorsoventral symmetry , resulting in dorsalized or ventralized flower phenotypes , respectively . although bilateral flower symmetry is not the norm in the mustard family , brassicaceae ( brassicales , figure 1b ) , a cyc - like gene has been implicated in the evolutionary transition to floral zygomorphy in the candytuft ( iberis ) , which is closely related to arabidopsis . the dorsal petals of iberis amara are reduced in size relative to the ventral petals , and a cyc - like gene in i. amara is specifically expressed in later stages of dorsal petal development as they differentiate in size from ventral petals . in i. amara peloric mutants ( radially symmetric flowers ) , dorsal petals are similar in size to ventral petals and lack the wild - type pattern of dorsal - petal - specific cyc - like gene expression . in addition , heterologous functional studies in arabidopsis demonstrate that i. amara cyc - like genes function to reduce petal growth , consistent with dorsal petal morphology in i. amara . beyond a role for establishing corolla zygomorphy in multiple eudicot lineages , changes in the expression of cyc - like genes are correlated with stamen number evolution . cyc expression during snapdragon flower develop ment is necessary for dorsal stamen abortion ( figure 1a ) . in the close relative of snapdragon , the desert ghost flower ( mohavea confertiflora , plantaginaceae ) , increased number of sterile staminodes ( dorsal plus lateral ) correlates with lateral expansion of the domain of cyc - like gene expression into lateral stamen primordia ( reviewed in ; figure 1a ) . these studies of corolla symmetry and stamen number evolution both illustrate that the function of cyc - like genes in core eudicots is generally related to the control of dorsal and lateral floral organs , with a possible exception in asteraceae , leaving open the question of whether cyc - like genes might just as easily be co - opted to pattern ventral flower morphology . recently , two studies explicitly addressed the question of whether changes in regulation of cyc - like genes might explain evolutionary novelty in ventral flower morphology , specifically abortion of ventral stamens . preston and hileman found no evidence that shifts in the expression of cyc - like genes correlate with ventral stamen abortion in veronica and gratiola ( plantaginaceae , figure 1a ) . provide the first evidence for a function of cyc - like genes in the abortion of ventral stamens . this ventral activity is associated with a new expression domain in ventral stamen primordia of opithandra ( gesneriaceae , figure 1a ) . by evolving a new domain of expression this cyc - like gene has acquired not a novel role but the ability to carry out the same role in a new position . the evidence that cyc - like genes have been recruited multiple times in the evolution of floral zygomorphy , along with these exciting new data from opithandra , open up the possibility that cyc - like genes may have a role in the evolution of diverse patterns of stamen abortion . these recent data suggest that cyc - dependent floral modifications may evolve without restriction to dorsal / lateral positioning - and this might even extend to a role for cyc - like genes in the development of unisexual flowers . indeed , there is a strong correlation between cyc - like gene expression and stamen loss in maize female flowers . the fact that cyc - like genes have been recruited for the evolution of ventral stamen abortion in the lineage leading to opithandra , but not in the lineages leading to veronica or gratiola , suggests that as additional , independently derived reductions in stamen number are explored , convergent genetic mechanisms affecting cell proliferation are likely to be identified . this stands in contrast to the growing body of evidence that transitions to floral zygomorphy recurrently involve the recruitment of a cyc - dependent developmental pathway . the recent studies discussed above illustrate how tcp genes , in particular class ii tcp genes , have greatly contributed to the evolution of novel morphological traits and the modification of existing ones . this may be due to their great capability to alter the growth patterns of tissues in which they are expressed ( reviewed in ) . extensive duplication and diversification during plant evolution may have facilitated their co - option multiple times in morphological transitions . it is now understood that groups of class ii tcp genes are transiently expressed in different developing tissues , such as flower and shoot meristems and leaf and floral organ primordia , where they help give shape to these structures . indeed , these genes not only control floral organ number , petal shape and stamen abortion ( cyc - like genes ) but they also have strong effects in leaf shape , size and curvature ( cincinnata genes ) and prevent branch outgrowth ( tb1/branched1 genes ; reviewed in ) . given that they control basic developmental processes related to tissue proliferation and differentiation , it is perhaps not surprising that tcp genes have been recruited many times independently in the evolution of plant developmental patterning . the field is now open for exploring how evolutionary changes in this critical gene family have affected other diverse aspects of plant form . pc 's work is supported by spanish micinn grants gen2006 - 27788-e , bio2008 - 00581 and csd2007 - 00057 .
among the etiologic agents of infectious disease , staphylococcus aureus , especially methicillin - resistant staphylococcus aureus ( mrsa ) strains are the main agents of human infections ( 1 ) . this pathogen can cause nosocomial infection in such a manner that it is the most common pathogen isolated from patients in hospitals that can cause wide range of infections from skin and soft tissue infections till toxic shock syndrome and life threatening syndromes . during the end of 1990s , increasing of s. aureus infections was led to isolation of mrsa ( 2 - 5 ) . nearly , all s. aureus isolates produce various toxins and enzymes that are important in the pathogenicity of this bacterium ( 6 , 7 ) . with the emergence of resistant strains at 1961 that had multidrug resistance , the mrsa stains are widely distributed around the world that shows their resistance to both penicillins and cephalosporins ( 8 , 9 ) . the mrsa produces a unique type of penicillin binding protein 2a ( pbp2a ) that has low affinity for -lactam antibiotics ( 10 , 11 ) . most of mrsa strains can also produce a leukotoxin as panton - valentine leukocidin ( pvl ) that increases their virulence and can cause severe necrotic pneumonia ( 8 , 12 ) . pathogenicity of s. aureus results from the surface cell wall structures and different exoproteins that pvl is one of the most important of them for overcoming host immunity and disease progress ( 13 ) . pore forming toxins perforate membranes of host cells , mainly the plasma membrane and also intracellular organelle membranes . they directly kill target cells , in order to intracellular delivery of other bacterial or external factors , releasing nutrients or escaping from phagosomal space . panton - valentine leukocidin is a member of pore forming toxins that targets host leukocytes . the pvl- producing strains are commonly involved in the skin and soft tissue infections as well as able to cause life threatening infections like pneumonia ( 7 , 8 , 12 ) . two open reading frames are responsible for coding pvl , i.e. luks - pv and lukf - pv . the presence of pvl gene is a genetic marker for the mrsa populations ( 14 ) . recently , reports about infections caused by pvl - positive strains have been increased that necessitate investigations about the prevalence of this virulent marker among mrsa strains in hospital and also community acquired infections . the aim of the present study was to discover the association of pvl and meca in mrsa strains isolated from hospitalized patients in three educational hospitals of ahvaz city , iran . fifty s. aureus isolates were collected from patients in three educational hospitals of ahvaz , including imam khomeini , golestan and taleghni hospitals . these samples were obtained from 200 samples collected from different sections of the above - mentioned hospitals including outpatient department ( opd ) , skin , men surgery , ophthalmology , nephrology , pediatric , women , orthopedic , ear , nose and throat ( ent ) , neonates and internal medicine . samples were collected from skin lesions , blood cultures , burns , intravenous catheters , wound drainages , abscesses , tracheal secretions , synovial fluids , ocular secretions and urines . these isolates were identified based on standard morphological and biochemical tests including catalse , oxidase , tube coagulase , mannitol fermentation , sensitivity to furazolidon , resistance to bacitracine , pyr test and voges - proskauer ( 15 ) . in order to determine methicillin resistance of isolates muller - hinton agar ( mha , merck , germany ) screening test was used according to clsi ( clinical and laboratory standards institute ) ( 16 ) . briefly , a 0.5 mcfarland suspension was prepared from pure culture of isolates and then cultured on mha containing oxacillin ( 6 g / ml ) . for this purpose using a sterile swab the bacterium was inoculated as a dot with a 10 - 15 mm diameter and a strike culture was prepared on the other part of plate and incubated at 35c . growth of only one colony on this culture medium revealed methicillin resistance of that isolate . staphylococcus aureus atcc 29213 as the negative control and sensitive to methicillin and s. aureus atcc 33591 as the positive control and resistant to methicillin were used in all experiments . furthermore , the susceptibility of isolates to oxacillin was surveyed using the standard kirby - bauer disc diffusion method ( 17 ) . the isolates were cultured in trypticase soy broth ( tsb , merck , germany ) for 18 h at 37c . then , 1.5 ml of culture was centrifuged ( 12000 rpm , 10 minutes ) and the precipitate was dissolved in one ml deionized water and boiled for 20 minutes . after centrifugation ( 5000 rpm , 5 minutes ) the supernatant was slowly mixed with phenol - chloroform - isoamyl alcohol ( 1 : 24 : 25 ) at 1 : 1 ratio and the aqueous phase was harvested by centrifugation at 13000 rpm , 30 seconds . cold isopropanol was added to aqueous phase ( 60.100 , v / v ) and stored overnight at -22c . finally , dna was air - dried at 37c and dissolved in 200 l sterile deionized water and stored at -22c till experiments ( 18 ) . polymerase chain reaction ( pcr ) was performed in a final 25 l volume reaction containing pcr buffer ( 10x ) , magnesium chloride ( mgcl2 ) ( 2 mm ) , deoxynucleotide triphosphates ( dntps ) ( 0.2 mm ) , forward and reverse primers ( 10 mol/l ) , template dna ( 1 l ) , taq dna polymerase ( 1.5 u ) and deionized water . the pcr method was performed in thermal cycler ( bio - rad , usa ) according to the following program : initial denaturation ( 94c , 2 minutes ) , 35 cycles each composed of initial denaturation ( 94c , 30 seconds ) , primer annealing ( 55c , 45 seconds ) and extension ( 72c , 75 seconds ) and a final extension ( 72c , 4 minutes ) ( 19 ) . positive control for meca and pvl genes and negative control ( distilled water ) were also regarded in each series of pcr reaction . the pcr product was subjected to electrophoresis in 1% agarose gel containing dna safe stain and documented using gel documentation ( uvi tec , cambridge , uk ) . fifty s. aureus isolates were collected from patients in three educational hospitals of ahvaz , including imam khomeini , golestan and taleghni hospitals . these samples were obtained from 200 samples collected from different sections of the above - mentioned hospitals including outpatient department ( opd ) , skin , men surgery , ophthalmology , nephrology , pediatric , women , orthopedic , ear , nose and throat ( ent ) , neonates and internal medicine . samples were collected from skin lesions , blood cultures , burns , intravenous catheters , wound drainages , abscesses , tracheal secretions , synovial fluids , ocular secretions and urines . these isolates were identified based on standard morphological and biochemical tests including catalse , oxidase , tube coagulase , mannitol fermentation , sensitivity to furazolidon , resistance to bacitracine , pyr test and voges - proskauer ( 15 ) . in order to determine methicillin resistance of isolates muller - hinton agar ( mha , merck , germany ) screening test was used according to clsi ( clinical and laboratory standards institute ) ( 16 ) . briefly , a 0.5 mcfarland suspension was prepared from pure culture of isolates and then cultured on mha containing oxacillin ( 6 g / ml ) . for this purpose using a sterile swab the bacterium was inoculated as a dot with a 10 - 15 mm diameter and a strike culture was prepared on the other part of plate and incubated at 35c . growth of only one colony on this culture medium revealed methicillin resistance of that isolate . staphylococcus aureus atcc 29213 as the negative control and sensitive to methicillin and s. aureus atcc 33591 as the positive control and resistant to methicillin were used in all experiments . furthermore , the susceptibility of isolates to oxacillin was surveyed using the standard kirby - bauer disc diffusion method ( 17 ) . the isolates were cultured in trypticase soy broth ( tsb , merck , germany ) for 18 h at 37c . then , 1.5 ml of culture was centrifuged ( 12000 rpm , 10 minutes ) and the precipitate was dissolved in one ml deionized water and boiled for 20 minutes . after centrifugation ( 5000 rpm , 5 minutes ) the supernatant was slowly mixed with phenol - chloroform - isoamyl alcohol ( 1 : 24 : 25 ) at 1 : 1 ratio and the aqueous phase was harvested by centrifugation at 13000 rpm , 30 seconds . cold isopropanol was added to aqueous phase ( 60.100 , v / v ) and stored overnight at -22c . finally , dna was air - dried at 37c and dissolved in 200 l sterile deionized water and stored at -22c till experiments ( 18 ) . polymerase chain reaction ( pcr ) was performed in a final 25 l volume reaction containing pcr buffer ( 10x ) , magnesium chloride ( mgcl2 ) ( 2 mm ) , deoxynucleotide triphosphates ( dntps ) ( 0.2 mm ) , forward and reverse primers ( 10 mol/l ) , template dna ( 1 l ) , taq dna polymerase ( 1.5 u ) and deionized water . the pcr method was performed in thermal cycler ( bio - rad , usa ) according to the following program : initial denaturation ( 94c , 2 minutes ) , 35 cycles each composed of initial denaturation ( 94c , 30 seconds ) , primer annealing ( 55c , 45 seconds ) and extension ( 72c , 75 seconds ) and a final extension ( 72c , 4 minutes ) ( 19 ) . positive control for meca and pvl genes and negative control ( distilled water ) were also regarded in each series of pcr reaction . the pcr product was subjected to electrophoresis in 1% agarose gel containing dna safe stain and documented using gel documentation ( uvi tec , cambridge , uk ) . from a total of 50 samples , 27 samples ( 54% ) were belonged to imam khomeini hospital , 16 samples ( 32% ) from golestan hospital and 7 samples ( 14% ) from taleghani hospital . according to the patient gender , 26 samples ( 52% ) were isolated from men while 24 samples ( 48% ) were from women . the number of samples from different clinical specimens and hospital sections are presented in tables 2 and 3 , respectively . the results of antibiotic susceptibility tests revealed that all isolates were resistant to methicillin . following pcr reaction with meca and pvl specific primers , 314 bp and 433 bp pcr products were produced for meca and pvl genes , respectively ( figure 1 ) . 1 : positive control for pvl ; 2 and 3 : positive pvl clinical isolates ; 4 , 5 , 6 , 8 and 9 : positive meca in clinical isolates ; 7:100 bp molecular weight marker ; 10 : negative control ( distilled water ) and 11 : positive control for meca . table 4 shows the total number of mrsa isolates and their meca and pvl markers . based on the obtained results , 30% ( 15 samples ) of mrsa strains were positive for meca gene while 6% ( 3 samples ) had pvl gene . staphylococcus aureus is one of the most common pathogens that have been isolated from patients in hospitals , especially mrsa strains are among important nosocomial infections for humans ( 1 ) . having knowledge about the prevalence of mrsa and their virulence factors is useful for treatment and control of community and hospital acquired s. aureus infections . following the emergence of mrsa strains , molecular studies showed that some of these isolates carry pvl gene ( 20 ) . the pvl positive strains lead to infections with different clinical appearance even in immunocompromised patients lead to necrotic pneumonia , which its mortality can be as much as 75% ( 8) . therefore , frequent monitoring of this pathogen , its antibiotic susceptibility and determining their virulence factors is of great importance in control and treatment of infections . according to the results of this study , this explains that it maybe meca positive mrsa isolates have different mechanism for methicillin resistance than meca . these results are in agreements with other reports such as reported by bagdonas et al . in lithuania ( 23.4% ) ( 12% ) ( 23 ) and also in different parts of iran , e.g. fatholahzadeh et al . in tehran ( 36% ) ( 24 ) and by japoni et al . in shiraz ( 43% ) ( 25 ) . however , the frequency of meca positive isolates in this study is more than the results of bagdonas et al . turnidge et al . and li et al . while is less than the results obtained by fatholahzadeh et al . ( this finding is a hopeful result that the frequency of meca in ahvaz is yet less than other studied areas of iran . the occurrence of pvl gene in previous studies has been reported from 2% - 35% ( 26 , 27 ) . in comparison the obtained results for pvl gene in the present study it is possible that meca and pvl genes but not both of them be present on the chromosome of clinical isolates of s. aureus . the obtained results in the present study are in agreement with the finding of okon et al . ( 9 ) that reported no pvl positive strain is there among tested mrsa strains . ( 27 ) have suggested it is maybe that resistance determinants be carried on other mobile elements , such as plasmids , transposons , and phages ; so , their elimination from bacterial cell would result in the absence of meca gene and consequently no association with pvl gene . ( 28 ) also reported no significant association between pvl and meca in s. aureus isolates from cotonou . however , the pvl gene has been reported in some mrsa in other studies ( 29 , 30 ) . according to the results of this study , pcr assay for mrsa gene can be useful for definite diagnosis of mrsa strains . identification of meca positive strains can be used as a guide for separating infected patients from others in hospital environment in order to prevent gene transfer among clinical strains and also distribution of virulent factors .
vogt koyanagi harada ( vkh ) syndrome is a rarely - seen multi - systemic , autoimmune and inflammatory disease . it observed frequently with neurologic , auditory and skin manifestations and characterized with bilateral , chronic and diffused granulomatous panuveitis . a 57 year - old female patient applied to a special center one year ago with a complaint of decrease in the sight acuity of the right eye . uveitis was developed firstly in the right eye and then in the left eye after the operation . having complaints about uveitis , tinnitus and hear loss , the patient was diagnosed with vkh syndrome . the pains started to be felt in small hand joints and both of the two ankles . being diagnosed with seronegative rheumatoid arthritis ( ra ) , our case is presented because vkh syndrome is rarely seen in turkey , and the joint findings are at the forefront . vogt koyanagi harada syndrome is a disease with chronic course characterized with neurologic and skin symptoms besides the bilateral granulomatous pan uveitis ( 1 ) . although the etiology of the disease is not known exactly , it is thought to be caused by autoimmunity created against antigen controlled via t - lymphocytes and associated with melanocytes ( 2 ) . vkh disease is more prevalent in certain racial and ethnic groups , particularly in asian individuals and certain latin - american groups . and individuals have certain hla groups in japan ( 3 , 4 ) . vogt koyanagi harada syndrome diagnosis is based on making distinguishing it from other uveitis reasons with clinic and laboratory findings ( 5 ) . besides neurologic and skin symptoms , the disease consists of typical bilateral exudative retina decollement in eyes , palillitis , choroiditis and iridocyclitis . in this article , the clinical course of a vkh case with delayed diagnosis having ocular and systemic findings suppressed due to corticosteroid treatment is presented . its etiology is not nown properly , but it is also known that it generally affects the joints . on the other hand , it has a significant extra - articular involvement with nearly all organ systems influenced . it has been proved that there is a genetic component transferred through hl a - dq and - dr alleles ( 6 ) . being diagnosed with seronegative rheumatoid arthritis diagnosis , our case is our case is presented because vkh syndrome is rarely seen in turkey . a 57 year - old female patient applied to a special center one year ago with a complaint of decrease in the sight acuity of the right eye . uveitis was developed firstly in the right eye and then in the left eye after the operation . the patient was suit for the vkh syndrome because of the ophthalmologists ' opinion , patients ' age , and skin and auditory involvements . she patient was diagnosed vkh syndrome according to uveitis , tinnitus and hearing sense loss complaints . the pains started to be felt in small hand joints and both of the two ankles . , there was redness in the cornea of left eye and the 3 cm alopecic region in hairy skin . there were pressure - induced pains in proximal interphalangeal joints of both hands , metacarpaphalangeal joints of the 4 and 5 fingers in the left hand , wrists , elbows , and both of the two achilles tendons . lumbar region , hip and sacroiliac joint examinations were normal . in neurologic examination , bilateral visual acuity and hear loss were identified . in laboratory evaluation , the hemogram values were normal , sedimentation was 36 mm / h and crp was 1.0 mg / dl ; rheumatoid factor , anti - nuclear antibody , anti ds - dna , anti - ccp , and hla b27 tests were negative . there were erosions in the pif joints in the hand radiography of patient ( figure 1 ) . , the patient diagnosed with seronegative rheumatoid arthritis diagnosis , and the consensus was reached with ophthalmic polyclinic about vkh and seronegative rheumatoid arthritis association . the patient was taken to follow - up with metothrexate 10 mg / week , prednisolone 5mg / day treatment schedule . vogt koyanagi harada syndrome is a multi - systemic disease characterized with granulomatous inflammation , and affecting the auditory system , the meninx and the skin ( 1 ) . despite that , it is rarely seen , has chronic course and the potential of leading to blindness ( 4 ) . although its prevalence in our country is not known , it has been reported that it constitutes 10% of uveitis in japan ( 7 ) , and its annual prevalence is 15.5 per million ( 8) . vogt koyanagi harada syndrome is generally seen in young middle aged individuals , and more frequently in women ( 8) . patients having autoimmune vestibulitis apply with tinnitus complaint , while patients with meninx involvement apply with inflammation , nausea and severe headache . patients with skin involvement have hypo - pigmentations around the hands and the face ( 1 , 5 , 8) . besides all of those findings , while wide choroid inflammation , exudative retina decollement and papillitis accompany the early stages of this disease , some ocular findings such as coroidal depigmentation are observed in late stages ( 9 ) . especially , because it develops bilateral granulomatous uveitis , although it can be confused with sympathetic ophthalmia , it is distinguished from that disease since it does not have vkh trauma background ( 10 ) . vogt koyanagi harada syndrome constitutes 8% of endogen and uveitis in japan and 14% in uveitis in mixed races in usa . it has been reported in a study that extra ocular findings in vkh syndrome vary depending on races and ethnic groups . while the prevalence values of meningismus , dysacousia , alopecia , polisis and vitilligo are 80% , 60% , 60% and 25% respectively , dermatological findings have been observed much less frequently . although the first symptoms of the disease appear generally between the ages of 20 and 50 , it has been reported in a 4 years old child . in genetic factor it is assumed that a t - cell - mediated autoimmune reaction is created against common membrane antigen in melanocyte and/or neural crest originated tissues ( 5 ) . this autoimmune response generally results especially with collapse of melanocytes in epidermis , cochlea , meninx and uvea ( 12 ) . the case had joint involvement and morning stiffness in her little joints in the hands and the feet . there was erosions in pif joints in the hand radiography of the patient ( figure 1 ) . although generally steroid is used in the treatment of vkh syndrome , methotrexate is added into the treatment because there are arthritic findings . as a result , the system involvement shows variation depending on geographic regions and ethnic origin . also , it is interesting that both of the two diseases have relationship with hla genes . with these results , it is a matter of debate provided that the vkh syndrome of our case is joint involvement or seronegative ra .